- Nov 2022
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Referee #4
Evidence, reproducibility and clarity
Summary:
The manuscript by Liang et al. shows that, while overall translation is reduced under hypoxic conditions, translation of Ldh mRNA substantially increases. This increase is demonstrated to depend upon the Ldh 3' UTR and the variant translation factor eIF4EHP. The manuscript further shows that eIF4EHP associates with polysomal fractions and with Ldh mRNA under hypoxic conditions and that it is enriched in cytoplasmic foci apparently distinct from stress granules and P bodies in hypoxia. Finally, the paper provides evidence that loss of eIF4EHP worsens the survival rate of flies in reduced oxygen conditions.
Major comments:
The experimental data largely support the conclusions that are drawn but I have some important reservations.
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In human cells eIF4E2 (the eIF4EHP orthologue) can activate translation in hypoxia by forming a complex with HIF2alpha, RBM4, and eIF4G3. The paper states in the discussion that inhibition of the Drosophila eIF4G3 counterparts eIF4G2 and NAT1 does not affect translation under hypoxia. However, the data behind this important conclusion are not shown, nor are details given as to how eIF4G2 and NAT1 were 'inhibited', whether or not both were inhibited at the same time, and whether the fly RBM4 orthologue Lark was investigated. While the mechanism of how eIF4EHP activates translation must be different in Drosophila from that in human cells because of the absence of HIF2alpha, not much more can be concluded than that in the absence of explicit experimental data.
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Fig 3B shows a large increase in LDH levels under hypoxic conditions in eIF4EHP KO cells, which is inconsistent with the narrative description of this result and the paper's conclusions. Ratios of LDH in normoxic and hypoxic conditions for eIF4EHP KO and control cells should be quantitated from multiple experiments, compared, and tested for statistical significance.
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Statistical significance should also be shown for the data in Fig 3E. I note that the empty vector control reduces puromycin incorporation by quite a lot.
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The number of survivors is very low for both control and eIF4EHP KD flies in Fig 7A. Are these comparisons statistically significant?
Minor comments:
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It should be explained why eIF4E6 KO was included in Fig 3.
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I assume that 6% oxygen was used for the viability experiment in Fig 7A because the lower concentration of 1% used in all other experiments would be lethal to both control and KD flies. This reasoning should be made explicit.
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There is a great deal of sloppiness about nomenclature. The FlyBase term for the molecule of interest is eIF4EHP. Within this manuscript I found it referred to as eIF4EHP, eiF4EHP, eIF4HP, eiF4HP, and ei4EHP. This requires correction.
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The references are also sloppy and presented in several different formats.
Significance
As mentioned above, it is established in human cells that the orthologue of eIF4EHP can activate translation under hypoxic conditions. So the basic result in this manuscript simply confirms that the same phenomenon occurs in flies. As mentioned in the previous section, in human cells eIF4EHP activates translation in a 3' UTR dependent manner as part of a complex containing HIF2alpha, RBM4, and eIF4G3. While a different mechanism is likely to exist in Drosophila, it is not elucidated by the experimental data presented in this paper. Therefore I find the work to be of limited significance.
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Referee #3
Evidence, reproducibility and clarity
Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. The authors present extensive, compelling results to support their conclusions. However, we still find this work can be improved in several aspects:
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The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact.
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One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and o eIF4EHP are not connected by experimental tests. Experiments aimed at determining the functional relation of the 3' UTR and eIF4EHP could enhance the paper and deliver a more complete story about the mechanism of selective translation under hypoxia.
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The authors refer to human cell studies showing that HIF2a is involved in mRNA translation in hypoxic conditions. They mentioned in the discussion that Drosophila Sima has not been identified to interact with eIF4EHP. To test whether Sima regulates to Ldh translation, the authors could test the involvement of Sima in experiments from Fig 6.
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In Fig 3A-B, induction of LDH by hypoxia is almost completely blocked by eiF4EHP knockdown in fly heads but not in S2 cells. We wonder whether this indicates tissue specific regulation of LDH translation, such that there might be alternative cap-binding proteins in S2 cells. Please comment.
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In Fig 3E, the authors present results of puromycin incorporation with eIF4EHP KD under hypoxia. It is actually not clear what the function of eIF4EHP is under normoxic conditions. The authors shall include a control of puromycin incorporation with eIF4EHP KD under 21% O2. In addition, the authors should include statistical tests for the comparisons.
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In Fig 4C, the authors did Western Blots to detect eIF4EHP, and find that there is protein signal under the condition with eIF4EHP KD + D. persimilis eIF4EHP overexpression. It is unclear whether the antibody detects both eIF4EHPs in Drosophila melanogaster and D. persimilis, or whether, alternatively, expressing the D. persimilis version induces cells to express endogenous eIF4EHP. Please comment.
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In Fig 7A, we don't see the point of including results of flies carrying balancers as control. Direct comparisons with mCherry-RNAi should suffice. Also, presenting the percentage of hatched embryos can be misleading. We would suggest the authors present the absolute numbers of embryos examined and indicate the number of larvae that hatched.
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In addition (Fig 7) "birth" is not an appropriate term for insects. Please state whether the numbers indicated larvae "hatched" from eggs, or adult flies "eclosed" from pupae. Please use these terms in the text, figure and figure legends.
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In Fig 6c, a statistical test is missing.
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The authors sometimes refer to knockdown as KO, please be accurate.
Significance
Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. These results are unique and significant, and should be of interest to researchers who work on hypoxia, stress responses in general, and mRNA translation. As noted above (#1, #2) the paper could go further mechanistically, to determine the relative roles of the LDH 3' UTR and eIF4EHP. But it already has an extensive array of impressive translation data.
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Referee #2
Evidence, reproducibility and clarity
Summary:
When cells are stressed, such as during hypoxia, most of the canonical mRNA translation is shut off. Nonetheless, stress-response genes need to be translated. One such example is the lactate dehydrogenase (Ldh) mRNA, which encodes for an enzyme needed by cells to resist hypoxia. Therefore, these mRNA employ non-canonical mechanisms of translation to escape the general repression. Here, Liang et al. study the molecular mechanism how the Ldh mRNA is translated in Drosophila during hypoxia. They discover that the Ldh 3'UTR contains sequences that enable efficient translation. They identify the non-canonical initiation factor eIF4HP as a factor required for Ldh translation, and show that it binds the Ldh mRNA. Interestingly, they show that the ability of the Ldh 3'UTR to promote translation during hypoxia depends on eIF4HP. They go on to show that unlike other canonical translation initiation factors, during hypoxia eIF4HP remains in polysomes and does not form translationally-repressed aggregates such as stress granules or P-bodies. Finally, Liang et al show that eIF4HP is required in flies to efficiently resist hypoxia.
Major comments:
This is a very nice and solid study. Overall, I found the data and the conclusions very convincing. Also nice is how they cover the topic broadly, starting at the molecular level with the 3'UTR of the Ldh mRNA and ending with physiological assays such as resistance of flies to hypoxia. In particular I found the experiments presented in Fig 2C and 4A to be very nice, showing that the 3'UTR is responsible for the resistance to translation inhibition, and that this is mediated by eiF4EHP. I have only a few minor comments (below) to strengthen further the study, but I think it could be published also without these additional experiments.
Minor comments:
I have only two minor suggestions for experiments that would further strengthen the conclusions presented in the paper:
• Fig 2A - it would be good to show the equivalent luciferase assays at 21% oxygen, to test whether the elevated activity of the Ldh 3'UTR is something specific to the hypoxic condition, or whether this is always the case, also under non-stressed conditions.
• Similarly, in Fig 6C it would be good to show the equivalent CLIP data for 21% oxygen. Presumably, sinc eIF4HP is not in polysomes in the normoxic condition, there should be no enrichment (or little enrichment) for the Ldh mRNA.
• For Fig 7B - it's a bit confusing to label the y-axis as "flies escaping" to mean flies that climb past a certain limit. I suggest relabeling the axis to something like "flies climbing past threshold".
Significance
• The mechanisms of translation that one can read when opening a standard molecular biology textbook are the canonical mechanisms that take place in cells when they are not stressed. Cells, however, often experience stress. For instance, animals in the wild are exposed to heat or cold stress, cancer cells in a tumor are exposed to hypoxia, low amino acids, low sugar, etc. In such conditions, the canonical mRNA translation systems are shut down, and non-canononical mechanisms are remain (or are turned on). So it is a very interesting topic to understand how these non-canonical mRNA translation mechanisms function. This study contributes significantly to this topic by identifying the molecular mechanism how Ldh is translated during hypoxia. Ldh is an important gene because it is the last step of anaerobic glycolysis in animals, needed for cells to produce ATP when oxidative phosphorylation in mitochondria is incapable of functioning. Hence, understanding how the Ldh mRNA is translated is important for cell metabolism, cell viability, and organismal physiology. Furthermore, discovering that a non-canonical form of eIF4E, called eIF4EHP in Drosophila, or eIF4E2 in humans, is responsible for this translation is an important finding that opens up new avenues for future research, asking more broadly which parts of the translatome depend on this factor for their translation. The fact that eIF4EHP knockdown flies fare less well than control flies in response to hypoxia shows that eIF4EHP is playing an important role.
• I think these findings will be interesting for a broad audience studying mRNA translation, hypoxia, cell stress responses, cell and organismal metabolism, and organismal physiology.
• My expertise is in Drosophila development, mRNA translation and tissue growth.
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Gueydan and collaborators examines the role of eiF4EHP in Drosophila. The major conclusion of the paper is that eIF4EHP believed to be a translation repressor in fact drives protein synthesis during hypoxia. The bulk of the study is focused on the lactate dehydrogenase (LDH) mRNA, a mRNA that undergoes efficient translation during hypoxia thereby going against the grain of the general protein synthesis inhibition by low oxygen tension. While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story. The paper also provides additional evidence that cells are equipped with multiple stimuli-specific cap-binding translation initiation factors that produce adaptive translatomes. The work is convincing, the paper well-written and the demonstration of eIF4EHP role in whole organism is important. Nonetheless, I do have a few comments that should be addressed to strengthen the conclusions of the authors.
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The work focuses mainly of LDH. It would be a missed opportunity to show the effect of eIF4EHP in hypoxic Drosophila on puromycin incorporation. The work on S2 cells shown in Fig. 3E, while convincing, only reproduces what is already known. Puromycin incorporation in larvae.
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In mammalian cells, blocking de novo transcription does not affect protein accumulation of eIF4E2 translated mRNA, even if these mRNA do not increase during hypoxia. It would be important to silence sima and test if this could block increased translation of ldh or other target in hypoxic conditions. This would suggest that sima is both a transcription and translation factor that evolved in HIF1a and HIF2a, the latter being a hypoxic translation regulator. This can be compared to cells treated with a general transcription inhibitor. These experiments would broaden the impact of the work
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Fig 5C. It is unclear what are the conclusions of the authors are on the lack of co-localization between poly(A)RNA and eIF4EHP. In principle, eIF4EHP should co-localize with poly(A)mRNA. Perhaps a proximity ligation assay should be done to clarify this question. The data shown in F5C is not convincing one way or the other and needs clear cut experiments. Idem for 5A and B. PLA would provide convincing results.
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Fig 6C is important but the data is hard to understand. First, the Y-axis is "enrichment relative to input". Is this hypoxia vs normoxia? Is the Y-axis log (probably)? The best would be to show that normoxia and hypoxia and see if eIF4EHP binds to ldh mRNA in both conditions perhaps acting as a translational repressor in normoxia and translational activator in hypoxia. Also, the authors could pool monosome and polysome factions and see in which fractions eIF4EHP binds to ldh mRNA. Finally, do the authors think that rpl32 remains associated with ldh mRNA in normoxia and hypoxia?
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The authors should integrate the emerging concept of adaptive cap-binding translation factors in the discussion. It is still generally assumed that inhibition of eIF4E results automatically to cap-independent protein synthesis. The discovery that eIF3D, 4E2 and 4E3, amongst others, can initiate stimuli-specific translation should be discussed in the context of the work from this paper.
Significance
While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story
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Reply to the reviewers
Manuscript number: RC-2022-01680
Corresponding author(s): Woo Jae, Kim
- General Statements The goal of this study is to provide the groundwork for future studies of genetically controlled neuronal regulation of ‘interval timing’ through the provision of a behavioral paradigm. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to integrate action sequences, thoughts, and behavior, detect developing trends, and predict future consequences.
Many researchers have tried to figure out how animals, including humans, can estimate time intervals with such precision. However, most investigations have been conducted in the realm of psychology rather than biology thus far. Because the study of interval timing was limited in its ability to intervene in the human brain, many psychologists concentrated on developing convincing theoretical models to explain the known occurrence of interval timing.
To overcome the limits of studying interval timing in terms of genetic control, we have reported that the time investment strategy for mating in Drosophila males can be a suitable behavioral platform to genetically dissect the principle of brain circuit mechanism for interval timing. For example, we previously reported that males prolong their mating when they have previously been exposed to rivals (Kim, Jan & Jan, "Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals" Nature Neuroscience, 2012), and this behavior is regulated by visual stimuli, clock genes, and neuropeptide signaling in a subset of neurons (Kim, Jan & Jan, “A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating” Neuron, 2013).
Throughout their lives, all animals must make decisions in order to optimize their utility function. Male reproductive success is determined by how many sperms successfully fertilize an egg with a restricted number of investment resources. To optimize male reproductive fitness, a time investment strategy has been devised. As a consequence, we believe that the flexible responses of mating duration to different environmental contexts in Drosophila males might be an excellent model to investigate neural circuits for interval timing.
One of the most well-known features of human interval timing is the association of different sensory inputs with perception of time intervals, which influences our estimate of time intervals. Therefore, the first step toward comprehending the neural regulation of interval timing is to dissect the role that numerous sensory inputs play in determining the time duration. In this article, we discuss a different time-investment strategy adopted by males, called "Shorter-Mating-Duration" (SMD). According to our findings, male Drosophila with more sexual experience had shorter mating duration. During our investigation into the sensory inputs for SMD behavior, we found a small number of cells that express sugar receptors and pheromone receptors (ppk25 and ppk29) and thus transmit the multisensory information from females in order to generate memories of sexual experiences, which will determine the final decision of mating duration.
Our discovery of sensory integration mechanisms associated with complex behavioral trait in male Drosophila at the brain circuit and genetic network levels will be a huge step forward in our knowledge of interval timing behavior.
Description of the planned revisions
REVIEWER #1
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- Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer. * Answer: During the revision process, we will make our text more legible for wide audiences.
REVIEWER #2
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- 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript. * Answer: We value the reviewer's concern. LUSH has been discovered as an odorant-binding protein; nevertheless, current research suggests that LUSH may be involved in the sensing of additional pheromones to cVA, implying the presence of a lush-independent cVA detection mechanism [1]. Billeter et al. demonstrated in their paper that LUSH detects a female stimulatory chemical and modifies male mating latency (Fig. 2 of Billeter at al.). As Billeter et al. stated, our present understanding of pheromonal recognition in Drosophila is insufficient, and we concur. As a result, we attempted to validate the expression of Snmp1 in the male leg by experiments (Fig. 7I-J) performing sncRNA seq analysis on the Fly SCope dataset, as shown in Fig. 12. As demonstrated in Fig.12, Snmp1 and LUSH is higly expressed fly leg and wing system. Future study will look at the roles of Snmp1 and LUSH in female pheromone sensing, as well as PPK receptors.
Following the reviewer's advice, we will repeat the electrophysiologically validated Orco2 mutant phenotype with proper control and attach it when we submit the complete revision to the journal.
- What is this (GustDx6)? I suggest using Poxn mutant line. *
Answer: We value the reviewer's recommendation. We believe we have previously demonstrated that the Gr5a-mediated gustatory pathway is essential for the generation of sensory input for SMD behavior, but we will test the Poxn mutant and Poxn-RNAi to replace the GustDx6 mutant result.
Description of the revisions that have already been incorporated in the transferred manuscript
REVIEWER #1
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My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.* Answer: We put page numbers and line numbers.
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A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact. *
Answer: We appreciate the reviewer's insight. We have previously separated this document from our original preprint [2] in response to a prior reviewer's advice; we believe we have included too much data, which may confuse readers. As a result, we will delete all of the mechanosensory/thermosensory receptor screening data from our present paper and write a second manuscript on sensory integration for the production of SMD behavior. We also removed the most of sncRNA seq data analysis except Fig.12 which confirms our finding in a single diagram.
- Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol). *
Answer: We recognize that there is a conflicting report concerning white mutation on LMD behavior, however because we are now reporting SMD rather than LMD behavior, we deleted the statement comparing white mutant results to earlier reports, as shown below;
“thus suggesting that the effect of the white mutant genetic background was not evident.” (line 97)
- A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).
A "sperm depletion" assay is described in the results that I cannot find any methodology for. *
Answer: We thank the reviewer for allowing us to clarify our lacking methodologies for a better comprehension of our manuscript.
We included the egg counting procedure to the EXPERIMENTAL PROCEDURES section to further illustrate our approach of egg laying assay as below;
“In short, wild type females mated with naïve or experienced males were transferred to a fresh new vial and allowed to lay eggs for 24 hr at 25°C. After 24 hr of egg laying, number of eggs were counted under the stereomicroscope. After we count the number of eggs, we kept vials in 25°C incubator and counted the total number of progenies ecolsed from them.” (line 956-960)
We included “Sperm Depletion from Males” section in EXPERIMENTAL PROCEDURES as below;
“To deplete sperm from males, 40 virgin Defexel6234 females which lacks SPR and shows multiple mating with males (Yang 2009) were placed in a vial containing four CS males for indicated time (2 h, 4 h, 8 h, and 24 h).” (line 880)
- Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.
It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration. *
Answer: We modified the sentence describing SPR mutant female experiment and added references as below;
“Sexual experiences with sex peptide receptor (SPR) mutant females which exhibit a delayed post-mating response and multiple mating with males [3] had no additional effect on SMD (Fig. 2I).” (line 135)
We clarify in which extent, fru>UAS-mSP virgin females behave like mated females as below;
“Virgin females behave like mated females by expressing a membrane-bound version of male sex-peptide in fruitless-positive neurons, hence rejecting the male's copulation attempt.” (line 136)
In the instance of feminized males, we assume that these feminine males can give adequate signals for inducing SMD and eliminated the term "successful copulation" since we are unsure if males can copulate these feminized males or not, despite the fact that males can mount and mate with them (Fig. 2O-P).
Tables S1 and S2 describe the conditions, genotypes, and descriptions of an experiments illustrated in Fig. 2. We believe that these tables may assist general audiences in comprehending our experimental design.
- Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue). *
Answer: We described our statistical analysis with mating duration previously [4–7] and followed the statistical analysis of copulation duration assay reported by Crickmore et al., published in CELL (2013) and NEURON (2020) [8,9]. To further validate our statistical analysis, we added estimation statistics which focuses on the effect size of one's experiment/intervention, as opposed to significance testing [10]. We already described our statistical analysis in EXPERIMENTAL PROCEDURES section in details. We also described our statistical analysis for mating duration will be same in all other figures in the Fig.1 legend.
We appreciate the reviewer's recommendation that the normal distribution of our mating duration data be validated. As a consequence, we performed the normailty test with Graphpad prism and added the histogram and QQ plot results to Fig. S1M and N. Table S3 also contains the results of the normality and lognormality tests.
- Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved? *
Answer: We accidentally left out the citation for that phrase and updated it with Wang et al.'s CELL (2004) paper. Wang et al. wrote in their article about taste representations in the Drosophila brain, “Our behavioral studies reveal that Gr5a cells recognize sugars and mediate acceptance/attractive behaviors whereas Gr66a cells recognize bitter compounds and mediate avoidance…. This suggests that Gr5a cells may be “acceptance” cells rather than “sweet” cells…. Our expression and behavioral studies reveal that Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognize bitter compounds and mediate avoidance.” [11]
As a result, we hypothesize that Gr5a and Gr66a-positive cells influence acceptance or avoidance of "taste." We also changed certain sentences to make them clearer, as seen below;
“Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance.” (line 173)
- As Orco was not found to affect the behaviour, why test Or67d? *
Answer: We appreciate the reviewer bringing this to our attention. We omitted the Or67d result from the present manuscript to simplify it and make it easier for readers to grasp.
- "Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?
In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation. *
Answer: We have defined “mate guarding” in the text now. The costs and benefits of mate guarding have been extensively studied in insects and demonstrated to shape the optimal mating duration of males. In our experiment, we cannot specify whether the shortened mating duration was caused by the adjustments in ejaculate expenditure or a shorted stay after the ejaculation. Instead, our model has a general assumption that the costs of mate guarding increase linearly at the same rate in both pre- and post-ejaculation periods, which is highlighted in the model text.
There exist many models for the optimal mating duration (earlier models include Grafen and Ridley, 1983. A model of mate guarding. J. Theor. Biol. 102: 549 – 567 [12]). While our model was not built upon a novel theoretical approach (it was built based on the classical Charnov’s marginal value theorem equation), our model was developed specifically for generating testable predictions for the observed SMD behaviors.
We have rephrased the text as follow;
“This model assumes that (i) the shortened (or prolonged) mating duration is controlled by males and shaped by a trade-off between the benefit of mate guarding (remaining with the female both before and after the sperm ejaculation) and opportunistic costs (e.g. searching for another mate).” (line 970)”
- I can't find a data accessibility statement. *
Answer: We added it in the manuscript.
- That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge. However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field. *
Answer: We believe the reviewer highlighted relevant past research that examined the influence of female experiences on mating duration. We agree with the reviewer that SMD behavior does not have to be original in order to contribute significantly to the field. As a result, we examined past reports and updated the introduction as follows;
“It has been reported that previous sexual experience with females influences the mating duration of male D. melanogaster [15,16,34]; however, the neural circuits and physiology underlying this behavior have not been deeply investigated. Here, we report the sensory integration mechanisms by which sexually experienced males exhibit plastic behavior by limiting their investment in copulation time; we refer to this behavior as "shorter mating duration (SMD)."” (line 85)
- Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere). *
Answer: We agree with the reviewer. In the introductory and discussion sections, we cited as many key publications explaining the plastic responses of male mating duration as we could.
__REVIEWER #2
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- Summary: The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.* Answer: We appreciate the reviewer's assessment, especially the statement that our statistical approaches were appropriate. We will revise our manuscript in response to the reviewer's suggestions.
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The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males? *
Answer: We value the reviewer's feedback. Several research have been conducted to investigate the mating duration of male fruit fly. For example, our lab [2,13–15] and others [13–30] have regularly reported that previous rival exposure increases male fruit fly mating duration. Bretman A et al. utilized 49-59 males in their studies to compare the variations in mating duration between circumstances. Crickmore et al. also reported the effect of mating duration differences caused by genetic or experimental modification [8]. They utilized 10-18 male flies in their study to compare the variations in mating duration across circumstances, as shown in Figs. 1G (n=15-18) and 2A (n=10-27). All of these findings indicate that our mating duration sample size is sufficient to examine the effect size variations between the naive and experienced conditions. To confirm our statistical analysis further, we incorporated estimate statistics, which focus on the effect size of one's experiment/intervention rather than significance tests [10]. We have already detailed our statistical analyses under the EXPERIMENTAL PROCEDURES section. We conducted hundreds of mating duration assays using this configuration and confirmed that all of our results are reproducible in a blind test. As a result, we believe our mating duration assay has been validated by other groups' findings, several analytic tools, and numerous blind tests conducted by us. We appreciate the reviewers' concerns, but our data meets the reproducibility requirements.
- I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L. *
Answer: We appreciate the reviewer's feedback. Yes, all of our tests comparing the differences in mating duration between naive and experienced conditions were conducted under the same conditions and at the same time. We replaced Fig.1B with new data (n=49-51) obtained lately in a new lab in China. As previously stated, SMD behavior could be reproduced by the same Canton S genotype in different locations by different experimenters.
- It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience? *
Answer: We appreciate the reviewer’s suggestion and added in the manuscript as follow;
“We found that the mating duration of various wild-type and w1118 naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 99)
- 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba. *
Answer: We appreciate the reviewer's feedback. We removed the D. simulans data because it is not required for the conclusion of this manuscript. In future research, we will look on the conservation of SMD behavior between species.
- The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005. *
Answer: We made the following changes and cited the article reviewer's suggestion.
“Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance (Wang et al, 2004; Dweck & Carlson, 2020).” (line 180)
- Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early. *
Answer: We appreciate the reviewer's feedback. Copulation duration in Drosophila melanogaster male is extremely variable and has been reported to be approximately 20 minutes. However, as other groups documented, male copulation duration can range from 10-15 minutes depending on sperm completion (Fig. 1a-c of Bretman A et al.) [30] and genetic background (Fig. 1C, Fig. 2E, Fig. 5D, and Fig. 7A and E of Crickmore et al) [8]. And, as previously stated, males dominate copulation duration [8,30], not females, and we always utilized the same genotype of females for mating duration experiment. As a result, we believe that these rather short mating duration outcomes are the product of a distinct genetic background. Because we employed the same genotype of males while altering the female experience condition, we believe our mating duration results are all equivalent and comparable.
- In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E. Why you test 100, 103 replicates in this exp fig 10 F? How you compare 47 replicates against 9 replicates in fig S10 I? *
Answer: We appreciate the reviewer's input. As we previously stated in response to Reviewer Question 2, the n number of males exhibited in Figs. 2F and 12E is statistically significant. To corroborate findings with replication, we examined 100, 103 duplicates of Fig. 10F, which represents pyx-RNAi screening results. The results of Fig. S10I are screening data, and we cannot rule out the possibility that TrpA1 knockdown in Gr5a neurons affects the mating success of sexually experienced males. We only placed it there because it was screening results and the differences between naive and experienced conditions were substantial despite the small sample size. However, we deleted Fig. 10F and Fig. S10I data from the current paper in response to Reviewer #1's advice, thus it will not be an issue for the manuscript's conclusion.
- 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25? **
*
Answer: As indicated by the reviewer, we included ppk25-GAL4/snmp1-RNAi data in Fig. S9I, indicating that snmp1 expression in ppk25-positive cells is similarly implicated in SMD behavior.
- There are no page or line numbers throughout the ms! *
Answer: We included page and line numbers.
- The use of subheadings in the results section makes reading much easier.*
Answer: We added subheadings in the results section.
- 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .*
Answer: The revised sentence is as follows:
“We found that the mating duration of Canton S, WT-Berlin, Oregon-R, and w1118 naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 102)
- Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time. *
Answer: We value the reviewer's recommendation. As seen in Fig. S4B of Kim et al., we have previously conducted experiments for examining the memory circuit of SMD [6]. Briefly, the male with a shorter mating duration recovers completely after 12 to 24 hours of isolation from females. As we are currently preparing the memory section of the SMD study, this information will be included in a future manuscript.
- General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration. *
Answer: We welcome the reviewer's idea, however in this situation we believe that the y axis of each data set is independent from one another and will thus retain the originals. We feel this would be more useful for the general audiences.
- You might move the number of replicates to the legend. *
Answer: We appreciate the reviewer's idea, however we feel that adding more information to the graphic will aid the general audience in comprehending our statistics.
- Latin name should be italic as example Drosophila simulans.*
Answer: We fixed it.
Description of analyses that authors prefer not to carry out
N/A
References
- Billeter J-C, Levine JD. The role of cVA and the Odorant binding protein Lush in social and sexual behavior in Drosophila melanogaster. Frontiers Ecol Evol. 2015;3: 75. doi:10.3389/fevo.2015.00075
- Kim WJ, Lee SG, Schweizer J, Auge A-C, Jan LY, Jan YN. Sexually experienced male Drosophila melanogaster uses gustatory-to-neuropeptide integrative circuits to reduce time investment for mating. Biorxiv. 2016; 088724. doi:10.1101/088724
- Yang C, Rumpf S, Xiang Y, Gordon MD, Song W, Jan LY, et al. Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons. Neuron. 2009;61: 519–526. doi:10.1016/j.neuron.2008.12.021
- Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876–883. doi:10.1038/nn.3104
- Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190–1205. doi:10.1016/j.neuron.2013.09.034
- Kim WJ, Lee SG, Auge A-C, Jan LY, Jan YN. Sexually satiated male uses gustatory-to-neuropeptide integrative circuits to reduce time investment for mating. Biorxiv. 2016; 088724. doi:10.1101/088724
- Wong K, Schweizer J, Nguyen K-NH, Atieh S, Kim WJ. Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv. 2019; 819045. doi:10.1101/819045
- Crickmore MA, Vosshall LB. Opposing Dopaminergic and GABAergic Neurons Control the Duration and Persistence of Copulation in Drosophila. Cell. 2013;155: 881–893. doi:10.1016/j.cell.2013.09.055
- Thornquist SC, Langer K, Zhang SX, Rogulja D, Crickmore MA. CaMKII Measures the Passage of Time to Coordinate Behavior and Motivational State. Neuron. 2020;105: 334-345.e9. doi:10.1016/j.neuron.2019.10.018
- Claridge-Chang A, Assam PN. Estimation statistics should replace significance testing. Nat Methods. 2016;13: 108–109. doi:10.1038/nmeth.3729
- Wang Z, Singhvi A, Kong P, Scott K. Taste Representations in the Drosophila Brain. Cell. 2004;117: 981–991. doi:10.1016/j.cell.2004.06.011
- Grafen A, Ridley M. A model of mate guarding. J Theor Biol. 1983;102: 549–567. doi:10.1016/0022-5193(83)90390-9
- Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190–1205. doi:10.1016/j.neuron.2013.09.034
- Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876–883. doi:10.1038/nn.3104
- Wong K, Schweizer J, Nguyen K-NH, Atieh S, Kim WJ. Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv. 2019; 819045. doi:10.1101/819045
- Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. doi:10.1098/rspb.2008.1878
- Bretman A, Westmancoat JD, Chapman T. Male control of mating duration following exposure to rivals in fruitflies. J Insect Physiol. 2013;59: 824–827. doi:10.1016/j.jinsphys.2013.05.011
- Bretman A, Gage MJG, Chapman T. Quick-change artists: male plastic behavioural responses to rivals. Trends Ecol Evol. 2011;26: 467–473. doi:10.1016/j.tree.2011.05.002
- Lizé A, Doff RJ, Smaller EA, Lewis Z, Hurst GDD. Perception of male–male competition influences Drosophila copulation behaviour even in species where females rarely remate. Biol Letters. 2012;8: 35–38. doi:10.1098/rsbl.2011.0544
- Rouse J, Bretman A. Exposure time to rivals and sensory cues affect how quickly males respond to changes in sperm competition threat. Anim Behav. 2016;122: 1–8. doi:10.1016/j.anbehav.2016.09.011
- Bretman A, Fricke C, Hetherington P, Stone R, Chapman T. Exposure to rivals and plastic responses to sperm competition in Drosophila melanogaster. Behav Ecol. 2010;21: 317–321. doi:10.1093/beheco/arp189
- Rouse J, Watkinson K, Bretman A. Flexible memory controls sperm competition responses in male Drosophila melanogaster. Proc Royal Soc B Biological Sci. 2018;285: 20180619. doi:10.1098/rspb.2018.0619
- Maguire CP, Lizé A, Price TAR. Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura. Plos One. 2015;10: e0123058. doi:10.1371/journal.pone.0123058
- Bretman A, Westmancoat JD, Gage MJG, Chapman T. COSTS AND BENEFITS OF LIFETIME EXPOSURE TO MATING RIVALS IN MALE DROSOPHILA MELANOGASTER. Evolution. 2013;67: 2413–2422. doi:10.1111/evo.12125
- Bretman A, Fricke C, Westmancoat JD, Chapman T. Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies. Behav Ecol. 2016;27: 452–461. doi:10.1093/beheco/arv170
- Price TAR, Lizé A, Marcello M, Bretman A. Experience of mating rivals causes males to modulate sperm transfer in the fly Drosophila pseudoobscura. J Insect Physiol. 2012;58: 1669–1675. doi:10.1016/j.jinsphys.2012.10.008
- Bretman A, Westmancoat JD, Gage MJG, Chapman T. Males Use Multiple, Redundant Cues to Detect Mating Rivals. Curr Biol. 2011;21: 617–622. doi:10.1016/j.cub.2011.03.008
- Fowler EK, Leigh S, Rostant WG, Thomas A, Bretman A, Chapman T. Memory of social experience affects female fecundity via perception of fly deposits. Bmc Biol. 2022;20: 244. doi:10.1186/s12915-022-01438-5
- Dore AA, Rostant WG, Bretman A, Chapman T. Plastic male mating behavior evolves in response to the competitive environment*. Evolution. 2021;75: 101–115. doi:10.1111/evo.14089
- Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. doi:10.1098/rspb.2008.1878
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Referee #2
Evidence, reproducibility and clarity
Summary:
The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.
Major comments:
- The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males?
- I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L.
- It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience?
- 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba.
- The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005.
- Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early.
- In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E
- Why you test 100, 103 replicates in this exp fig 10 F?
- How you compare 47 replicates against 9 replicates in fig S10 I?
- 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript.
- 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25?
Minor comments:
- There are no page or line numbers throughout the ms!
- The use of subheadings in the results section makes reading much easier.
- 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .
- Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time.
- What is this (GustDx6)? I suggest using Poxn mutant line.
- General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration.
- You might move the number of replicates to the legend.
- Latin name should be italic as example Drosophila simulans.
Referees cross-commenting
I found the comments of the other reviewer reasonable and fair. I agree that the time for fixing all these comments is about six months.
Significance
The idea of the work is interesting, but the design of experiments in some places is inappropriate (see above). The discussion mainly depends on doi: 10.1016/j.neuron.2013.09.034. I study chemoreception and sexual communication in Drosophila and insect vectors of human disease.
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Referee #1
Evidence, reproducibility and clarity
My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.
Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer.
Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol).
A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).
A "sperm depletion" assay is described in the results that I cannot find any methodology for.
Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.
It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration.
Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue).
Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved?
As Orco was not found to affect the behaviour, why test Or67d?
"Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?
In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation.
I can't find a data accessibility statement.
Significance
A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact.
That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge.
However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field.
Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere).
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Reply to the reviewers
Reviewer #1:
Minor edits
- Line 91. Is a bit misleading to say "many other vibrios" possess T3SS. This conveys that this is perhaps the majority, but T3SS in vibrios is at best 50/50. I think best just to delete this sentence.
We deleted this comment, as suggested.
- Revised to "Thus, in this study, we set out to..." Since the entire paragraph starts with "recent study" I missed that this was summary of new data rather than preview of new results.
The sentence was revised as suggested.
- Line 503. Correct "xxx-584" or more detail on what this means.
We Thank the reviewer for pointing out this typo._ This refers to the deletion made in tie1, in the region corresponding to nucleotides 485-584 of this gene. The text was corrected accordingly.
- Line 603. Salmonella should be italicized.
Corrected.
- The labelling of the figures is pretty complicated with the long genetic designations. Is it reasonable to for example name the ∆vprh/∆hns1 strain with an abbreviation (such as ∆VH)? Or instead create a strain name, common used approaches would be HC## (for Hadar Cohen) or TAU# for Tel Aviv University. If you go this route, be sure to update the strain list. The current method can be followed, the figures are just complicated.
We thank the reviewer for raising this concern. We acknowledge the difficulty in following the many different strains and mutations. Nevertheless, after considering the proposed modifications to the strain names, we believe that they will not add much clarity, and may even cause some confusion. Therefore, we respectfully decided to keep the current nomenclature in place.
Reviewer #2:
Minor edits
- The authors used a hyperactive T6SS (HNS mutant) to investigate its toxicity. Would the authors be able to use a wild type strain to reproduce the function of T6SS?
We have yet to reveal the external cues that lead to full activation of T6SS3 in vitro. Therefore, in the current study we used genetic tools, such as hns deletion or Ats3 over-expression, to monitor the effect of this system on immune cells. We will dissect the activating conditions in future studies, but we believe that the use of genetic tools should not affect the validity of the results in the current study, nor their timely publication.
- The authors showed that Tie1 and Tie2 are secreted by T6SS3. It is important to show if they are actually delivered into the host cells during infection. Otherwise it is hard to conclude that they are truly effectors. The primary concern is the lack of in vivo studies to show that Tie1 and Tie2 are actually effectors that play a role in activation of NLRP3 inflammasome._
We present 3 pieces of evidence that, when taken together, support the conclusion that Tie1 and Tie2 are T6SS3 effectors: 1) the proteins are secreted in a T6SS3-dependent manner; 2) their deletion does not hamper overall T6SS3 activity; and 3) their deletion causes the same loss of NLRP3-mediated inflammasome activation and pyroptosis as does inactivation of T6SS3 by deletion of its structural component, tssL3. Although we agree with the reviewer that directly showing delivery of Tie1 and Tie2 into host cells will further strengthen our conclusion, such experiments are quite challenging and difficult to interpret, especially with T6SS effectors that can use diverse mechanisms for secretion through the system. This point was also noted by reviewer #3: “…I believe they were suggesting to demonstrate secretion in host cells. Although this would be nice, it is non-standard and technically not feasible. These types of experiments require genetically fusing the effector with either an enzymatic moiety (e.g. Beta lactamase) or fragment of split GFP. Although such approaches have been previously performed, they often result in either blocked or aberrant secretion due to the presence of the added fragment."
Regarding the reviewer’s comment on the lack of in vivo studies: we agree that these are extremely important, yet they are beyond the scope of the current work, as concurred by reviewers #1 and #3:
Reviewer#1 with regard to Reviewer#2: "I don't think mouse (or aquatic animal) studies are essential for this study. The work contributes nicely to our understanding molecular mechanisms of this T6SS system. As noted in my review, there are many additional lines of study that can be pursued from this work, including animal studies, but this should not preclude publication of this work that is itself an intact unit."
Reviewer#3 regarding reviewer #1's comment on Reviewer#2: "I don't believe that reviewer #2 was suggesting to perform mouse or aquatic animal studies by suggesting in vivo demonstration of secretion…”
Reviewer #3:
Major comments:
- If the authors believe that GSDME partially compensates in the absence of GSDMD, have they infected a GSDME/GSDMD double knockouts to see if there is an additive effect?
Indeed, this is a very interesting and specific question for the cell death field. We do not currently possess such a GSDME/GSDMD double knockout mouse, and generating one will be a long endeavor. Since its absence does not diminish the importance or the conclusions of the current work, we think that it should not warrant a delay in publication. We do plan to address this question in future studies.
- It is clear that Ats3 regulates T6SS3, but not the T6SS1; however, there no evidence suggesting that Atg3 does not regulate other gene clusters. For example, have the authors performed RNA seq to compare the transcriptomes of WT and an Ats3 mutant? If not, the authors should refrain using the words "specific activation".
We thank the reviewer for this important note. Indeed, we lack additional data indicating that Ats3’s effect is indeed restricted only to T6SS3. Therefore, we modified the text accordingly and removed mentions of specific T6SS3 activation.
- In figure 6B, it's unclear why the bacteria infecting cytochalasin D-treated cells grow more than the T6SS3 mutants in the absence of cytochalasin D.
The difference probably stems from the fact that phagocytosis, the major mechanisms by which BMDMs kill bacteria, is hampered in the presence of cytochalasin D, thus allowing bacteria to grow more than when the BMDMs phagocytose them. The results show that in the absence of cytochalasin D, an active T6SS3 counteracts the killing effect by BMDMs with functional phagocytosis.
Minor comments:
- Figure 1A and other secretion assays: The Western blots include loading control (LC) blots. These are non-standard, non-informative, and not required with the inclusion of the western blots on the "cells" fraction. I would suggest removing these as they may confuse the reader.
We respectfully disagree. Loading controls are standard in bacterial secretion assays, and they are important since they confirm comparable loading and allow proper analysis of the results, especially since we aim to determine whether certain mutations affect the expression of T6SS components. Notably, some groups choose to blot for a cytoplasmic protein (e.g., RpoB in Allsop et al., PNAS, 2017; Liang et al., PLoS Pathogens, 2021) instead of showing overall loaded proteins, as shown in our figures.
- Line 503: "xxx" should reflect the actual nucleotide nubmers_
We thank the reviewer for pointing out this typo._ This refers to the deletion made in tie1, in the region corresponding to nucleotides 485-584 of this gene. The text was corrected accordingly.
- Since V. proteolyticus is an aquatic pathogen, have the authors tried to infect corals, fish, and crustaceans (or derived cells) with WT and effector mutants?
This is an interesting point, and indeed we are setting up such systems and we plan to perform such experiments in the future as part of follow up projects. However, these in vivo studies are beyond the scope of the current manuscript, as also noted by the reviewer in the cross-consultation comments: “…my previous comment on infecting aquatic animals or cells derived from them is non-standard and not necessary…”
- Are the targeted host proteins in this study (performed with murine BMDM) conserved in the natural hosts for V. proteolyticus?
We hypothesize that the conservation is not in the pathway components that are activated upon infection, but rather in the ability of the host cell to sense danger (i.e., to sense the effect of T6SS3 effectors on the host cell or one of its components), which is the role of the NLRP3 inflammasome in mammalian cells. It is well documented that major differences in immune mechanisms exist between mammals and the potential natural marine hosts of V. proteolyticus (e.g., corals, arthropods, and fish); therefore, the conservation at the protein level is low. Nevertheless, basic signaling pathways, such as programed cell death, are conserved between the different phyla. For example, a caspase-1 homolog which was found in arthropods (Chu, B. et al. PLoS One (2014). doi:10.1371/journal.pone.0085343) probably induces an apoptotic-like cell death mechanism, similar to apoptosis in C. elegans. We now provide further discussion on this point in the text (lines 648-659).
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Referee #3
Evidence, reproducibility and clarity
Bacterial type VI secretion systems (T6SSs) are best-characterized in their ability to drive interbacterial competition; however, several systems target host cells and are important for pathogen-host interactions. Based on the presence of T6SS in almost a quarter of sequenced Gram-negative bacterial genomes, including many well-known pathogens, the T6SS is an understudied pathway that is worthy of further inspection. This manuscript by Cohen et al. investigates the role three T6SSs in Vibrio proteolyticus by 1) identifying two of these systems play a role in targeting host cells, 2) characterizing the host pyroptotic pathways targeted by these systems, and 3) identifying two secreted toxins responsible for the host response. Furthermore, this manuscript identifies a specific transcriptional regulator of the T6SS3 and suggests that the host cell responds with an alternative/compensatory pathway upon inhibition of the canonical pyroptosis pathway.
Major comments:
- If the authors believe that GSDME partially compensates in the absence of GSDMD, have they infected a GSDME / GSDMD double knockouts to see if there is an additive effect?
- It is clear that Ats3 regulates T6SS3, but not the T6SS1; however, there no evidence suggesting that Atg3 does not regulate other gene clusters. For example, have the authors performed RNA seq to compare the transcriptomes of WT and an Ats3 mutant? If not, the authors should refrain using the words "specific activation".
- In figure 6B, it's unclear why the bacteria infecting cytochalasin D-treated cells grow more than the T6SS3 mutants in the absence of cytochalasin D.
Minor comments:
- Figure 1A and other secretion assays: The Western blots include loading control (LC) blots. These are non-standard, non-informative, and not required with the inclusion of the the western blots on the "cells" fraction. I would suggest removing these as they may confuse the reader.
- Line 503: "xxx" should reflect the actual nucleotide nubmers
- Since V. proteolyticus is an aquatic pathogen, have the authors tried to infect corals, fish, and crustaceans (or derived cells) with WT and effector mutants?
- Are the targeted host proteins in this study (performed with murine BMDM) conserved in the natural hosts for V. proteolyticus?
Referees cross-commenting
Regarding Reviewer #1's comment on 2022-07-26, I don't believe that reviewer #2 was suggesting to perform mouse or aquatic animal studies by suggesting in vivo demonstration of secretion. (At least, I hope they weren't.) I believe they were suggesting to demonstrate secretion in host cells. Although this would be nice, it is non-standard and technically not feasible. These types of experiments require genetically fusing the effector with either an enzymatic moiety (e.g. Beta lactamase) or fragment of split GFP. Although such approaches have been previously performed, they often result in either blocked or aberrant secretion due to the presence of the added fragment.
Likewise, my previous comment on infecting aquatic animals or cells derived from them is non-standard and not necessary. It would however be interesting to note (either through a supplementary figure or in the text) if the targeted host proteins are conserved between mice and aquatic animals.
Significance
This manuscript provides both novel and innovative details of a previously uncharacterized secretion system. The interested audience includes readers who are interested in host-targeted secretion systems, bacterial effectors, and mechanisms of inflammasome activation. As with any key discovery, there are many avenues of investigation that will follow from this study.
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Referee #2
Evidence, reproducibility and clarity
The authors used a hyperactive T6SS (HNS mutant) to investigate its toxicity. Would the authors be able to use a wild type strain to reproduce the function of T6SS?<br /> The authors showed that Tie1 and Tie2 are secreted by T6SS3. It is important to show if they are actually delivered into the host cells during infection. Otherwise it is hard to conclude that they are truly effectors.<br /> The primary concern is the lack of in vivo studies to show that Tie1 and Tie2 are actually effectors that play a role in activation of NLRP3 inflammasome.
Significance
The authors demonstrated in vitro that T6SS of Vibrio plays an important role in inflammasome activation. This is potentially important as this may suggest that T6SS may be a virulence factor. However, as mentioned above, it is important to show in vivo data to demonstrate this.
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Referee #1
Evidence, reproducibility and clarity
This paper by Cohen et al described discovery of the function of novel genes in the T6SS operon of Vibrio proteolyticus, a Vibrio isolated from corals. V. proteolyticus also impacts other sea animals. The T6SS3 in particular is found to kill eukarytoic phagocytic cells following engulfment of bacteria into the phagocyte. This strategy of killing phagocytic cells following entry has been shown for other Vibrios. The net goal is protection of the population by the bystander effector. The study first shows that deletion of H-NS (a global negative regulator) stimulates T6SS facilitating ease of work by pushing the system to great cell killing. This allowed them to probe the mechanism of cell death and reveal it as NLRP3 dependent, capase 1 dependent pyroptosis via pore formation by Gasdermin D. Activation of the inflammasome is also linked to cleavage and release of IL-1beta. When GSDMD is absent, there was a slower cell killing by GSDME via capsase 3 activation. The stimulation of this system is additive by two newly recognized T6SS effectors Tie1 and Tie2.
The study is complete, the experiments are well conducted and well controlled. The experiments show reproducibility. The manuscript text is clear, Overall. I suggest no changes in the results or experiments and suggest only a few minor edits of the text.
Minor edits
Line 91. Is a bit misleading to say "many other vibrios" possess T3SS. This conveys that this is perhaps the majority, but T3SS in vibrios is at best 50/50. I think best just to delete this sentence.
Line 102. Revised to "Thus, in this study, we set out to..." Since the entire paragraph starts with "recent study" I missed that this was summary of new data rather than preview of new results.
Line 503. Correct "xxx-584" or more detail on what this means.
Line 603. Salmonella should be italicized.
Figures. The labelling of the figures is pretty complicated with the long genetic designations. Is it reasonable to for example name the ∆vprh/∆hns1 strain with an abbreviation (such as ∆VH)? Or instead create a strain name, common used approaches would be HC## (for Hadar Cohen) or TAU# for Tel Aviv University. If you go this route, be sure to update the strain list. The current method can be followed, the figures are just complicated.
Referees cross-commenting
With regard to Reviewer#2, I don't think mouse (or aquatic animal) studies are essential for this study. The work contributes nicely to our understanding molecular mechanisms of this T6SS system. As noted in my review, there are many additional lines of study that can be pursued from this work, including animal studies, but this should not preclude publication of this work that is itself an intact unit.
Significance
The work is significant in that it links T6SS to a eukaryotic killing system and discovers novel details regarding the mechanisms of death, that may impact our knowledge of other Vibrio T6SS (including V. cholerae) that also target eukaryotic cell actin. There are remaining questions that could be probed, but these are in my opinion major studies that would easily themselves comprise new papers if done properly and thus are not essential for this paper. These include the struture and biochemical activity of Tie1 and Tie2 and the mechanism of caspase-8 independent activation of caspase-3 to then cleave GSDME. Why NLRP3 is required for capase 3 activation is also an open question. I look forward to following this work for some time to come. The authors have revealed very interesting effectors and interesting cell biological process that will merit multiple years and multiple manuscripts to unravel. This work will be of interest to the community interested in bacterial toxin systems (microbial pathogenesis), the bacterial effector mechanism field (biochemistry and cell biology), and the inflammasome activation field (immune systems). The work will be of interest (with essentially no modification) directed at these fields of interest.
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Reply to the reviewers
Reviewer #1:
Major comments:
In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure. Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.
Response: As suggested by Referees 1 and 3 we have modified the colour channels in all figures. We have also enlarged the figures taking away the uninformative region and focused around the enlarged gonads and added scale bars. For Fig 2F-G, we have added a close up of the region of interest both in colour and in black and white. These changes have increased the contrast and facilitate the data interpretation to non-expert readers.
The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.
Response: We have changed the names to maintain nomenclature uniformity.
I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.
Response: The methods are well described, statistics adequate and the data well described._
Minor Comments:
My only suggestion for the text is to provide a more through description of the StART domain in the introduction.
Response: We have included the following paragraph in the introduction describing the StART domain:
“This family of proteins share different domains: besides the Rho GTPase Activating Protein domain (GAP), they present a protein-protein interacting Sterile Alpha Motif (SAM) at the N terminal end and a Steroidogenic Acute Regulatory protein (StAR)-related lipid transfer (StART) domain at the C terminal. StART domains have been shown in other proteins to be involved in lipid interaction, protein localization and function.”
Reviewer #2:
My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.
The reviewer says:
“As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility.”
Response: This sentence does not represent the spirit of our findings accurately and this probably reflects the fact that we stressed the interaction between somatic mesodermal cells and germ cells in Drosophila which probably concealed that the main defects in Cv-c mutants are caused by the abnormal interaction of the mesodermal cells with germ cells but also among themselves. Our study provides insights about a new conserved pathway required in the mesodermal cells for the maintenance of an already formed testis, and only indirectly can be considered to deal with sterility. We show that Cv-c is required in the mesodermal cells for the correct maintenance of the testis structure, that when it fails leads to the testis dysgenesis which, among other defects, releases the germ cells. We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells. Thus, our proposal is that DLC3/STARD8 plays a role in testis maintenance through its function in mesodermal cells which will probably affects both Sertoli and Leydig cell function.
To clarify the issue raised by the referee we have modified both, the introduction and the discussion to highlight that although humans and Drosophila diverged millions of years ago there are similarities regarding gonad stabilisation.
We have modified the introduction to clarify this issue:
“Gonadogenesis can be subdivided into three stages: specification of precursor germ cells, directional migration towards the somatic gonadal precursors and gonad compaction. In mammals, somatic cells, i.e. Sertoli cells in male and Granulosa cells in females, play a central role in sex determination with the germ cells differentiating into sperms or oocytes depending on their somatic mesoderm environment. In humans, Primordial Germ Cells (PGCs) are formed near the allantois during gastrulation around the 4th gestational week (GW) and migrate to the genital ridge where they form the anlage necessary for gonadal development (GW5-6). Somatic mesodermal cells are required for both PGCs migration and the formation of a proper gonad. Once PGCs reach their destination, the somatic gonadal cells join them (around GW 7-8 in males, GW10 in females) and provide a suitable environment for survival and self-renewal until gamete differentiation {Jemc, 2011 #413}. Thus, mutations in genes regulating somatic Sertoli and Granulosa support cell function in humans are often associated with complete or partial gonadal dysgenesis in both sexes and sex reversal in males {Zarkower, 2021 #430; Knower, 2011 #418; Brunello, 2021 #399}. Other mesodermal cells, the Leydig cells, also play an important role in the testis by being the primary source of testosterone and other androgens and maintaining secondary sexual characteristics.”
Also we have added a paragraph in the discussion to emphasize this argument:
“We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed from the testis. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells”.
I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.
Response: We have modified the last part of the discussion to introduce referee 2’s suggestion:
“Our work points to DLC3/Cv-c as a novel gene required specifically in testis formation. Adding DLC3 to the list of genes involved in 46X,Y complete dysgenesis opens up a new avenue to analyse the molecular and cellular mechanisms behind these disorders that could help in diagnosis and the development of future treatments”.
Reviewer #3 :
Major comments:
- This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
- In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.
Response: We don’t think quantifying the stainings will add much to the results. We believe that the changes performed increasing the images’ contrast and their amplification are sufficient to illustrate our statement about cv-c being expressed in testis but not in ovaries.
- In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.
Response: As referee suggested, we have quantified bleb phenotype. The results are presented in figure 3, panel J.
- In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?
Response: The percentages are quantified considering if the testes have any germ cell outside the gonad. We have added a line to clarify this point in the figure legend: “…quantified as encapsulated gonads with all germ cells inside the testis as assessed by Fisher-test”.
Nevertheless, we are going to quantify the number of ECM breaks and show the results in the reviewed manuscript.
- Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?
Response: cv-c mutants are embryonic lethal because of Cv-c’s widespread requirement on various embryonic tissues during development. Induction of FRT clones in the embryonic testis mesoderm was unsuccessful because of the low number of divisions during embryogenesis. We also tried to knock down cv-c expression with 3 different RNAi lines. Unfortunately, overexpression of these RNAi with different testis Gal4 drivers did not decrease cv-c mRNA levels significantly in the mesoderm or in other tissues where cv-c is expressed. Despite these experiments unsatisfactory outcome, our finding that cv-c is expressed in the testis mesoderm cells, and the fact that we can rescue the testis phenotypes by expressing Cv-c with gonadal mesodermal specific Gal4 lines supports a testis mesoderm requirement of cv-c for its gonadal function.
- Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.
Response: We agree with the referee that the molecular characterisation of the StART-mediated GAP-independent Cv-c function we have uncovered in this work is a very interesting finding that should be addressed by future work. However, such biochemical characterisation requires a complex approach to distinguish between the already known StART function regulating the GAP activity shown before (Sotillos Scientific Reports) and the new GAP-independent function we describe in the testes that falls beyond this work.
The central point of this manuscript is the demonstration that both DLC3/Cv-c are involved in male gonad formation, an important conserved function for both of them that had been overlooked by previous publication. Thus, DLC3 should be considered a new gene to be analysed in the future when studying gonadal dysgenesis. A second important point raised by our work is the demonstration that DLC3/Cv-c can perform RhoGAP independent functions, something that had never been described for these proteins.
Not withstanding this, in the revised version, we have added a new supplementary figure (1) related to the StART domain-lipid interaction analysed in-silico. The in-silico model shows that the DLC3-StART domain Ω1-loop structure displays the highest frequency of interaction with the membrane. This loop is conserved in the StART domains of several other STARD proteins and seems to modulate access to the ligand binding cavity. Ω-loops play multiple roles in protein function, often related to ligand binding, stability and folding. In this context, mutations in the proximity of the Ω1-loop, like the ones carried by the patients, may have drastic effects on overall protein stability that could affect the interaction between gonadal precursor cells.
- Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?
Response: Unfortunately, all studied cv-c mutants are embryonic lethal.
- Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.
Response: This is an interesting suggestion. JAK/STAT signalling has a male specific function that could explain why cv-c gonadal defects are male specific. We are going to study how cv-c affects STAT signalling in the male gonad. We are currently preparing stocks combining 10XSTAT::GFP reporter with cv-c mutants and preparing samples for anti-STAT labelling. We will also analyse if embryos lacking STAT activation, activate cv-c expression in the testes.
- I was also wondering if the authors have examined the number of germ cells in the mutant gonads.
Response: Yes, we have counted the number of germ cells in cv-c mutants and, if anything, there are more. We initially considered that an excess of GC proliferation could be the cause of gonad disruption. However, we have discarded this hypothesis as phospho-histone 3 stainings did not show a significant increase of GC divisions. Moreover, when we blocked cell proliferation in cv-c’ mutant gonads using UAS-p21, the testes phenotype was not rescued. We are unsure what could be responsible for the slight increase of germ cells observed.
- In addition, I think the quality of the images should be improved.
Response: We have changed the colours used in the confocal images and amplified the relevant regions in all panels. We thank both referees for this suggestion as these changes have improved the figure contrast.
Minor comments:
- cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.
Response: We have changed it.
- There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.
Response: We have added scale bars to all panels.
- In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.
Response: Corrected
- Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.
Response: Corrected
- Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.
Response: Corrected
- What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.
Response: The meaning of the asterisk was explained in the figure legend.
- There is no grey channel (sagittal view) for the panels Figure 3I and J.
Response: We have already included sagittal views in the figure.
- Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
- Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
- In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
- In line 723, it should be anti-beta catenin.
Response: As suggested, we have unified figure labelling.
- The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.
Response: We have already done the quantification and added the data as a graph in figure (3J).
- The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.
Response: We are repeating stainings to quantify Perlecan disruption in cv-c mutants and we will display the results as a graph in figure 6.
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Referee #3
Evidence, reproducibility and clarity
Summary:
This manuscript uses genetics, cell biology and confocal imaging to study the loss-of-function phenotypes of Cv-c, the Drosophila ortholog of DLC3. DLC3 mutation has been recently associated with male gonad dysgenesis in DSD patients. This work showed that Cv-c acts in the somatic gonadal cells to regulate male gonad development. Interestingly, the Cv-c mutant phenotypes result in testicular dysgenesis, marked by defective germ cell ensheathment and blebbing. Using different variants of the Cv-c protein and genetics based analyses, the authors further identified that the gonad development is dependent on StART domain but is independent of the GAP domain. Overall, these results should be of interest to researchers in disorders of sexual development, germ cell biology and developmental biology fields. However, at several places in order to reach the conclusions, more rigorous experiments should be performed (see revision details). In conclusion, this work has the potential but requires statistical quantification to support the central claims.
Major comments:
- This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
- In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.
- In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.
- In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?
- Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?
- Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.
- Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?
- Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.
- I was also wondering if the authors have examined the number of germ cells in the mutant gonads.
- In addition, I think the quality of the images should be improved.
Minor comments:
- cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.
- There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.
- In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.
- Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.
- Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.
- What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.
- There is no grey channel (sagittal view) for the panels Figure 3I and J.
- Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
- Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
- In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
- In line 723, it should be anti-beta catenin.
- The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.
- The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.
Significance
The significance of this paper is the identification of a conserved protein that has a conserved function in regulating spermatogenesis. The Drosophila embryonic testis is an ideal system to study the role of cv-c in gonadogenesis. It also sets the stage for studying the potential roles of cv-c in adult testes.
The existing published knowledge about somatic ensheathment of germ stem cells is sufficiently covered and cv-c is a new player in this process.
The audience for this study is developmental biologists and researchers studying disorders of sex development.
Our expertise is somatic sex identity in adult Drosophila gonads and DSD.
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Referee #2
Evidence, reproducibility and clarity
This is an interesting and generally convincing study demonstrating the potential role of DLC3 (STARD8)/Cv-c in testicular development, and in particular its role in early (fetal in human) germ cell development. The genetic manipulations and associated techniques involving Drosophila appear to be state of the art, although I would not class myself as expert enough in such techniques to be able to give a truly informed opinion.
My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.
I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.
Significance
This represents a significant advance in our understanding and identifies model systems in which to gain further insight.
I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders in humans.
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Referee #1
Evidence, reproducibility and clarity
Summary:
The authors have utilised Drosophila to provide evidence that a DLC3 mutation identified in 46,XY patients with male gonadal dysgenesis is likely causative for the phenotype. They demonstrated that mutation of the Drosophila ortholog of DLC3, Cv-c, was associated with defects in embryonic testis development and that the phenotype could be rescued by expression of wildtype human DLC3 but not the patient variant. DLC3/Cv-c are members of the RhoGAP family of proteins but GAP activity was not required for in the testis while mutation of the StART domain disrupted gonad morphogenesis. Mutations in this domain were associated with human gonadal dysgenesis suggesting a conservation of function during gonad development.
Major comments:
In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure.<br /> Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.<br /> The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.<br /> I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.<br /> The methods are well described, statistics adequate and the data well described.
Minor Comments:
My only suggestion for the text is to provide a more through description of the StART domain in the introduction.
Referees cross-commenting
Quantification of dispersed low level punctate expression levels will be very difficult to achieve and I do not believe will alter the conclusions. I agree that quantification of the percentage of gonads that display the illustrated phenotypes would be beneficial. The manuscript nicely describes a phenotype that has been defined to be due to mutation of the StART domain. Defining the mechanism of how the StART domain regulates protein function would take the study to a new level but may not be necessary for publication of the findings.
Significance
The significance of this work is two-fold.
- A demonstration that Cv-c and DLC3 have similar functions in regulation of gonad morphogenesis, and that the patient mutations almost certainly correlate with the observed phenotypes.
- An intriguing demonstration that this family of proteins is not simply functioning as RhoGAPs, and that there is a specific roles for the StART domain in male gonad development.
DLC3 has already been shown to rescue Malpighian tubule defects associated with Cv-c mutants so the functional equivalence of these proteins has already been established. The novelty of the present study relates to a demonstration that the StART domain is specifically required for male gonad morphogenesis, and the utility of experiments in Drosophila to assist in associating a human mutation with a clinical phenotype.
This work should find a willing audience from clinical geneticists as an example of how model organism genetics can assist genetic diagnoses. It will also be of great interest to the field of reproductive biologists who wish to understand how the sex determination pathways are resolved by differential tissue development. The obvious next steps are to determine why mutations in Cv-c only affect male gonads and not females, and what is the specific role of the StART domain in this process.<br /> My field of study has focussed on adult gonad development and regeneration, and I found the manuscript presented in a style that was simple to follow with my suggestion that contrast of some of the colour panels could be improved.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The rapid syncytial nuclear cycles that occur during the first ~2.5 hours of Drosophila embryogenesis and give rise to the blastoderm are supported by large amounts of maternally deposited histone proteins which are stored in the egg cytoplasm for deposition into replicating DNA during each round of S phase. Although the H2A/H2B storage chaperone Jabba was identified by Michael Welte's lab several years ago, maternal H3/H4 storage chaperones have not been identified. Tirgar et al provide evidence that the Drosophila NASP protein provides histone H3 and H4 storage function during these earliest stages of Drosophila embryogenesis. The data include genetic analyses that NASP function is required maternally, but not zygotically, and molecular analyses that NASP binds H3 and that H3 and H4 levels are reduced in the embryo and late-stage oocytes in the absence of NASP. These data are convincing and support the conclusion that NASP is a maternally acting H3/H4 storage chaperone needed in the early embryo.
Two additional lines of investigation would strengthen this conclusion and perhaps increase the impact and appeal of the manuscript.
The first is a microscopic analysis of the nuclear division cycles in eggs derived from NASP mutant mothers. The authors report DAPI staining and assessment of nuclear cycles, but do not show these data. In fact, the two embryos shown in Figure 4B do not look like DAPI stained embryos-there are no nuclei apparent in the images. Loss of maternal histone causes defects in chromosome morphology that result in characteristic defects such as lagging chromosomes and the failure of sister chromatid segregation leading to fused daughter nuclei (see PMID: 11157774 for an example). These defects should not be difficult to detect via DNA staining or even using fluorescently labeled H2 type histones. Characterizing such defects would lend support to the hypothesis and I think is important for this paper.
We thank the reviewer for their constructive review and feedback. We have switched to Propidium Iodide (PI) staining to increase the signal-to-noise for DNA staining in early embryos. Given the improved signal we see with PI over DAPI, we will be able to provide both improved images of nuclear staining and assay for defects in chromosome morphology as suggested. We will include this data in the revised version of the manuscript. Second, determining the location of NASP in the early embryo might provide further insight into the mechanism of storage. i.e. is NASP located in the cytoplasm rather than the nucleus, perhaps in association with lipid droplets like Jabba? Do the antibodies the authors developed work in IF experiments to ask this question? At the moment what is shown is that NASP is present in 0-2 hour embryos via western blot analysis, supporting the conclusion that it functions in the early embryo as a storage chaperone. This analysis would be nice to have but is not essential in my view.
We have tried to use our antibody to monitor the localization of NASP in the early embryo. Unfortunately, the staining has yet to work. We will continue to alter fixation and permeabilization conditions in the early embryo with the goal of including this data in the revised manuscript. We have, however, been able to monitor NASP localization in Drosophila S2 cultured cells with our antibody. If we are unable to get the antibody staining to work in embryos, we will include the NASP localization data in S2 cells in combination with EdU labeling to mark cells in S phase.
Small points: Is NASP really a maternal effect "lethal"? Some of the eggs do hatch, and so some develop to stages where maternal histones are no longer necessary and zygotic production takes over (i.e. cycle 15). Perhaps consider the language used here.
We see the reviewers point with respect to the term ‘lethal’. We do see a very small fraction of progeny laid by NASPmutant mothers make it to adulthood, although they die shortly after hatching. We’ve removed the term ‘lethal’ and refer to NASP solely as a maternal effect gene. On this point, do NASP mutant females lay the same number of eggs as wild type? i.e. is there a requirement for oogenesis/egg production (other than depositing H3/H4 into the egg), or just for the early zygotic cycles?
We have noticed that NASP mutant mothers have lower fecundity. We have included this data in the revised manuscript as Supplemental Figure 2A.
The first paragraph of the results is redundant with much of the introduction, which I think could do a better job at describing in more detail the syncytial cycles and the special needs they have for histone storage and chaperone function versus the post-blastoderm embryonic cycles and the rest of development. i.e. make a better distinction between the first two hours of embryogenesis versus the rest of embryogenesis, and the when the switch from maternal to zygotic control of development and histone production occurs (cycle 15 at 3-4 hours AED).
We appreciate the reviewer for this suggestion. The manuscript has been edited to be less redundant and include details of embryogenesis as suggested. CROSS-CONSULTATION COMMENTS Seems like all reviewers are in general agreement, particularly about providing additional data regarding chromosome/nuclear behavior in the NASP mutants and NASP localization in the early embryo to increase impact of the study. While rescue of the NASP mutant phenotype with a transgene would be nice, as suggested by referee #2, I don't think it's essential given the genetic approaches employed.
Reviewer #1 (Significance (Required)):
see above
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Tirgar et al. report on a functional characterization of the Drosophila homolog of the histone H3/H4 chaperone NASP. They generated a loss of function allele of NASP by CRISPR/Cas9, which induces a partial maternal effect embryo lethal phenotype. Using quantitative mass spectrometry, they demonstrate that NASP stabilizes reservoirs of H3 and H4 in the early embryo. The manuscript is very clear and confirms the functional importance of maternal NASP for the early embryo. Genetic analyses are well conducted (but see my comments below) and the impact of NASP maternal mutant on H3 and H4 stockpiles is convincingly established by both quantitative mass spectrometry and Western-blotting.
Major comments:
- Although the authors used two independent deficiencies of the NASP genetic region to characterize their NASP CRISPR alleles, it is relatively standard in this type of functional analyses to perform rescue experiments using a transgene expressing the WT protein.
We thank the reviewer for this suggestion. As discussed in the cross consultation, we agree that the use of the two different deficiency lines and the NASP1 CRISPR control are clear lines of evidence that the phenotypical data are due to lack of NASP.
- In WB analyses, NASP appears systematically shorter in the NASP[1]/Df genotype compared to WT. Can the authors comment on this?
While we reproducibly see this change in migration, we can only guess as to why this may be. One possible reason is that the NASP1 mutant protein could be missing a post-translational modification. Proteomic data from Krauchunas et al. (Dev Biol. 2012; PMC3441184) shows that NASP has the potential to be regulated by phosphorylation. Therefore, the NASP1 mutant protein could be missing a phosphorylation. Intriguingly, the 6bp insertion is next to a Thr residue that could affect its ability to be phosphorylated (if it is phosphorylated at all). Since we can only offer speculation, we do not feel comfortable adding this to the manuscript.
- The authors do not mention the centromeric histone H3 variant Cid in their analyses. Do they have evidence that it is not affected by loss of maternal NASP?
We thank the reviewer for raising this great point. Our mass spec data reveals that Cid levels stay the same in the absence of NASP in both embryos and stage 14 egg chambers. We have edited Figures 3D and 3E to include Cid. Unfortunately, we did not identify any Cid-specific peptides in our IP-mass spec data.
- The authors could have chosen to explore in more details the phenotypic defects of embryos derived from NASP mutant mothers. Instead, a single abnormal embryo is shown with no cytological details. This is a bit problematic since an earlier study (Zhang et al 2018, cited in the manuscript) actually provided more phenotypic details of embryos from NASP KD mothers.
This issue was also raised by Reviewer 1. We have switched to Propidium Iodide (PI) staining to increase the signal-to-noise for DNA staining in early embryos. Given the improved signal we see with PI over DAPI, we will be able to provide both improved images of nuclear staining and assay for defects in chromosome morphology as suggested. We will include this data in the revised version of the manuscript. - Similarly, the authors could have used their anti-NASP antibody to analyze the distribution of NASP during cleavage divisions. Does it behave like ASF1, for instance, which enters S phase nuclei at each cycle or does it remain in the cytoplasm? These are relatively simple experiments/analyses that could increase the significance of the study.
This point was also raised by Reviewer 1. We have tried to use our antibody to monitor the localization of NASP in the early embryo. Unfortunately, the staining has yet to work. We will continue to alter fixation and permeabilization conditions in the early embryo with the goal of including this data in the revised manuscript. We have, however, been able to monitor NASP localization in Drosophila S2 cultured cells with our antibody. If we are unable to get the antibody staining to work in embryos, we will include the NASP localization data in S2 cells in combination with EdU labeling to mark cells in S phase.
Minor comments:
- line 60: I suggest to introduce Drosophila in the next sentence, where it seems more appropriate (not all embryos develop "extremely rapidly").
We have edited the second sentence to state “the early Drosophila embryo”.
- line 68: the 50% estimation of free histones does not really make sense without defining the embryonic stage.
We have edited the manuscript to state the specific cell cycle in which there has been 50% free histones measured. - line 89: Are the authors specifically referring to Drosophila NASP?
Yes, we have edited the text to include Drosophila in this instance. - lines 99-106: I found this paragraph redundant with the introduction.
We appreciate this suggestion. It was also pointed out by Reviewer 1. We have made changes to the manuscript to address the redundancy.
- line 142: H3-H4
Thank you for noticing this. We have edited the text to include 4.
- line190-191: It seems to me that data of Figure S2C are already included in Fig. 2E.
The data in FigureS2C was performed with virgin females compared to the data in Figure 2E that was generated with non-virgin mothers. This was important to control the genotype of the embryos.
- line 232: it is surprising that the Zhang et al paper (reporting maternal KD of NASP) is only mentioned here. As a reader, I would certainly prefer to have it presented right from the introduction.
We have edited the manuscript to include this reference in the introduction.
- Figure 4B needs a scale bar.
Figure 4B will be replaced with better images of the embryo stained with PI. It will also include images of chromosome morphology/segregation. We will be sure to include scale bars.
- line 302: Mentioning the identity and function of known H3/H4 histone chaperones acting in the early embryo (ASF1, HIRA, CAF-1, ...) could provide perspective to the present study.
Thank you for this suggestion. We have edited the manuscript to include functions of other histone chaperones in the early embryo to provide context.
- line 304: in contrast to this statement, I found quite surprising and interesting that NASP is not absolutely essential for embryo development considering its role. This should be discussed.
In the absence of Jabba alone, upregulation of translation can compensate for the destabilization of H2A, H2B, and H2Av. It is only when translation is inhibited in embryos laid by Jabba mutant mothers that embryos die (Li.Z, et al. Curr Biol 2013). Therefore, it is possible that translation can partially compensate for the degradation of H3 and H4 in the absence of NASP. This may be why a fraction of embryos laid by NASP mutant mothers are able to hatch and why we still detect some H3 in embryos laid by NASP mutant mothers. We have edited the manuscript to discuss this more in depth.
CROSS-CONSULTATION COMMENTS I fully agree with the other reports. The NASP rescue experiment is just a suggestion but is not essential.
Reviewer #2 (Significance (Required)):
This work clarifies the identity and function of Drosophila NASP and clearly demonstrates that NASP is important for the stabilization of maternal stockpiles of H3 and H4 during early embryo development. The conservation of NASP function as a histone H3/H4 chaperone in Drosophila is not really a surprise but the merit of this study is to establish this assumption as a fact. It also establishes useful tools (mutant lines and antibody) for the fly community interested in this topic. The study however does not provide new insights about the dynamic distribution of NASP and the cytological consequences of its maternal depletion on the amplification of cleavage nuclei.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary: Rapid cell cycles in early embryogenesis is driven from maternally supplied stockpiles of RNA and protein, including histones H3 and H4. This study uses sequence homology searches, biochemical approaches (immunoprecipitation and mass spectrometry) and genetics to identify NASP (CG8223) as the H3-H4 chaperone in Drosophila. Using CRISPR technology, the authors generate a NASP mutant fly line and show using genetic crosses that NASP is a maternal lethal gene. Furthermore the study shows that NASP stabilises H3-H4 during oogenesis and embryogenesis and is required for early embryogenesis.
Major comments: The key conclusions of this study are very convincing. For example, the authors use multiple approaches to show H3-H4 specific interactions with NASP and that H3-H4 protein levels are reduced in mutants (Western analyses, quantitative MS). Analysis is carried out on two individual NASP mutant lines (one deletion that produces no protein, one insertion that still produces some protein acting as a control). All experiments are well controlled, executed and presented. Genetic crossing schemes are well presented and statistical analysis of progeny is clear.
- We thank the reviewer for their positive feedback of our manuscript. Minor comments: In Figure 1B - Authors could indicate amino acids shown or are they full length proteins?
We have edited the methods to include specific amino residues that are included for each structure.
In Figure 2B - Authors could (semi) quantify reduction in NASP1 mutant to show this is a gene dose effect?
We have now included the quantification of the Western blot in Figure 2B.
CROSS-CONSULTATION COMMENTS I agree with the other reports. Although I did not indicate it in my original report, I agree that more in depth analysis of nuclear or chromosomal defects in NASP mutant embryos would enhance the study.
Thank you for this suggestion. We are repeating the DNA staining in embryos and will include this new data in the revised version of the manuscript.
Reviewer #3 (Significance (Required)):
Excess soluble histones can be toxic and must be bound to chaperones. Until this study the chaperone responsible for H3-H4 stabilisation in rapidly cycling cells in Drosophila embryos was not known. Moreover, the NASP homolog had not yet been identified in Drosophila nor had its function been characterised. The findings are of interest to Drosophila researchers, the field of chromatin assembly, as well as those interested in early embryogenesis in animals.
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Referee #3
Evidence, reproducibility and clarity
Summary:
Rapid cell cycles in early embryogenesis is driven from maternally supplied stockpiles of RNA and protein, including histones H3 and H4. This study uses sequence homology searches, biochemical approaches (immunoprecipitation and mass spectrometry) and genetics to identify NASP (CG8223) as the H3-H4 chaperone in Drosophila. Using CRISPR technology, the authors generate a NASP mutant fly line and show using genetic crosses that NASP is a maternal lethal gene. Furthermore the study shows that NASP stabilises H3-H4 during oogenesis and embryogenesis and is required for early embryogenesis.
Major comments:
The key conclusions of this study are very convincing. For example, the authors use multiple approaches to show H3-H4 specific interactions with NASP and that H3-H4 protein levels are reduced in mutants (Western analyses, quantitative MS). Analysis is carried out on two individual NASP mutant lines (one deletion that produces no protein, one insertion that still produces some protein acting as a control). All experiments are well controlled, executed and presented. Genetic crossing schemes are well presented and statistical analysis of progeny is clear.
Minor comments:
In Figure 1B - Authors could indicate amino acids shown or are they full length proteins?
In Figure 2B - Authors could (semi) quantify reduction in NASP1 mutant to show this is a gene dose effect?
Referees cross-commenting
I agree with the other reports. Although I did not indicate it in my original report, I agree that more in depth analysis of nuclear or chromosomal defects in NASP mutant embryos would enhance the study.
Significance
Excess soluble histones can be toxic and must be bound to chaperones. Until this study the chaperone responsible for H3-H4 stabilisation in rapidly cycling cells in Drosophila embryos was not known. Moreover, the NASP homolog had not yet been identified in Drosophila nor had its function been characterised. The findings are of interest to Drosophila researchers, the field of chromatin assembly, as well as those interested in early embryogenesis in animals.
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Referee #2
Evidence, reproducibility and clarity
Tirgar et al. report on a functional characterization of the Drosophila homolog of the histone H3/H4 chaperone NASP. They generated a loss of function allele of NASP by CRISPR/Cas9, which induces a partial maternal effect embryo lethal phenotype. Using quantitative mass spectrometry, they demonstrate that NASP stabilizes reservoirs of H3 and H4 in the early embryo. The manuscript is very clear and confirms the functional importance of maternal NASP for the early embryo. Genetic analyses are well conducted (but see my comments below) and the impact of NASP maternal mutant on H3 and H4 stockpiles is convincingly established by both quantitative mass spectrometry and Western-blotting.
Major comments:
- Although the authors used two independent deficiencies of the NASP genetic region to characterize their NASP CRISPR alleles, it is relatively standard in this type of functional analyses to perform rescue experiments using a transgene expressing the WT protein.
- In WB analyses, NASP appears systematically shorter in the NASP[1]/Df genotype compared to WT. Can the authors comment on this?
- The authors do not mention the centromeric histone H3 variant Cid in their analyses. Do they have evidence that it is not affected by loss of maternal NASP?
- The authors could have chosen to explore in more details the phenotypic defects of embryos derived from NASP mutant mothers. Instead, a single abnormal embryo is shown with no cytological details. This is a bit problematic since an earlier study (Zhang et al 2018, cited in the manuscript) actually provided more phenotypic details of embryos from NASP KD mothers.
- Similarly, the authors could have used their anti-NASP antibody to analyze the distribution of NASP during cleavage divisions. Does it behave like ASF1, for instance, which enters S phase nuclei at each cycle or does it remain in the cytoplasm? These are relatively simple experiments/analyses that could increase the significance of the study.
Minor comments:
- line 60: I suggest to introduce Drosophila in the next sentence, where it seems more appropriate (not all embryos develop "extremely rapidly").
- line 68: the 50% estimation of free histones does not really make sense without defining the embryonic stage.
- line 89: Are the authors specifically referring to Drosophila NASP?
- lines 99-106: I found this paragraph redundant with the introduction.
- line 142: H3-H4
- line190-191: It seems to me that data of Figure S2C are already included in Fig. 2E.
- line 232: it is surprising that the Zhang et al paper (reporting maternal KD of NASP) is only mentioned here. As a reader, I would certainly prefer to have it presented right from the introduction.
- Figure 4B needs a scale bar.
- line 302: Mentioning the identity and function of known H3/H4 histone chaperones acting in the early embryo (ASF1, HIRA, CAF-1, ...) could provide perspective to the present study.
- line 304: in contrast to this statement, I found quite surprising and interesting that NASP is not absolutely essential for embryo development considering its role. This should be discussed.
Referees cross-commenting
I fully agree with the other reports.
The NASP rescue experiment is just a suggestion but is not essential.
Significance
This work clarifies the identity and function of Drosophila NASP and clearly demonstrates that NASP is important for the stabilization of maternal stockpiles of H3 and H4 during early embryo development. The conservation of NASP function as a histone H3/H4 chaperone in Drosophila is not really a surprise but the merit of this study is to establish this assumption as a fact. It also establishes useful tools (mutant lines and antibody) for the fly community interested in this topic. The study however does not provide new insights about the dynamic distribution of NASP and the cytological consequences of its maternal depletion on the amplification of cleavage nuclei.
-
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Referee #1
Evidence, reproducibility and clarity
The rapid syncytial nuclear cycles that occur during the first ~2.5 hours of Drosophila embryogenesis and give rise to the blastoderm are supported by large amounts of maternally deposited histone proteins which are stored in the egg cytoplasm for deposition into replicating DNA during each round of S phase. Although the H2A/H2B storage chaperone Jabba was identified by Michael Welte's lab several years ago, maternal H3/H4 storage chaperones have not been identified. Tirgar et al provide evidence that the Drosophila NASP protein provides histone H3 and H4 storage function during these earliest stages of Drosophila embryogenesis. The data include genetic analyses that NASP function is required maternally, but not zygotically, and molecular analyses that NASP binds H3 and that H3 and H4 levels are reduced in the embryo and late-stage oocytes in the absence of NASP. These data are convincing and support the conclusion that NASP is a maternally acting H3/H4 storage chaperone needed in the early embryo.
Two additional lines of investigation would strengthen this conclusion and perhaps increase the impact and appeal of the manuscript.
The first is a microscopic analysis of the nuclear division cycles in eggs derived from NASP mutant mothers. The authors report DAPI staining and assessment of nuclear cycles, but do not show these data. In fact, the two embryos shown in Figure 4B do not look like DAPI stained embryos-there are no nuclei apparent in the images. Loss of maternal histone causes defects in chromosome morphology that result in characteristic defects such as lagging chromosomes and the failure of sister chromatid segregation leading to fused daughter nuclei (see PMID: 11157774 for an example). These defects should not be difficult to detect via DNA staining or even using fluorescently labeled H2 type histones. Characterizing such defects would lend support to the hypothesis and I think is important for this paper.
Second, determining the location of NASP in the early embryo might provide further insight into the mechanism of storage. i.e. is NASP located in the cytoplasm rather than the nucleus, perhaps in association with lipid droplets like Jabba? Do the antibodies the authors developed work in IF experiments to ask this question? At the moment what is shown is that NASP is present in 0-2 hour embryos via western blot analysis, supporting the conclusion that it functions in the early embryo as a storage chaperone. This analysis would be nice to have but is not essential in my view.
Small points:
Is NASP really a maternal effect "lethal"? Some of the eggs do hatch, and so some develop to stages where maternal histones are no longer necessary and zygotic production takes over (i.e. cycle 15). Perhaps consider the language used here. On this point, do NASP mutant females lay the same number of eggs as wild type? i.e. is there a requirement for oogenesis/egg production (other than depositing H3/H4 into the egg), or just for the early zygotic cycles?
The first paragraph of the results is redundant with much of the introduction, which I think could do a better job at describing in more detail the syncytial cycles and the special needs they have for histone storage and chaperone function versus the post-blastoderm embryonic cycles and the rest of development. i.e. make a better distinction between the first two hours of embryogenesis versus the rest of embryogenesis, and the when the switch from maternal to zygotic control of development and histone production occurs (cycle 15 at 3-4 hours AED).
Referees cross-commenting
Seems like all reviewers are in general agreement, particularly about providing additional data regarding chromosome/nuclear behavior in the NASP mutants and NASP localization in the early embryo to increase impact of the study. While rescue of the NASP mutant phenotype with a transgene would be nice, as suggested by referee #2, I don't think it's essential given the genetic approaches employed.
Significance
see above
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
An exciting development in our knowledge about how the Arp2/3 complex controls the assembly of actin networks has come from the discovery that in addition to forming branched networks, Arp2/3 can nucleate linear filaments when it is activated by WISH/DIP/SPIN90. However, despite some excellent work largely done by the Nolen lab in yeast, many questions remain about how Arp2/3-mediated assembly of branched vs. linear actin filament. This is especially true in the complex environment of cells, were synergy and competition of different actin networks is used to control biological processes. Knowing the biochemical and physical properties of these different Arp2/3 assemblies will be key to figuring out how they work in cells. Here Cao et al. use an elegant microfluidics based single filament assay system to perform a comparative analysis of the stability of linear and branched Arp2/3 networks. They find interesting differences in how they respond to stabilizing and destabilizing factors. The most striking differences happens when force or aging is applied- both cause debranching of branched networks but have little effect on Spin90-Arp2/3 nucleated filaments.
We thank the reviewer for their positive comments.
Major comments:
As a comparative study on the stability of branched vs. linear Arp2/3 nucleated filaments, this manuscript is fairly complete. The key conclusions are well supported by rigorous experiments which can be reproduced by others based on the information provided. However, I am not seeing explicit information on performing biological replicates. This should be included in the manuscript. The use of statistics is largely fine; however I question the use of one statistical test on one figure (see minor comments below).
The revised manuscript is now explicit about biological replicates. We now specify the biological repeats of all our experiments in the figure legends, and we now show the results from new repeats in Fig 4 and Supp Fig S2 (please see also our response to the minor comments below, for more details).
I would not ask for additional experiments at this time. However, there is an analysis that would be important for interpreting the authors' claims- branch/filament length at the time of dissociation or destabilization of Arp2/3. This would help address if there was a physical tipping point for each type of structure that could explain potential differences they see. The authors should already have this data and the time to complete it would be negligible in delaying publication.
If we understand correctly, the “physical tipping point” mentioned by the reviewer would be a threshold force, where the Arp2/3-filament interface would become unstable. This is an interesting idea. Indeed, the applied force scales with the length of the filament (or branch), as well as with the flow velocity. In most of our experiments, however, the force applied to SPIN90-Arp2/3 and to branch junctions was kept constant and below 0.2 pN. This was done by exposing the filaments (or branches) to G-actin at the critical concentration, in order to minimize variations of their lengths. Therefore, by design, dissociation events in these experiments take place at the same length, ruling out the existence of a “tipping point”.
Our data provide another test of the reviewer’s hypothesis, thanks to the experiments where we specifically address the question of the impact of force (Fig 5 and Supp Fig S6), by varying length and flow rate. We found that the stability of SPIN90-Arp2/3 linear filaments was unaffected by force, and that debranching was steadily accelerated by force. In both cases, it thus appears that there is no detectable threshold.
One additional major comment is that the manuscript's title and abstract hint that this paper explores the differences in nucleation of branched vs. linear filaments by Arp2/3. However, the only figure that deals explicitly with nucleation in the paper is Figure 1, which is really just a confirmation that the mammalian proteins used in this study perform similarly to their yeast homologues (Balzer et al, Current Biology 2019). The authors might think about rewording the title/abstract to better reflect that paper really explores the differences in the stability of the two networks
This is a fair point. We have now modified the title into “Regulation of branched versus linear Arp2/3-generated actin filaments”.
Minor comments:
1 in 12 men and 1 in 200 women are red/green colorblind. Please change the coloring of the schematics and images so that they can be easily seen by all people. This is especially true of the schematics, which are important for understanding exactly what each assay is measuring.
We thank the reviewer for pointing this out. We have now made the schematics and images in Figs 1A, 2A, 2D and 4D colorblind-friendly.
The Introduction is a bit choppy and unfocused. It was difficult to deduce exactly where the paper was going from it. Please consider re-writing it for better clarity. The Discussion on the other hand was fantastic. Great job on interpreting your results in a larger context.
We have re-written large parts of the Introduction to make it clearer. We are glad the reviewer liked the Discussion, where we have nonetheless made some small changes in response to comments from the other reviewers.
Many figures- while the use of different lightness values of the same color is appreciated in conveying different concentrations of reagents used, there were several instances where it was very hard to read the one on the very bottom (ex. 2B, E; 3A; 5C, G).
We have now changed the colors in these figures, to make them clearer.
Figure 1- since this is a confirmation of previous results performed using the same proteins from other species, the title should reflect that (ex. VCA domains accelerate the nucleation of filaments by mammalian SPIN90-Arp2/3). Also, to me this figure is supplementary to the main message of the paper. The authors might think of moving it to Supplementary Information.
We have modified the title of Figure 1, now specifying “mammalian”, following the reviewer’s suggestion. However, we prefer to keep this figure as a main figure, rather than move it to Supplementary as proposed. Indeed, this figure does more than simply confirm previous results with mammalian proteins, since it compares different VCAs, which is new. These results are important because they are put in perspective with our results on the acceleration of linear filament detachment by different VCAs, later in the manuscript.
Figure 1- If the goal was to verify that G-actin recruitment by VCA was important for Spin90-Arp 2/3 nucleation by performing a competition experiment with profilin, why was the concentration of G-actin AND profilin increased between the experiments in 1B vs. 1C. It makes it hard to directly compare the results.
We now provide new data in Fig 1C, which can be directly compared to Fig 1B (only the profilin concentration was increased). It clearly shows that the effect of VCA disappears when the profilin concentration is increased.
Figure 4B-F- Here, it would be nice to see the distribution of all the individual results, which are hidden by the bar graph. Additionally, the Chi-square test is not the appropriate test for evaluating statistical significance between multiple groups. ANOVA followed by an appropriate post hoc test should be used here.
We now show the individual results in the bar graphs of figure 4. In this situation, we agree that the statistical significance should not be evaluated by a Chi-square test. We now indicate the p-values obtained from a paired t-test, which seems appropriate since we are comparing averages in pairs.
Figure 4G- Please quantify and show reproducibility.
We now show quantified repeats (shown in Fig 4, new panels H and I).
Figure 5- the piconewton forces used for these experiments is in line with measured forces that are applied to actin in cells (ex. Mehida et al, Nature Cell Biology 2021; Jiang et al, Nature 2003). The text would benefit if this was explicitly stated.
We now state this explicitly, when presenting these results.
Reviewer #1 (Significance (Required)):
The real significance of this work is in characterizing the differential stabilities of linear vs. branched Arp2/3 filaments in response to actin-binding proteins, mechanical stress, and aging. While both types of filaments respond similarly to actin-binding proteins, with nuanced differences, the most striking results came from applied force and aging experiments, with Spin90-Arp2/3 filaments being much more resistant to both. This has some very interesting implications for how these two types of assemblies might synergize in cells. Additionally, the results also have some exciting implications for the pointed-end regulation of actin filaments, which is still poorly understood in complex systems. Since the manuscript is A) more of a survey study on the factors that influence filament stability that does not go particularly deep into any particular mechanism of regulation and B) has no direct applicability to how the physical properties of branched and linear Arp2/3 nucleated actin filaments influence actin network activity in cells, the audience will likely by limited to actin enthusiasts. However, the work is still important in both what it reveals and implies.
We thank the reviewer for pointing out the novelty and the importance of our work. We agree that the significance of our paper lies in the characterization of the differential stabilities of linear vs. branched Arp2/3 filaments, in response to different physiological factors. One of the strengths of our approach is that we do not focus on one regulatory mechanism in particular. Rather, we reveal fundamental differences between the Arp2/3-generated filaments and how they can be regulated. Understanding these basic mechanisms is a prerequisite to understand the regulation of entire cytoskeletal networks.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.
Indeed, we argue that the two forms of activated Arp2/3 differ in their sensitivity to different VCA motifs, based on how these VCA motifs rank in their ability to destabilize branched and linear filaments (the VCA motifs also rank differently in their activation and co-activation of Arp2/3 to nucleate branches and linear filaments, but this result does not contribute to our discussion of how proteins interact with the activated Arp2/3). Following the reviewer’s suggestion, we now show repeats of these experiments (new Supp Fig S2), clearly showing that N-WASP is the most effective in dissociating linear filaments while the differences are milder for dissociating branches, with WASH being at least as effective as NWASP. We now also discuss how this observation could relate to differences in sequence between VCAs (Discussion section and new Supp Fig S9).
Also, please note that, following a suggestion from Reviewer 3, we have now performed experiments with the CA-domains of NWASP (new Supp Fig S4C and S4D), which show that the V-domain plays an important role in debranching but plays no role in destabilizing SPIN90-Arp2/3 at filament pointed ends. These new results reinforce our statement that VCA affects branched and linear Arp2/3-generated filaments differently.
Reviewer #2 (Significance (Required)):
Arp2/3 complex is a 7-protein complex implicated in actin filament nucleation and branching. Arp2/3 complex-nucleated branched networks are found at several locations in cells and are responsible for processes such as cell motility.
Cao et al. compare the effect of several proteins on the filament nucleation activity of Arp2/3 complex, and the stabilization or destabilization of actin filament branches as well as linear actin filaments nucleated by SPIN90-Arp2/3 complex. The proteins tested include the VCA regions of three NPFs (N-WASP, WASP, and WASH) that activate Arp2/3 complex, GMF (a debranching protein) and cortactin (a branch stabilizing protein). For the most part, the study uses a single method, microfluidics-TIRF microscopy.
The main findings are:
- VCA domains enhance nucleation of linear filaments by SPIN90-Arp2/3 complex in the presence of actin monomers.
- However, VCA domains can also destabilize existing SPIN90-Arp2/3 complex linear filaments and branches, and this effect depends on the presence of of V-domain (WH2 domain that binds actin monomers).
- The debranching factor GMF also destabilizes SPIN90-Arp2/3 complex linear filaments. Both GMF and VCA generate free pointed ends by dissociating Arp2/3 complex from pointed ends and SPIN90.
- SPIN90-Arp2/3 complex linear filaments are less susceptible to force and aging than filament branches.
- Cortactin stabilizes SPIN90-Arp2/3 complex linear filaments to higher degree than it does branches. These are novel and very interesting new observations of significant interest to the actin cytoskeleton field. Therefore, I recommend publication of this paper in EMBO J.
We thank the reviewer for their positive evaluation of our work.
I have one recommendation and one suggestion for improvement:
Major:
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The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.
This comment is identical to the reviewer’s first paragraph. We copy our answer here again, for convenience:
Indeed, we argue that the two forms of activated Arp2/3 differ in their sensitivity to different VCA motifs, based on how these VCA motifs rank in their ability to destabilize branched and linear filaments (the VCA motifs also rank differently in their activation and co-activation of Arp2/3 to nucleate branches and linear filaments, but this result does not contribute to our discussion of how proteins interact with the activated Arp2/3). Following the reviewer’s suggestion, we now show repeats of these experiments (new Supp Fig S2), clearly showing that N-WASP is the most effective in dissociating linear filaments while the differences are milder for dissociating branches, with WASH being at least as effective as NWASP. We now also discuss how this observation could relate to differences in sequence between VCAs (Discussion section and new Supp Fig S9).
Also, please note that, following a suggestion from Reviewer 3, we have now performed experiments with the CA-domains of NWASP (new Supp Fig S4C and S4D), which show that the V-domain plays an important role in debranching but plays no role in destabilizing SPIN90-Arp2/3 at filament pointed ends. These new results reinforce our statement that VCA affects branched and linear Arp2/3-generated filaments differently.
Minor:
In GST-pull-down experiments (Fig. 4G), the amount of Arp2/3 complex bound is analyzed by Western, which is rather unprecise. Is the amount of Arp2/3 complex so little that it cannot be quantified using regular SDS-PAGE? If that is the case, this would suggest rather low affinity of SPIN90 for Arp2/3 complex. How does this affect the proposed mechanism and experiments in the microfluidics chamber?
Indeed, the amount of pulled-down Arp2/3 is low and difficult to quantify by SDS-PAGE. This is consistent with previous reports which indicate a low affinity of SPIN90 for the Arp2/3 complex (Wagner et al. Current Biology 2013, Balzer et al. eLife 2020). This does not affect our conclusions, which we now confirm by showing quantified repeats of our pull-down experiments (new panels H and I, in Figure 4). In spite of this low affinity, which makes it difficult to saturate SPIN90 with Arp2/3, the SPIN90-Arp2/3 interaction is very stable and allows us to carry out our experiments in the microfluidics chamber over several tens of minutes (as was already the case in our previous study, Cao et al. NCB 2020).
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
In this study, Cao and collaborators investigate the biochemical and mechanical differences between branched actin filaments nucleated by WASP-activated Arp2/3 complex and linear actin filaments nucleated by SPIN90-activated Arp2/3 complex. They use TIRF microscopy in a microfluidic chamber to show that the mammalian proteins, SPIN90 and WASP (or N-WASP or WAVE), like their yeast homologues, co-activate Arp2/3 complex to nucleate linear actin filaments. Using the same assays, they find the surprising result that the VCA segment of WASP proteins destabilizes the interaction between SPIN90 and Arp2/3 complex in linear actin filaments nucleated by Arp2/3 complex. They then show that VCA also destabilizes actin filament branches. The remainder of the study explores the influence of branch stabilizing/destabilizing proteins or mechanical stress on the stability of the interaction between SPIN90 and Arp2/3 complex on the pointed end of the actin filament. They find that like branch junctions, SPIN90-bound Arp2/3 is destabilized at the end of linear filaments by GMF and stabilized by cortactin. However, unlike branch junctions, SPIN90-Arp2/3 complex is not destabilized on filament ends by piconewton forces or by aging. They conclude that SPIN90- versus VCA-activated Arp2/3 complex adopt similar but non-identical conformations.
Overall, the paper is well written and the experiments, which are very challenging, are rigorously executed. The biochemical results are convincing, novel and unexpected. However, the work could be strengthened by more strongly connecting the biochemical observations to biological implications. In addition, there are some interpretations/conclusions that seem somewhat weakly supported, and the authors should consider revising. Nonetheless, given the quality of the work and the importance of the system, this manuscript will appeal to a broad audience.
We thank the reviewer for their positive comments. We have rewritten parts of the Discussion in order to better connect our observations to implications in cells. We address the concerns regarding our interpretations in the point-by-point, below.
Comments on evidence, reproducibility, clarity and significance:
The differences in the stability of SPIN90-Arp2/3 on linear filaments verses branch junctions led the authors to conclude that SPIN90- versus VCA-activated complexes adopt similar yet non-identical conformations. There are two problems with this conclusion:
1) This conclusion rests on the idea that the biochemical differences can only be due to differences in the "ground state" active conformations of the complex. Another possible scenario would be that the active conformations are the same, but the transition state or intermediate state structures within the debranching reactions are different, thus changing the kinetics of the debranching reactions.
We thank the reviewer for this remark, and we agree that conformational differences may also arise in the intermediate states, during dissociation (of the branch from the mother, or of the linear filaments from SPIN90). We now mention this possibility in our Discussion.
2.) There are already structural data showing conformational differences between the Dip1-bound Arp2/3 complex on the end of a linear filament and Arp2/3 complex at a branch junction. While there are some caveats to comparisons of the structures (e.g., the Dip1 structure includes the fission yeast SPIN90 protein (Dip1) and the fission yeast Arp2/3 complex while the branch junction contains mammalian proteins), these data offer much stronger evidence that the active states adopt (somewhat) different conformations than the data presented here.
We agree that the available structural data (in particular, Ding et al. PNAS 2022, which was not yet published when we submitted our manuscript, and which we now cite) provide a clear indication that active Arp2/3 adopts different conformations in branches and linear filaments. We have modified our text to make this point clearer.
The authors make comparisons between the Fäβler branch junction structure and the Shaaban Dip1-Arp2/3-filament structure. The Fäβler branch junction structure is a low resolution structure (9 angstroms) and should be interpreted with caution (see below). A much higher resolution of a branch junction structure was recently solved (Ding et al, PNAS 2022) and should be used for comparisons between the structures.
Ding et al. PNAS 2022 was not yet published when we submitted our manuscript. We now use it to compare the structures of active Arp2/3, and we have modified the text accordingly.
Pg 14 - The authors say differences between ARPC3-Arp2 and ARPC5-Arp2 contacts in the two structures are likely to cause the differences in interactions with GMF and VCA. Two concerns with this statement are: 1.) The basis for the conclusion that the ARPC5-Arp2 contacts are different (in Fäβler, et al.) is not solid (see Ding, et al) and 2.) The analysis is vague. To reasonably conclude that differences in the contacts would influence GMF and VCA interactions would require mapping out the structural connection between the ARPC3-Arp2 interaction site and the GMF or VCA binding sites. If there is no obvious connection between these sites, the conclusion that the differences in the ARPC3-Arp2 interface cause differences in VCA and GMF binding should be far more circumspect.
We have re-written this part of the Discussion section. In light of the new data by Ding et al., we agree with the reviewer that the conclusion that the ARPC5-Arp2 contacts are different is not solid. Our revised text makes it clear that we are not making any claims involving interactions within the Arp2/3 complex. Our point is simply that recent cryo-EM reports indicate conformational differences in Arp2 and Arp3 between the two activated forms of the Arp2/3 complex and that, since the CA-domain of NPFs bind to Arp2 and Arp3, it appears reasonable to make a connection with our results.
Pg 6. "These observations suggest that the ability of VCA to destabilize Arp2/3-nucleated filaments relies on the availability of its V-domain." It's possible that G-actin binding to V blocks the CA from accessing the branch junction. Therefore, it seems important to test whether N-WASP-CA can destabilize Arp2/3-nucleated actin filaments.
We thank the reviewer for this suggestion. We now present results from new experiments performed with the CA-domain of NWASP (new Supp Fig S4C,D). We find that the V-domain participates in the enhancement of debranching, but that it appears to play no role in the destabilization of SPIN90-Arp2/3 from the pointed end. It thus seems that the reviewer’s proposal is correct, and that G-actin binding to the V-domain blocks the CA-domain from accessing the branch junction. We now propose this interpretation in the text.
Pg 1 - The authors state that "It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences". It is worth considering if one should make a blanket statement that linear and branched filaments respond similarly to regulatory proteins when they have tested 3 in total.
We have rephrased this sentence. It now reads “… respond similarly to the regulatory proteins we have tested…”
Pg 3 - "More generally, the stability of SPIN90-Arp2/3 at the pointed end, which is important to understand the reorganization and disassembly of actin filament networks, remains to be established." In some ways this statement not quite accurate because Balzer et al previously showed that Dip1-Arp2/3 complex is very stable at the pointed end. Is the question here whether that stability is also conserved in mammalian systems? If so, that should be more directly stated.
We meant that, beyond observing that SPIN90 remains visible at the pointed end for some time (as in Balzer et al.), a lot remained unknown: its lifetime had not been quantified, and its sensitivity to the factors that affect branch junctions (proteins, aging, mechanical tension) had not been studied. We have rephrased the sentence in the manuscript to clarify this point.
The observation that VCA accelerates debranching and SPIN90-Arp2/3 dissociation is very interesting. However, it is uncertain if this biochemical activity has biological relevance, given that once nucleation occurs, Arp2/3 complex will move away from the membrane. While the authors mention in the discussion that debranching by VCA could be relevant when the network is compressed near the membrane, this argument is not particularly strong. Are there ways to strengthen this argument, or find another impact this finding might have on our understanding of Arp2/3 complex regulation?
We now mention another situation where branch junctions could encounter membrane-bound VCA domains: on the dorsal and ventral membrane surfaces of lamellipodia. We now cite the recent Kage et al. J Cell Science 2022 and Mehidi et al. NCB 2021, where WAVE has been observed in lamellipodia away from the leading edge.
The observation that SPIN90+Arp2/3-nucleated filaments are not sensitive to piconewton forces is also very interesting. The authors focus on the differences in the amount of surface area buried when discussing this result. However, if seems a key factor in the stability of the linear filaments would be the direction of the force relative to the complex and attached filament(s), which would be very different for a branch versus a linear filament. The authors should consider addressing this in their discussion.
The orientation of the applied force is an interesting point. In their study on debranching, Pandit et al. (PNAS 2020) report that their results are not affected by the angle of the applied force relative to the mother filament (their Fig S1D). We now specify this in our manuscript, when introducing our results on mechanical tension. Similarly, we found that anchoring SPIN90 to the coverslip surface by its N-terminus rather than its C-terminus, which likely affects the orientation of the applied force, had no impact on our results (Supp Fig S6A). We have now also added a sentence regarding this aspect in our manuscript, after presenting this result.
Fig 4, D-F: It is unclear how the authors determined which filaments were spontaneously nucleated versus those that were nucleated by SPIN90-Arp2/3 complex in these experiments. In reactions containing SPIN90 and Arp2/3 complex what fraction of the filaments will be spontaneously nucleated?
In our conditions, there is no detectable spontaneous nucleation. In control experiments where we flow in the same concentration of G-actin, in the absence of Arp2/3 or in the absence of SPIN90, we observe no filaments at all on the surface, over several fields of view, after 5 minutes. We now specify this in the Methods section.
Pg 9 - The observation that VCA negatively influences binding of SPIN90 to the complex is unexpected. What implications does this have for understanding how SPIN90 and VCA synergize to activate the complex?
It appears that the outcome depends on the context. The main role of VCA during co-activation of the Arp2/3 complex with SPIN90 seems to be to supply G-actin, as already proposed (Balzer, 2020) and confirmed by our results (Fig 1C). In the absence of G-actin, VCA is more likely to remove Arp2/3 from SPIN90 (Fig 4G,I). Similarly, when a filament is already formed, the presence of G-actin mitigates the removal of SPIN90-Arp2/3 from the pointed end by VCA (Supp Fig S4).
Fig 4B - Why is there greater nucleation when Arp2/3 complex and GMF are added together compared to renucleation in reactions that don't have any GMF? This is surprising, especially considering that GMF decreases binding of Arp2/3 complex to SPIN90.
Indeed, there is a small yet statistically significant difference in the re-nucleation fraction we measured in the presence of Arp2/3, with or without GMF (Fig 4B). This may be due to the different timescales of the two situations. In the absence of GMF, the detachment of filaments is slow and new filaments are nucleated from the initial Arp2/3 complexes, which remained bound to SPIN90 upon detachment of the first filaments. In contrast, in the presence of GMF, detachment is faster and accompanied by the departure of the initial Arp2/3, and a fresh Arp2/3 then binds to SPIN90 to nucleate a new filament. It is thus possible that, in the absence of GMF, a small fraction of the SPIN90 and/or their initially bound Arp2/3 complexes would denature over the time they spend at the bottom of the microchamber at 25°C, thereby leading to a slightly smaller re-nucleation fraction. A similar mechanism could be at play in the experiments with or without VCA, in addition to the enhancement of nucleation by VCA (Fig 4C).
Minor Corrections/Comments
Pg 3 "We show that Arp2/3 nucleation is similarly stabilized by cortactin and destabilized by GMF" Do the authors mean branches and linear filaments nucleated by Arp2/3 complex?
Yes, that is what we meant. This sentence has now been modified.
Pg 6- The cyan 3uM data and legend in figure 2B and E is probably too dim to see clearly.
The colors have been changed to improve readability.
Fig 4 B,C,E,F: It would be best to show the individual data points here if possible.
We now show individual data points in all these figure panels.
Pg 16 Please specify which antibody was used to anchor SPIN90.
The antibodies are Anti-GST for Nter anchoring of GST-SPIN90, and anti-His for Cter anchoring of SPIN90-His. We now specify this in the Methods section.
CROSS-CONSULTATION COMMENTS I agree with the points that the other reviewers raised.
Reviewer #3 (Significance (Required)):
Comments on significance are in the above section.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this study, Cao and collaborators investigate the biochemical and mechanical differences between branched actin filaments nucleated by WASP-activated Arp2/3 complex and linear actin filaments nucleated by SPIN90-activated Arp2/3 complex. They use TIRF microscopy in a microfluidic chamber to show that the mammalian proteins, SPIN90 and WASP (or N-WASP or WAVE), like their yeast homologues, co-activate Arp2/3 complex to nucleate linear actin filaments. Using the same assays, they find the surprising result that the VCA segment of WASP proteins destabilizes the interaction between SPIN90 and Arp2/3 complex in linear actin filaments nucleated by Arp2/3 complex. They then show that VCA also destabilizes actin filament branches. The remainder of the study explores the influence of branch stabilizing/destabilizing proteins or mechanical stress on the stability of the interaction between SPIN90 and Arp2/3 complex on the pointed end of the actin filament. They find that like branch junctions, SPIN90-bound Arp2/3 is destabilized at the end of linear filaments by GMF and stabilized by cortactin. However, unlike branch junctions, SPIN90-Arp2/3 complex is not destabilized on filament ends by piconewton forces or by aging. They conclude that SPIN90- versus VCA-activated Arp2/3 complex adopt similar but non-identical conformations.
Overall, the paper is well written and the experiments, which are very challenging, are rigorously executed. The biochemical results are convincing, novel and unexpected. However, the work could be strengthened by more strongly connecting the biochemical observations to biological implications. In addition, there are some interpretations/conclusions that seem somewhat weakly supported, and the authors should consider revising. Nonetheless, given the quality of the work and the importance of the system, this manuscript will appeal to a broad audience.
Comments on evidence, reproducibility, clarity and significance:
The differences in the stability of SPIN90-Arp2/3 on linear filaments verses branch junctions led the authors to conclude that SPIN90- versus VCA-activated complexes adopt similar yet non-identical conformations. There are two problems with this conclusion:
- This conclusion rests on the idea that the biochemical differences can only be due to differences in the "ground state" active conformations of the complex. Another possible scenario would be that the active conformations are the same, but the transition state or intermediate state structures within the debranching reactions are different, thus changing the kinetics of the debranching reactions.
- There are already structural data showing conformational differences between the Dip1-bound Arp2/3 complex on the end of a linear filament and Arp2/3 complex at a branch junction. While there are some caveats to comparisons of the structures (e.g., the Dip1 structure includes the fission yeast SPIN90 protein (Dip1) and the fission yeast Arp2/3 complex while the branch junction contains mammalian proteins), these data offer much stronger evidence that the active states adopt (somewhat) different conformations than the data presented here.
The authors make comparisons between the Fäβler branch junction structure and the Shaaban Dip1-Arp2/3-filament structure. The Fäβler branch junction structure is a low resolution structure (9 angstroms) and should be interpreted with caution (see below). A much higher resolution of a branch junction structure was recently solved (Ding et al, PNAS 2022) and should be used for comparisons between the structures.
Pg 14 - The authors say differences between ARPC3-Arp2 and ARPC5-Arp2 contacts in the two structures are likely to cause the differences in interactions with GMF and VCA. Two concerns with this statement are: 1.) The basis for the conclusion that the ARPC5-Arp2 contacts are different (in Fäβler, et al.) is not solid (see Ding, et al) and 2.) The analysis is vague. To reasonably conclude that differences in the contacts would influence GMF and VCA interactions would require mapping out the structural connection between the ARPC3-Arp2 interaction site and the GMF or VCA binding sites. If there is no obvious connection between these sites, the conclusion that the differences in the ARPC3-Arp2 interface cause differences in VCA and GMF binding should be far more circumspect.
Pg 6. "These observations suggest that the ability of VCA to destabilize Arp2/3-nucleated filaments relies on the availability of its V-domain." It's possible that G-actin binding to V blocks the CA from accessing the branch junction. Therefore, it seems important to test whether N-WASP-CA can destabilize Arp2/3-nucleated actin filaments.
Pg 1 - The authors state that "It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences". It is worth considering if one should make a blanket statement that linear and branched filaments respond similarly to regulatory proteins when they have tested 3 in total.
Pg 3 - "More generally, the stability of SPIN90-Arp2/3 at the pointed end, which is important to understand the reorganization and disassembly of actin filament networks, remains to be established." In some ways this statement not quite accurate because Balzer et al previously showed that Dip1-Arp2/3 complex is very stable at the pointed end. Is the question here whether that stability is also conserved in mammalian systems? If so, that should be more directly stated.
The observation that VCA accelerates debranching and SPIN90-Arp2/3 dissociation is very interesting. However, it is uncertain if this biochemical activity has biological relevance, given that once nucleation occurs, Arp2/3 complex will move away from the membrane. While the authors mention in the discussion that debranching by VCA could be relevant when the network is compressed near the membrane, this argument is not particularly strong. Are there ways to strengthen this argument, or find another impact this finding might have on our understanding of Arp2/3 complex regulation?
The observation that SPIN90+Arp2/3-nucleated filaments are not sensitive to piconewton forces is also very interesting. The authors focus on the differences in the amount of surface area buried when discussing this result. However, if seems a key factor in the stability of the linear filaments would be the direction of the force relative to the complex and attached filament(s), which would be very different for a branch versus a linear filament. The authors should consider addressing this in their discussion.
Fig 4, D-F: It is unclear how the authors determined which filaments were spontaneously nucleated versus those that were nucleated by SPIN90-Arp2/3 complex in these experiments. In reactions containing SPIN90 and Arp2/3 complex what fraction of the filaments will be spontaneously nucleated?
Pg 9 - The observation that VCA negatively influences binding of SPIN90 to the complex is unexpected. What implications does this have for understanding how SPIN90 and VCA synergize to activate the complex?
Fig 4B - Why is there greater nucleation when Arp2/3 complex and GMF are added together compared to renucleation in reactions that don't have any GMF? This is surprising, especially considering that GMF decreases binding of Arp2/3 complex to SPIN90.
Minor Corrections/Comments
Pg 3 "We show that Arp2/3 nucleation is similarly stabilized by cortactin and destabilized by GMF" Do the authors mean branches and linear filaments nucleated by Arp2/3 complex?
Pg 6- The cyan 3uM data and legend in figure 2B and E is probably too dim to see clearly.
Fig 4 B,C,E,F: It would be best to show the individual data points here if possible.
Pg 16 Please specify which antibody was used to anchor SPIN90.
Referees cross-commenting
I agree with the points that the other reviewers raised.
Significance
Comments on significance are in the above section.
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Referee #2
Evidence, reproducibility and clarity
The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.
Significance
Arp2/3 complex is a 7-protein complex implicated in actin filament nucleation and branching. Arp2/3 complex-nucleated branched networks are found at several locations in cells and are responsible for processes such as cell motility.
Cao et al. compare the effect of several proteins on the filament nucleation activity of Arp2/3 complex, and the stabilization or destabilization of actin filament branches as well as linear actin filaments nucleated by SPIN90-Arp2/3 complex. The proteins tested include the VCA regions of three NPFs (N-WASP, WASP, and WASH) that activate Arp2/3 complex, GMF (a debranching protein) and cortactin (a branch stabilizing protein). For the most part, the study uses a single method, microfluidics-TIRF microscopy.
The main findings are:
- VCA domains enhance nucleation of linear filaments by SPIN90-Arp2/3 complex in the presence of actin monomers.
- However, VCA domains can also destabilize existing SPIN90-Arp2/3 complex linear filaments and branches, and this effect depends on the presence of of V-domain (WH2 domain that binds actin monomers).
- The debranching factor GMF also destabilizes SPIN90-Arp2/3 complex linear filaments. Both GMF and VCA generate free pointed ends by dissociating Arp2/3 complex from pointed ends and SPIN90.
- SPIN90-Arp2/3 complex linear filaments are less susceptible to force and aging than filament branches.
- Cortactin stabilizes SPIN90-Arp2/3 complex linear filaments to higher degree than it does branches.
These are novel and very interesting new observations of significant interest to the actin cytoskeleton field. Therefore, I recommend publication of this paper in EMBO J. I have one recommendation and one suggestion for improvement:
Major:
- The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.
Minor:
- In GST-pull-down experiments (Fig. 4G), the amount of Arp2/3 complex bound is analyzed by Western, which is rather unprecise. Is the amount of Arp2/3 complex so little that it cannot be quantified using regular SDS-PAGE? If that is the case, this would suggest rather low affinity of SPIN90 for Arp2/3 complex. How does this affect the proposed mechanism and experiments in the microfluidics chamber?
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Referee #1
Evidence, reproducibility and clarity
An exciting development in our knowledge about how the Arp2/3 complex controls the assembly of actin networks has come from the discovery that in addition to forming branched networks, Arp2/3 can nucleate linear filaments when it is activated by WISH/DIP/SPIN90. However, despite some excellent work largely done by the Nolen lab in yeast, many questions remain about how Arp2/3-mediated assembly of branched vs. linear actin filament. This is especially true in the complex environment of cells, were synergy and competition of different actin networks is used to control biological processes. Knowing the biochemical and physical properties of these different Arp2/3 assemblies will be key to figuring out how they work in cells. Here Cao et al. use an elegant microfluidics based single filament assay system to perform a comparative analysis of the stability of linear and branched Arp2/3 networks. They find interesting differences in how they respond to stabilizing and destabilizing factors. The most striking differences happens when force or aging is applied- both cause debranching of branched networks but have little effect on Spin90-Arp2/3 nucleated filaments.
Major comments:
As a comparative study on the stability of branched vs. linear Arp2/3 nucleated filaments, this manuscript is fairly complete. The key conclusions are well supported by rigorous experiments which can be reproduced by others based on the information provided. However, I am not seeing explicit information on performing biological replicates. This should be included in the manuscript. The use of statistics is largely fine; however I question the use of one statistical test on one figure (see minor comments below).
I would not ask for additional experiments at this time. However, there is an analysis that would be important for interpreting the authors' claims- branch/filament length at the time of dissociation or destabilization of Arp2/3. This would help address if there was a physical tipping point for each type of structure that could explain potential differences they see. The authors should already have this data and the time to complete it would be negligible in delaying publication.
One additional major comment is that the manuscript's title and abstract hint that this paper explores the differences in nucleation of branched vs. linear filaments by Arp2/3. However, the only figure that deals explicitly with nucleation in the paper is Figure 1, which is really just a confirmation that the mammalian proteins used in this study perform similarly to their yeast homologues (Balzer et al, Current Biology 2019). The authors might think about rewording the title/abstract to better reflect that paper really explores the differences in the stability of the two networks
Minor comments:
1 in 12 men and 1 in 200 women are red/green colorblind. Please change the coloring of the schematics and images so that they can be easily seen by all people. This is especially true of the schematics, which are important for understanding exactly what each assay is measuring.
The Introduction is a bit choppy and unfocused. It was difficult to deduce exactly where the paper was going from it. Please consider re-writing it for better clarity. The Discussion on the other hand was fantastic. Great job on interpreting your results in a larger context.
Many figures- while the use of different lightness values of the same color is appreciated in conveying different concentrations of reagents used, there were several instances where it was very hard to read the one on the very bottom (ex. 2B, E; 3A; 5C, G).
Figure 1- since this is a confirmation of previous results performed using the same proteins from other species, the title should reflect that (ex. VCA domains accelerate the nucleation of filaments by mammalian SPIN90-Arp2/3). Also, to me this figure is supplementary to the main message of the paper. The authors might think of moving it to Supplementary Information.
Figure 1- If the goal was to verify that G-actin recruitment by VCA was important for Spin90-Arp 2/3 nucleation by performing a competition experiment with profilin, why was the concentration of G-actin AND profilin increased between the experiments in 1B vs. 1C. It makes it hard to directly compare the results.
Figure 4B-F- Here, it would be nice to see the distribution of all the individual results, which are hidden by the bar graph. Additionally, the Chi-square test is not the appropriate test for evaluating statistical significance between multiple groups. ANOVA followed by an appropriate post hoc test should be used here.
Figure 4G- Please quantify and show reproducibility.
Figure 5- the piconewton forces used for these experiments is in line with measured forces that are applied to actin in cells (ex. Mehida et al, Nature Cell Biology 2021; Jiang et al, Nature 2003). The text would benefit if this was explicitly stated.
Significance
The real significance of this work is in characterizing the differential stabilities of linear vs. branched Arp2/3 filaments in response to actin-binding proteins, mechanical stress, and aging. While both types of filaments respond similarly to actin-binding proteins, with nuanced differences, the most striking results came from applied force and aging experiments, with Spin90-Arp2/3 filaments being much more resistant to both. This has some very interesting implications for how these two types of assemblies might synergize in cells. Additionally, the results also have some exciting implications for the pointed-end regulation of actin filaments, which is still poorly understood in complex systems. Since the manuscript is A) more of a survey study on the factors that influence filament stability that does not go particularly deep into any particular mechanism of regulation and B) has no direct applicability to how the physical properties of branched and linear Arp2/3 nucleated actin filaments influence actin network activity in cells, the audience will likely by limited to actin enthusiasts. However, the work is still important in both what it reveals and implies.
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Reply to the reviewers
We thank all three reviewers for their thoughtful and rigorous critique of our manuscript, which we feel has significantly improved the presentation of our work. Below we detail point-by-point responses to comments made by the three reviewers as well changes we have already made addressing the majority of minor and some major points.
Specification of the eye-field during gastrulation represents the earliest known stage of eye development. Using an optic-vesicle organoid model system, the overall goal of our work is to provide an unbiased characterisation of this critical, early developmental event in mammals and to gain insights into relevant gene regulatory mechanisms. A common theme to some of the reviewer comments is that this work doesn't provide much of an advance to the field and our findings are not particularly original. We feel that these comments are slightly harsh for the following reasons. Firstly, although some of our findings are not unexpected, to our knowledge, this is the first unbiased characterisation of the eye-field in a mammalian model system, and not based on knowledge gained through previous work in other non-mammalian vertebrate systems, e.g. Xenopus. Secondly, by generating both RNA-seq and ATAC-seq from a timecourse of organoid development we have been able to quantify dynamic patterns of gene-expression as the eye-field is established and simultaneously gain insights to the regulatory role of some of the key transcription factors, both of which are not present in the literature. Thirdly, by constructing careful, integrated analyses of our RNA-seq and ATAC-seq datasets we were able to generate specific hypotheses regarding cis-regulation of key genes, which we have then demonstrated are possible to efficiently test within the organoid system. In all, although we have been purposely careful not to overinterpret our results, we feel our work does represent a significant step towards understanding the mammalian eye field and additionally provides important datasets as well as an analysis framework to begin to quantitatively probe the regulatory mechanisms underlying the transition to an ocular fate. Given the relevance of this developmental event to clinical genetics research as well as to developmental biology we are confident that this work represents an important and significant advance to the literature.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary Owen et al. characterize the transcriptome and chromatin accessibility of mouse retinal organoids at early stages during which eye field-like cells are specified. Since cell specification and differentiation in retinal organoids largely mimic those processes in vivo, retinal organoids are viable models for studying the mechanisms of early eye development. Owen et al. utilize a previously established Rx-GFP cell line, bulk RNA sequencing, and bulk ATAC sequencing to dissect the mechanisms of early eye development in mice. Their findings are generally consistent with previous studies. Overall, the study is interesting for the field, but its conceptual and technical advances are moderate. In addition, a few major points need to be clarified.
Major points 1. The authors did not show any analysis of retinal organoids at stages when Vsx2 is expressed. This is a significant weakness since the chemically defined medium (CDM) used in Owen et al.'s study was previously shown to induce rostral hypothalamic differentiation (Wataya et al., 2008). Related to this notion, several eye-field transcription factors, such as Rax and Six3, are also expressed in the hypothalamus. Therefore, Owen et al. need to demonstrate that organoids in their modified differentiation system efficiently produce Vsx2-positive retinal progenitors, and samples of organoids at stages when Vsx2 is expressed should be included for RNA sequencing. If Vsx2 is not efficiently expressed in their organoids, the interpretation of results will be very different.
We thank the reviewer for their important comments here. There are several reasons why we are confident that our data and conclusions regarding the organoid eye-field are robust. Firstly, our RNA-seq data, in particular the differences between GFP-positive and GFP-negative cells, clearly show a coordinated up-regulation of the set of canonical eye-field TFs (not individually), which previous studies in Xenopus have shown is a prerequisite for differentiation into anterior eye structures (including retina). Secondly, we have checked that some of the later (in development) eye markers, including Vsx2, are differentially up-regulated (DeSeq2, logfc>1.5, FDRIn all, we are very confident that our approach of using the optic-vesicle organoids and generating molecular data from an organoid developmental timecourse (including sorting), is unpicking the ocular-fate transition event that we are interested in.
- The authors state that "two differentiation medias were used for this work due to the differentiation becoming unstable after the initial experiments had been performed. The organoids used for RNA and ATAC-seq were grown in CDM media and the organoids with mutations introduced in potential CREs were grown in KSR media". Why the differentiation becomes unstable after the initial experiments? Differences in the two media cause additional complexities. Related to this notion, "WT Rx-GFP" in Figure 4B and 4E appears to show a different expression pattern compared to that in Figure 1A.
We were unable to identify the reason behind the destabilisation of differentiation in CDM media after the cell lines had been through CRISPR despite thorough testing. The differentiation of these cell lines was stabilised enough using KSR media such that every batch of organoids grown contained some organoids that expressed GFP in a pattern similar to what we had seen before and we carried on our experiments using this. We recognise that using two different media adds complexity, however we see the same patterns of organoid growth and GFP expression when differentiating untransfected WT Rax-GFP cells in both of these medias. We have edited Fig.S1 to include representative images of organoids grown in KSR media which can be directly compared to those grown in CDM shown in Figure 1A.
The reviewer has pointed out that the WT Rx-GFP organoids in Figure 6B and 6E show a different expression pattern to those in figure 1A. With the addition of the supplemental figure mentioned above it becomes apparent that these differences are not due to the change of media. We have clarified in the text that these WT cells have also been transfected so as to act as appropriate controls that have been treated identically to the CRISPR edited cell lines and that this has affected their differentiation capacity.
- Is the deletion of Rax and Six6 regulatory elements homozygous? Sanger sequencing or amplicon sequencing is needed to show the deletion.
The deletions are homozygous (we have stated this in the manuscript text) and as suggested we have added a supplementary figure showing the Sanger sequencing traces for the WT and mutant cell lines used in this study.
- The deletion of Rax and Six6 regulatory elements appears to cause minor changes in the expression of Rax and Six6 (Figure 6C, F). Therefore, the impact of findings in bulk RNA seq and bulk ATAC seq in this study is still unclear.
We have added a sentence to the text underlining that developmental genes are expected to be regulated by multiple enhancers. Our expectation is therefore, that in perturbing a single putative regulatory element for Rax/Six6, we will very likely not see the complete ablation of Rax/Six6 expression.
- Retinal organoids and sorted cells are composed of heterogeneous cell populations. Bulk RNA seq and bulk ATAC seq do not have the power to dissect the complexity of heterogeneous cell populations. Single-cell RNA seq and single-cell ATAC seq are more powerful for this study.
We agree with the referee about the fact that the organoids are likely composed of relatively heterogeneous cell populations. We have added this limitation of our generated datasets in a “limitations” paragraph in the discussion.
- Numerous motifs in the JASPAR database are identified using in vitro assays and have not been validated using in vivo assays. Unexpected results in motif analysis could be due to the differences in DNA binding motifs between in vitro and in vivo conditions. This notion should be added in the discussion.
We have added a couple of sentences in the discussion section, highlighting that TF-motif and footprinting analyses of ATAC-seq data provide indirect evidence of TF binding, and to validate these findings experiments such as ChIP-seq or Cut&Run could be performed in the future.
Minor points
Numerous labels in figures are too small.
We have adjusted the size of a number of the figures to increase the size of the labels, which are now mostly the same size as the text in the corresponding figure captions. We are very happy to make further increases in the sizes of figure labels/text upon recommendation.
CROSS-CONSULTATION COMMENTS
My fellow reviewers identify similar major weaknesses and additional points. I agree with the other reviewers' comments.
Reviewer #1 (Significance (Required)): Nature and Significance of the advances In Owen et al.'s study, the Rx-GFP cell line and retinal differentiation protocol were established in previous studies (Wataya et al., 2008; Eiraku et al., 2011); bulk RNA sequencing and bulk ATAC sequencing are standard procedures. Although candidate regulatory elements for early eye development are identified, deletions of two prioritized elements using CRISPR/Cas9 only cause minor changes in the expression of targeted genes. Overall, conceptual and technical advances in Owen et al.'s study are moderate. Compare to existing published knowledge The datasets could be useful for the field, but conceptual and technical advances are moderate.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The authors grow eye organoids from cells with a reporter driving GFP in the Rax locus, a gene that is expressed in the eye field in many animal model systems. They show that expression of GFP picks up by day 4 and performed FACS sorting of GFP+ cells on day 4 and day 5 organoids to compare gene expression by RNAseq comparing with earlier day organoids. The data shows 37 genes with a differential expression on days 4 and 5, compared to day 3, and enriched in GFP+ cells, which they define as EF-up genes. It is notable that some of these genes had already been identified as canonical eye field gene regulatory network transcription factors. In the same way, they identify a group of differentially expressed regulated genes, EF-down, and state that 'many' of them are involved in pluripotency. However, they do not mention how many, or the proportion of these genes in the whole list.
The number of EF-down genes with GO terms linked to pluripotency has now been added to the text.
It would be useful if they could provide the number to understand how many of these genes are related to pluripotency, the whole list of genes mentioned to be downregulated in a supplementary file.
We appreciate that this list was missing and will include it now as a supplemental file.
The authors also note that genes known to be required for eye specification like Sox2 and Otx2 are not differentially expressed across the day 3-4 timepoint (Ln 190). However, this is not surprising considering that both genes are broadly expressed in the anterior neural ectoderm and required for its specification, which should be noted by the authors.
We have amended the aforementioned sentence to reflect this: “It is noteworthy that Sox2 and Otx2, known to be crucial in eye development are not differentially expressed across this critical time-point (Fig.2A), consistent with these genes being more broadly expressed in the anterior neuroectoderm in vivo.”
The authors then go on and cluster the EF-up, EF-down and genes deferentially expressed between days 2 and 3, and identify 6 discreet trajectory groups. From this analysis, they identify a third group of genes which shows a peak on day 3 but whose expression falls on days 4 and 5. It is interesting to see that this group includes Wnt and Fgf morphogenes. The authors should provide a list of the genes in the different clusters for the readers to inspect and analyse.
We note that there was a typo in the original manuscirpt and the genes that were clustered were the EF-up and EF-down genes. This typo has been fixed and the requested information is now available in a supplementary file.
Aiming to generate insight into the cis-regulatory elements that regulate of the genes the authors found differentially expressed in their model system they performed a series of ATAC-seq experiments. When linking the genomic regions with differential ATAC-seq accessibility to gene locus using the GREAT analysis, they identified association to 22 of the EF-up and 161 of the EF-down genes. This suggests a functional link between the ATAC-seq genomic regions and the gene regulation of the differentially expressed genes.
The authors later screened the ATAC-seq regions of increased accessibility for TF binding motifs and found that these regions were enriched with motifs for EFTF genes Rax, Lhx2 and Pax6. When assessing motifs in the ATAC-seq regions in EF-up TADs, Rax and Lhx2 motifs scored highly associated to open chromatin positions. Authors also observe a positive gene expression-accessibility correlation between in Pax6, Lhx2, Six3 and Otx2, and suggest this could mean these genes activate transcription of the EF-up group of genes. The same analysis, but focusing on EF-down genes, suggests that EFTFs repress the expression of EF-down genes which include those involved in pluripotency.
Further interrogating the ATAC-seq data, the authors use TOBIAS footprinting analysis to identify changes in TF binding in EF-TADs and EF-up motifs. Remarkably, whole genome analysis reveals that the largest increase in motif binding corresponds to EF-up genes Rax, Pax6 and Lhx2. The authors then narrow down on specific gene regulation by studying the ATAC-seq data within the TAD of Rax and Six6. However, they do not explain the rationale for which these two genes were highlighted, and why Pax6 or Lhx2 were excluded. This explanation should be added to the manuscript.
We have expanded this section of the manuscript to explain that Rax and Six6 were prioritised due to the GFP readout of Rax expression and Six6 being located in a smaller and thus less complex TAD than Pax6, Six3 and Lhx2 after the initial analysis was performed for all five TADs.
The analysis identifies three regulatory elements in the Rax TAD and two for Six6. They then go on and study one putative regulatory element of each gene and generate CRISPR deletions in cell lines. The rationale for the choice of these particular elements is not clear, nor if the cell lines are the same used for the RNAseq experiments. This information should be explicit in the results and in the methods section.
The manuscript has been updated to include the rationale behind our choice of the regulatory elements deleted.
The authors mention that the CRISPR cell lines are "considerably more variable" (Ln 822) compared to the previously studied organoids and suggest that no conclusions can be driven from GFP expression or morphology alone. However, they do not specify which is the variable trait. This information should be added to the text.
We have amended the text to include that the organoids are more variable in terms of the OV like structures produced and GFP expression level.
The authors also miss out on specifying the time stage of the organoids in figure 6 which should be stated.
We thank the reviewer for pointing this out and have updated the manuscript to contain the stage of the organoids.
Regardless, the wildtype organoids in figure 6 and figure S7 show a very different morphology and GFP expression compared to those in figure 1, suggesting that the conclusions from this last set of experiments are not reliable or comparable to those in figure 1. This, together with the fact that different reagents were used to grow the organoids for the RNAseq and the CRISPR experiments, is a weakness of this work that must be addressed.
We recognise this weakness however our amendments detailed above in response to reviewer 1’s comments, including adding a figure showing WT organoids grown in the KSR media that closely resemble the organoids in Fig.1A, removes the uncertainty that it is the change in media producing these differences in morphology and GFP expression.
Our aim in this section was to specifically test the hypotheses regarding the regulatory nature of the distal genomic regions identified by our intra-TAD analyses of ATAC-seq data. To do this it was important to compare organoids derived from wildtype and mutant cells that had been subjected to the same growth conditions and genomic-editing protocols. The stress associated with the latter is what we expect has resulted in the differences in morphology and GFP expression compared with the original Fig1. organoids (which have not been through this procedure).
The last part of the results section belongs to the discussion as no results generated by the researchers are included.
Although no new data was generated for this section, we have used the data generated in our work, together with existing ChIP-seq datasets to construct a new plausible hypothesis regarding the activation of Rax-expression through changes in TF-binding at an enhancer displaying little/no change in accessibility. As this section ties in with previous results sections discussing the regulation of eye-field genes, we feel it belongs in the results section rather than in the discussion.
The discussion in this paper is a good opportunity to state the limitation of this study.
As requested, we have added a paragraph discussing the main limitations to our study in the discussion section.
Major comments to address
- One of the main issues identified is that the morphology of the control conditions in the CRISPR experiments (Fig.6) do not look is that those used for the RNAseq experiments (Fig.1) and the authors should address this issue. The fact that CDM media was used on the RNA extraction and ATACseq experiments and then KSR media was used for the CRISPR experiments is worrying and makes one wonder whether the second set of experiments is at all comparable to the first. This should be somehow controlled carefully by at least replicating one set of RNA experiments with the KSR media.
We have addressed this in response to the reviewer’s summary above. Unfortunately, it is not possible for us to replicate the RNA experiments in the KSR media due to the research group closure upon Professor FitzPatrick’s retirement.
- The requirement of Wnt signalling inhibition has been well established as a requirement for forebrain specification, including the eye field. Considering the link of the Wnt/beta-catenin pathway to eye specification and that TCFs, the transcription factors that mediate Wnt pathway transcription regulation, have known and well-studied DNA motifs, it is surprising that authors do not include the analysis of TCF motifs in their study. Also considering that TCF7l1 (TCF3, old nomenclature) has recently been shown to be cell-autonomously required for the expression of rx3 (Rax homologue) in zebrafish. One would expect TCFs to be included in the analysis as it was done with Sox2 and Otx2, which were studied due to the known relevance in forebrain specification rather than from the direct analysis of the differential gene expression experiments.
We thank the referee for their valuable comment here. Our current analyses indeed do not consider TCFs and are therefore likely incomplete. We plan to address this by further analysing our data to quantify the patterns and effects of the TCF genes, and will appropriately amend our manuscript to reflect our findings.
Minor comments to address
- The authors should clearly state the day timepoint used in the organoids experiments in the results section and figure legends, not just in the methods.
We have updated the text and figure legends to include the time point of all organoids.
- The report by Agnes et al Development 2022 should be cited in the introduction as it is an excellent paper related to this topic, including a comprehensive analysis of the EFTFs expression pattern.
We thank the reviewer for pointing us to this very interesting paper. Although we feel it doesn’t fit in with our introduction that is currently tailored to the set of genes that has historically defined the eye-field (and which was discovered in non-mammalian models), we do recognise that the 3D organisation of the eye-field and in particular the patterns of gene-expression defining different regions of this is important to disentangle in mammalian systems. We have therefore inserted a reference to the Agnes at al 2022 study on the dimorphic teleost in our extended discussion.
- Ln 41. Mutations in these genes do not always cause severe bilateral eye malformations. Probably best to moderate and mention that they 'can' cause these malformations.
As suggested we have softened this sentence to: “ Mutations in at least three of the genes encoding orthologs of the Xenopus EFTF can cause severe bilateral eye malformations in humans (OTX2, PAX6 and RAX) (Fitzpatrick and van Heyningen, 2005).”
- Ln 146. Authors mention that in vitro organoid systems "closely mimic the in vitro regulatory dynamics". This statement should be moderated as we do not know if this is true. In fact, one of the positive aspects of this study is that it contributes to supporting this statement.
We agree with the referee regarding the strength of this original statement. We have changed this to:
“We have exploited a reproducible, in vitro organoid model system enabling us to generate data from this cell-state transition and through computational analysis gain a quantitative understanding of the underlying regulatory mechanisms.”
- Ln 150. Rax homologue Rx3 is also expressed in cells that give rise to the hypothalamus in zebrafish and cavefish, and probably in Xenopus too. It could well be the case in mice too.
We have corrected this to indicate that Rax is also expressed in the hypothalamus in mice.
- I do not think the GO term data adds much to Figure 1. If possible, I would move it to the supplementary section.
We have moved the GO visualisations to supplementary, Fig.S2.
- It should be made clear which set of experiments was performed as biological replicates and which did not.
We have added details on the number of replicates used in each experiment.
- Based on the heatmap in Fig1A, expression of Rax is significant in GFP- cells at days 4 and 5. The authors should comment or discuss this.
We have amended the text and supplemental methods section to include more details of our FACS protocol. The limitations of our sorting procedure include the fact that cells are not sorted into pure GFP expressing and non-expressing populations. Rather the GFP negative sample may contain some cells with low Rax expression or cells that have just begun to express Rax that were not excluded by our sorting. Our aim was to collect sufficient numbers of cells for each condition and separate out cells that expressed GFP to get a more uniform population of cells to study. It is also of note that the heatmap shows Rax expression by day 3. Although it was not detectable by imaging there were around 100 cells per organoid that FACS marked as GFP positive but were retained within the day 3 sample to ensure we had a complete picture of the gene expression at this time point.
- Ln 99 of materials and methods mentions that the sorting of GFP+ was performed "when possible". The authors should state the differences in the conditions in the different experiments.
This has been expanded to detail exactly how cells were sorted.
- The sentence closing the first section of the results (Ln 270) is an overstatement and should be moderated. I cannot see how the results shown in this section on their own could reflect and drive solid conclusions on brain cell fate specification.
We agree with the referee and have changed this sentence to: “In summary, these first analyses of RNA-seq data generated from the timecourse of optic vesicle organoid development, show that this is a robust and relevant model system with which to study the gene dynamics underlying mammalian eye field specification.”
- Appropriate citations should be added to back up the argument that opens the second part of the results section (starting Ln 279).
We have added several citations that discuss and review the current knowledge regarding gene regulation via TF-binding at accessible cis-regulatory elements.
- Ln 342-343. I suggest being consistent and using the EF-up or EF-down nomenclature on the whole manuscript unless referring to a different subset of genes.
We have modified the text to consistently use “EF-up” or “EF-down” terminology.
- Ln 692 Refers to Fig.S4F, but this figure has only panels A-D.
This was a typo and has been corrected in the text.
- Figures 6B and E and the figure legend do not indicate the differences between the panels, or the time stage of the experiments.
The figure legend has been updated to include these details.
CROSS-CONSULTATION COMMENTS I agree with the comments and suggestions made by the other two reviewers, which identify similar and also specific issues in the manuscript. I believe they are all pertinent and should be acknowledged before re-submitting.
Reviewer #2 (Significance (Required)): The manuscript by Owen et al, presents the analysis of in vitro eye vesicle organoids derived from mouse ESCs at stages equivalent to when the eye field is specified in vivo. This work is pertinent and necessary as detailed data on gene expression in early eye organoids was missing in the field and is necessary for the interpretation of experiments in these systems.
Although the computational data provided in this manuscript is based on consensus TF motifs, the functional relevance of the specific motifs must be proven before being able to drive any significant conclusions, and one should be moderate about the conclusion that can be driven from this kind of analysis. Still, the analysis put forward is a good reference and starting point for future functional studies. One possible limitation of this study is that the quantification of the expression of genes is based on the RNAseq data, and the expression data should be further confirmed using a proper quantitative method like qPCR.
This study will be of interest to the audience studying eye development and disease in animal model systems and humans.
My lab studies the genetic, cellular, and molecular aspects of eye specification, development and disease in zebrafish, and study mutations identified patients with eye globe defects.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Studies in Xenopus embryos have established that the specification of the eye field requires a core set of transcription factors (TFs) that impose eye identity to anterior neural plate progenitors. In this manuscript the authors have used mouse embryonic stem cells-derived optic vesicle organoid to ask if the acquisition of mammalian eye identity requires the same set of TFs. They further use different genomic approaches to identify the cis-regulatory elements involved in the expression of these genes and analyses the consequences of altering the sequence of some of the identified regulatory elements. Their results confirm that in mammals the acquisition of eye field identity requires the upregulation of the expression of the same core set of TFs described in Xenopus, with a particularly important role for three of them: Rax, Pax6 and Lhx2. This upregulation is associated to the downregulation of pluripotency genes.
This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known. For example, they could enlarge the composition of the gene regulatory network that controls eye field specification, given than one of their argument is that their analysis can predict the composition of such a network. Perhaps, they could also address some of the questions that are posed in the discussion. This will strengthen the manuscript and valorize their work.
Additional points that could be taken into consideration are the following:
1) According to the text, the authors identify only 53 CREs with decreased chromatin accessibility (ATACseq signal) between the 3 day and 5 days timepoints, versus the 7752 CREs with increased signal. However, this contrasts with the proportion of genes upregulated/ downregulated in their RNAseq analysis (37 vs 448) and with the notion that specification of the eye field involves the concomitant repression of other neural fates. This also suggests that at least an important fraction of the dynamic ATACseq peaks associated with 161 of the 448 downregulated genes increase their accessibility and allow the recruitment of transcriptional repressors. However, the role of TF binding and chromatin accessibility dynamics on gene repression is poorly discussed and the authors need to provide some interpretation of these observations. Also, authors interpret the fact that the presence of BS for EF downregulated genes, such as En2 and GATA6, correlates with increased chromatin accessibility as a consequence of the fact that TFBS can be bound by different TF paralogs but do not seem to consider that these TFs have been reported to work as transcriptional repressors, so that their downregulation could well explain the changes in chromatin accessibility.
We thank the reviewer for their interesting comments here. We have added short discussions on both main points above (EF-down genes linked to peaks with increasing accessibility and En2/Gata as transcriptional repressors) in the text related to the analysis of our ATAC-seq data. The notion that a loss of repression leading to the activation of gene-expression is indeed a very exciting one and one that we have thought about in the context of the switch-on of the eye-field TFs. This certainly deserves further future work, however in the present study we wanted to be careful not to overinterpret our data. To robustly gain insights into the loss of repression, experiments such as En1/Gata6 ChIP-seq would be very useful, though we are unable to perform these in the near future.
2) ATACseq signal analysis is an indirect measure of TF binding. The authors demonstrate the predictive nature of this analysis of TF dynamics and have use an available Sox2 ChIP dataset. However, this does not allow assessing dynamic changes in the occupancy of this TF and its correlation with ATACseq. Therefore, at least for few of the TF stressed in this work (e.g. Sox2 and Otx2 and for which good antibodies exist) they could attempt ChIP-seq analysis. This would considerably strengthen the work and provide support to an idea that the authors have particularly emphasized in their manuscript.
We agree with the referee that not having generated ChIP-seq data does not allow us to validate some of the hypotheses and evidence provided by the computational analysis of our ATAC-seq data – we have added a discussion of this limitation in the discussion section of our manuscript. We do note however, as observed in Bentsen et al, 2020, that compared to simple TF-motif occurrence analyses, TF-footprinting analyses (such as those we have performed) yield results on putative TF binding that are much closer to more direct measurements of TF binding via e.g. ChIP-seq. We fully agree that it would be very interesting to perform ChIP-seq/Cut&Cut experiments on the organoid system for a set of interesting TFs identified in our study. Unfortunately, because the lab of Prof FitzPatrick has now closed, it is not possible for us to perform further wet-lab experiments in the very near future. However, we plan to further explore the literature to try to find additional publicly-available ChIP-seq datasets (including for Otx2) which would help reinforce some of the hypotheses we make, and will report any relevant findings in our final manuscript.
3) Previous studies (i.e. 10.1242/dev.067660; 10.1093/hmg/ddt562) have shown the importance of gene dosage in eye field specification and repression of other fates. These studies could be included in the discussion, which, in its current version is a quite brief and leaves out many of the reported analysis.
We thank the referee for pointing us to this very relevant question – we have added this to the further research questions in the discussion.
CROSS-CONSULATION COMMENTS
The comments from the other reviewers complement the aspects that we have underscored and should be fully considered as they will contribute to improve the manuscript.
Reviewer #3 (Significance (Required)): This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known.
Developmental neurobiologists, genome
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Referee #3
Evidence, reproducibility and clarity
Studies in Xenopus embryos have established that the specification of the eye field requires a core set of transcription factors (TFs) that impose eye identity to anterior neural plate progenitors. In this manuscript the authors have used mouse embryonic stem cells-derived optic vesicle organoid to ask if the acquisition of mammalian eye identity requires the same set of TFs. They further use different genomic approaches to identify the cis-regulatory elements involved in the expression of these genes and analyses the consequences of altering the sequence of some of the identified regulatory elements. Their results confirm that in mammals the acquisition of eye field identity requires the upregulation of the expression of the same core set of TFs described in Xenopus, with a particularly important role for three of them: Rax, Pax6 and Lhx2. This upregulation is associated to the downregulation of pluripotency genes.
This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known. For example, they could enlarge the composition of the gene regulatory network that controls eye field specification, given than one of their argument is that their analysis can predict the composition of such a network. Perhaps, they could also address some of the questions that are posed in the discussion. This will strengthen the manuscript and valorize their work. Additional points that could be taken into consideration are the following:
- According to the text, the authors identify only 53 CREs with decreased chromatin accessibility (ATACseq signal) between the 3 day and 5 days timepoints, versus the 7752 CREs with increased signal. However, this contrasts with the proportion of genes upregulated/ downregulated in their RNAseq analysis (37 vs 448) and with the notion that specification of the eye field involves the concomitant repression of other neural fates. This also suggests that at least an important fraction of the dynamic ATACseq peaks associated with 161 of the 448 downregulated genes increase their accessibility and allow the recruitment of transcriptional repressors. However, the role of TF binding and chromatin accessibility dynamics on gene repression is poorly discussed and the authors need to provide some interpretation of these observations. Also, authors interpret the fact that the presence of BS for EF downregulated genes, such as En2 and GATA6, correlates with increased chromatin accessibility as a consequence of the fact that TFBS can be bound by different TF paralogs but do not seem to consider that these TFs have been reported to work as transcriptional repressors, so that their downregulation could well explain the changes in chromatin accessibility.
- ATACseq signal analysis is an indirect measure of TF binding. The authors demonstrate the predictive nature of this analysis of TF dynamics and have use an available Sox2 ChIP dataset. However, this does not allow assessing dynamic changes in the occupancy of this TF and its correlation with ATACseq. Therefore, at least for few of the TF stressed in this work (e.g. Sox2 and Otx2 and for which good antibodies exist) they could attempt ChIP-seq analysis. This would considerably strenghen the work and provide support to an idea that the authors have particularly emphasized in their manuscript.
- Previous studies (i.e. 10.1242/dev.067660; 10.1093/hmg/ddt562) have shown the importance of gene dosage in eye field specification and repression of other fates. These studies could be included in the discussion, which, in its current version is a quite brief and leaves out many of the reported analysis.
Referees cross-commenting
The comments from the other reviewers complement the aspects that we have underscored and should be fully considered as they will contribute to improve the manuscript
Significance
This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known.
Developmental neurobiologists, genome
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Referee #2
Evidence, reproducibility and clarity
The authors grow eye organoids from cells with a reporter driving GFP in the Rax locus, a gene that is expressed in the eye field in many animal model systems. They show that expression of GFP picks up by day 4 and performed FACS sorting of GFP+ cells on day 4 and day 5 organoids to compare gene expression by RNAseq comparing with earlier day organoids. The data shows 37 genes with a differential expression on days 4 and 5, compared to day 3, and enriched in GFP+ cells, which they define as EF-up genes. It is notable that some of these genes had already been identified as canonical eye field gene regulatory network transcription factors. In the same way, they identify a group of differentially expressed regulated genes, EF-down, and state that 'many' of them are involved in pluripotency. However, they do not mention how many, or the proportion of these genes in the whole list. It would be useful if they could provide the number to understand how many of these genes are related to pluripotency, the whole list of genes mentioned to be downregulated in a supplementary file. The authors also note that genes known to be required for eye specification like Sox2 and Otx2 are not differentially expressed across the day 3-4 timepoint (Ln 190). However, this is not surprising considering that both genes are broadly expressed in the anterior neural ectoderm and required for its specification, which should be noted by the authors.
The authors then go on and cluster the EF-up, EF-down and genes deferentially expressed between days 2 and 3, and identify 6 discreet trajectory groups. From this analysis, they identify a third group of genes which shows a peak on day 3 but whose expression falls on days 4 and 5. It is interesting to see that this group includes Wnt and Fgf morphogenes. The authors should provide a list of the genes in the different clusters for the readers to inspect and analyse. Aiming to generate insight into the cis-regulatory elements that regulate of the genes the authors found differentially expressed in their model system they performed a series of ATAC-seq experiments. When linking the genomic regions with differential ATAC-seq accessibility to gene locus using the GREAT analysis, they identified association to 22 of the EF-up and 161 of the EF-down genes. This suggests a functional link between the ATAC-seq genomic regions and the gene regulation of the differentially expressed genes.
The authors later screened the ATAC-seq regions of increased accessibility for TF binding motifs and found that these regions were enriched with motifs for EFTF gens Rax, Lhx2 and Pax6. When assessing motifs in the ATAC-seq regions in EF-up TADs, Rax and Lhx2 motifs scored highly associated to open chromatin positions. Authors also observe a positive gene expression-accessibility correlation between in Pax6, Lhx2, Six3 and Otx2, and suggest this could mean these genes activate transcription of the EF-up group of genes. The same analysis, but focusing on EF-down genes, suggests that EFTFs repress the expression of EF-down genes which include those involved in pluripotency.
Further interrogating the ATAC-seq data, the authors use TOBIAS footprinting analysis to identify changes in TF binging in EF-TADs and EF-up motifs. Remarkably, whole genome analysis reveals that the largest increase in motif binging corresponds to EF-up genes Rax, Pax6 and Lhx2. The authors then narrow down on specific gene regulation by studying the ATAC-seq data within the TAD of Rax and Six6. However, they do not explain the rationale for which these two genes were highlighted, and why Pax6 or Lhx2 were excluded. This explanation should be added to the manuscript. The analysis identifies three regulatory elements in the Rax TAD and two for Six6. They then go on and study one putative regulatory element of each gene and generate CRISPR deletions in cell lines. The rationale for the choice of these particular elements is not clear, nor if the cell lines are the same used for the RNAseq experiments. This information should be explicit in the results and in the methods section. The authors mention that the CRISPR cell lines are "considerably more variable" (Ln 822) compared to the previously studied organoids and suggest that no conclusions can be driven from GFP expression or morphology alone. However, they do not specify which is the variable trait. This information should be added to the text. The authors also miss out on specifying the time stage of the organoids in figure 6 which should be stated. Regardless, the wildtype organoids in figure 6 and figure S7 show a very different morphology and GFP expression compared to those in figure 1, suggesting that the conclusions from this last set of experiments are not reliable or comparable to those in figure 1. This, together with the fact that different reagents were used to grow the organoids for the RNAseq and the CRISPR experiments, is a weakness of this work that must be addressed.
The last part of the results section belongs to the discussion as no results generated by the researchers are included. The discussion in this paper is a good opportunity to state the limitation of this study.
Major comments to address
- One of the main issues identified is that the morphology of the control conditions in the CRISPR experiments (Fig.6) do not look is that those used for the RNAseq experiments (Fig.1) and the authors should address this issue. The fact that CDM media was used on the RNA extraction and ATACseq experiments and then KSR media was used for the CRISPR experiments is worrying and makes one wonder whether the second set of experiments is at all comparable to the first. This should be somehow controlled carefully by at least replicating one set of RNA experiments with the KSR media.
- The requirement of Wnt signalling inhibition has been well established as a requirement for forebrain specification, including the eye field. Considering the link of the Wnt/beta-catenin pathway to eye specification and that TCFs, the transcription factors that mediate Wnt pathway transcription regulation, have known and well-studied DNA motifs, it is surprising that authors do not include the analysis of TCF motifs in their study. Also considering that TCF7l1 (TCF3, old nomenclature) has recently been shown to be cell-autonomously required for the expression of rx3 (Rax homologue) in zebrafish. One would expect TCFs to be included in the analysis as it was done with Sox2 and Otx2, which were studied due to the known relevance in forebrain specification rather than from the direct analysis of the differential gene expression experiments.
Minor comments to address
- The authors should clearly state the day timepoint used in the organoids experiments in the results section and figure legends, not just in the methods.
- The report by Agnes et al Development 2022 should be cited in the introduction as it is an excellent paper related to this topic, including a comprehensive analysis of the EFTFs expression pattern.
- Ln 41. Mutations in these genes do not always cause severe bilateral eye malformations. Probably best to moderate and mention that they 'can' cause these malformations.
- Ln 146. Authors mention that in vitro organoid systems "closely mimic the in vitro regulatory dynamics". This statement should be moderated as we do not know if this is true. In fact, one of the positive aspects of this study is that it contributes to supporting this statement.
- Ln 150. Rax homologue Rx3 is also expressed in cells that give rise to the hypothalamus in zebrafish and cavefish, and probably in Xenopus too. It could well be the case in mice too.
- I do not think the GO term data adds much to Figure 1. If possible, I would move it to the supplementary section.
- It should be made clear which set of experiments was performed as biological replicates and which did not.
- Based on the heatmap in Fig1A, expression of Rax is significant in GFP- cells at days 4 and 5. The authors should comment or discuss this.
- Ln 99 of materials and methods mentions that the sorting of GFP+ was performed "when possible". The authors should state the differences in the conditions in the different experiments.
- The sentence closing the first section of the results (Ln 270) is an overstatement and should be moderated. I cannot see how the results shown in this section on their own could reflect and drive solid conclusions on brain cell fate specification.
- Appropriate citations should be added to back up the argument that opens the second part of the results section (starting Ln 279).
- Ln 342-343. I suggest being consistent and using the EF-up or EF-down nomenclature on the whole manuscript unless referring to a different subset of genes.
- Ln 692 Refers to Fig.S4F, but this figure has only panels A-D.
- Figures 6B and E and the figure legend do not indicate the differences between the panels, or the time stage of the experiments.
Referees cross-commenting
I agree with the comments and suggestions made by the other two reviewers, which identify similar and also specific issues in the manuscript. I believe they are all pertinent and should be acknowledged before re-submitting.
Significance
The manuscript by Owen et al, presents the analysis of in vitro eye vesicle organoids derived from mouse ESCs at stages equivalent to when the eye field is specified in vivo. This work is pertinent and necessary as detailed data on gene expression in early eye organoids was missing in the field and is necessary for the interpretation of experiments in these systems.
Although the computational data provided in this manuscript is based on consensus TF motifs, the functional relevance of the specific motifs must be proven before being able to drive any significant conclusions, and one should be moderate about the conclusion that can be driven from this kind of analysis. Still, the analysis put forward is a good reference and starting point for future functional studies. One possible limitation of this study is that the quantification of the expression of genes is based on the RNAseq data, and the expression data should be further confirmed using a proper quantitative method like qPCR.
This study will be of interest to the audience studying eye development and disease in animal model systems and humans.
My lab studies the genetic, cellular, and molecular aspects of eye specification, development and disease in zebrafish, and study mutations identified patients with eye globe defects.
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Referee #1
Evidence, reproducibility and clarity
Summary
Owen et al. characterize the transcriptome and chromatin accessibility of mouse retinal organoids at early stages during which eye field-like cells are specified. Since cell specification and differentiation in retinal organoids largely mimic those processes in vivo, retinal organoids are viable models for studying the mechanisms of early eye development. Owen et al. utilize a previously established Rx-GFP cell line, bulk RNA sequencing, and bulk ATAC sequencing to dissect the mechanisms of early eye development in mice. Their findings are generally consistent with previous studies. Overall, the study is interesting for the field, but its conceptual and technical advances are moderate. In addition, a few major points need to be clarified.
Major points
- The authors did not show any analysis of retinal organoids at stages when Vsx2 is expressed. This is a significant weakness since the chemically defined medium (CDM) used in Owen et al.'s study was previously shown to induce rostral hypothalamic differentiation (Wataya et al., 2008). Related to this notion, several eye-field transcription factors, such as Rax and Six3, are also expressed in the hypothalamus. Therefore, Owen et al. need to demonstrate that organoids in their modified differentiation system efficiently produce Vsx2-positive retinal progenitors, and samples of organoids at stages when Vsx2 is expressed should be included for RNA sequencing. If Vsx2 is not efficiently expressed in their organoids, the interpretation of results will be very different.
- The authors state that "two differentiation medias were used for this work due to the differentiation becoming unstable after the initial experiments had been performed. The organoids used for RNA and ATAC-seq were grown in CDM media and the organoids with mutations introduced in potential CREs were grown in KSR media". Why the differentiation becomes unstable after the initial experiments? Differences in the two media cause additional complexities. Related to this notion, "WT Rx-GFP" in Figure 4B and 4E appears to show a different expression pattern compared to that in Figure 1A.
- Is the deletion of Rax and Six6 regulatory elements homozygous? Sanger sequencing or amplicon sequencing is needed to show the deletion.
- The deletion of Rax and Six6 regulatory elements appears to cause minor changes in the expression of Rax and Six6 (Figure 6C, F). Therefore, the impact of findings in bulk RNA seq and bulk ATAC seq in this study is still unclear.
- Retinal organoids and sorted cells are composed of heterogeneous cell populations. Bulk RNA seq and bulk ATAC seq do not have the power to dissect the complexity of heterogeneous cell populations. Single-cell RNA seq and single-cell ATAC seq are more powerful for this study.
- Numerous motifs in the JASPAR database are identified using in vitro assays and have not been validated using in vivo assays. Unexpected results in motif analysis could be due to the differences in DNA binding motifs between in vitro and in vivo conditions. This notion should be added in the discussion.
Minor points
Numerous labels in figures are too small.
Referees cross-commenting
My fellow reviewers identify similar major weaknesses and additional points. I agree with the other reviewers' comments.
Significance
Nature and Significance of the advances
In Owen et al.'s study, the Rx-GFP cell line and retinal differentiation protocol were established in previous studies (Wataya et al., 2008; Eiraku et al., 2011); bulk RNA sequencing and bulk ATAC sequencing are standard procedures. Although candidate regulatory elements for early eye development are identified, deletions of two prioritized elements using CRISPR/Cas9 only cause minor changes in the expression of targeted genes. Overall, conceptual and technical advances in Owen et al.'s study are moderate.
Compare to existing published knowledge
The datasets could be useful for the field, but conceptual and technical advances are moderate.
Audience
Developmental biologists, stem cell biologists, vision researchers.
Your expertise
Developmental biology, stem cell biology, vision research
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Reply to the reviewers
The authors do not wish to provide a response at this time.
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Referee #3
Evidence, reproducibility and clarity
The authors have utilised single-cell RNA seq to profile cells in the Drosophila female germline cells and somatic cells within the ovary. Using a candidate approach, the authors assessed whether some of the candidate genes identified in this study play a role in germ cell differentiation. Amongst these, they found that an uncharacterized gene called eggplant is required for differentiation. On rich diets, Eggplant expression is reduced. Furthermore, Mmps and Timp are required to regulate Eggplant expression level.
Major comments:
Overall, the conclusions are supported by experimental evidence. The sc-RNA seq provides an important resource for the community, and some of the reagents generated such as the antibody against Eggplant or the tagged lines would provide valuable resource for future studies. The main criticism I have for this manuscript is that it lies half way between a resource paper and a mechanistic paper. If it was just a resource paper, then the sc data should suffice. Instead, the authors studied the effects of Eggplant and MMP/TIMP on GSC differentiation, however, not enough mechanism was gained through these studies.
- It is not clear what kind of protein Eggplant is, and its mode of action is unclear, despite that it was shown by the authors that Eggplant is required for GSC differentiation. The authors further showed that animlas fed a high yeast diet promotes GSC proliferation and increased egg production by inhibiting the expression of eggpl. The authors suggested that this may lie downstream of insulin signaling, however no genetic experiments were conducted. In order to support this line of conclusions, essential experiments should include: is there a genetic interaction between Eggplant and insulin signaling? Is this happening autonomously within the GSCs or at the GSC niche? In the discussion the authors mentioned that since egg production can occur upon Eggpl knockdown even on normal diet, thus protein is not involved in this process. So, what is the mechanism?
- Similarly, the authors mentioned that MMPs and TIMP are involved in GSC differentiation, and they discussed that it could be mediated via tissue stiffness. However, no experimental data was presented to support this. It would be good to offer some mechanistic insights. TIMP and Eggplant genetic interaction should be done.
Minor comments:
Line 376 and Line 437 are repetitive in explaining why the gene is called eggplant. I did not find the pictures in Figure7 F,G and Figure 8 B informative. Some quantifications should also proof the same point.
Referees cross-commenting
I also agree with the comments from both reviewers 1 and 2.
Significance
- The significance of this study lies in presentation of a thorough characterization of gene expression at the single cell level in the GSC and in identification of novel regulators of GSC differentiation.
- My field of expertise is Drosophila stem cells and nutrition/metabolism.
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Referee #2
Evidence, reproducibility and clarity
Brief summary
The authors conducted single cell RNA sequencing on adult fly ovaries to survey the transcriptomic profile of individual ovarian cells. The datasets resulted in classification of 24 discrete cellular populations, including different types of stem cells, progenitors and differentiated cells. Genes that differentially expressed during germline differentiation were selected and then examined with RNA interference to determine their specific roles in regulating germline differentiation. Meanwhile, using the single cell transcriptomic profiles acquired from specific types of germ cells, gene regulatory networks were further characterized. Among the genes examined, the gene eggplant (eggpl) was found to act as a novel regulator mediating germline differentiation with its protein expression predominantly detected in germline stem cells (GSCs), CBs and early cysts. Interestingly, since the protein expression of Eggplant was decreased upon rich dietary intake, eggpl may function as a novel molecular link coupling the nutritional status and the process of germline differentiation during fly oogenesis.
Specific comments
Major points:
- The authors chose a specific group of differentially upregulated genes (in GSCs) and performed germline specific RNA interference (RNAi) to determine their function in regulating germline differentiation. The RNAi results were then compiled and presented in Figure 3B-C. However, details shall be provided for the readers/reviewers to understand the phenotypic analyses. Specifically, as stated in text that "we found that RNAi knockdown of 19 upregulated genes induced disruption of GSCs/CB homeostasis. Of these, 12 genes were classified as "changes to the number of CSC/CB", 6 genes as "empty germarium" and 4 genes exhibited "differentiation defects"... ", there was neither specific descriptions nor representative examples (with proper labelling) explaining the bases of such classification. It was particularly difficult to appreciate the assorted phenotypes presented in Figure 3C without proper descriptions. Not to mention that the immunostaining needs to be optimized in some of the representative pictures. Meanwhile, it was not clearly stated in the text about how 19 candidate genes were then classified into (12+6+4=22) genes. Along the same line, it will be helpful to include a supplementary table of summarizing the genes tested and their corresponding phenotypes.
- The authors also performed germline specific RNAi to knockdown the expression of 21 out of 39 most highly expressing genes in GSCs to determine their function in regulating oogenesis. Some of the RNAi effects were presented in Figure S2B. Similarly, the effects of RNAi experiments will be better appreciated if some more descriptions on specific RNAi phenotypes are added. Also, inclusion of a supplementary table summarizing the RNAi lines utilized in this experiment is required.
- The authors performed in situ hybridization and immunostaining to discover that the eggpl transcripts were enriched in GSCs and CBs (Bam-GFP- germ cells), while the Eggpl::GFP expression (in the knock-in line, KI) was detected throughout region 1 within germarium. The authors then concluded that protein level of Eggpl was "actually highest in cells that have no detectable eggpl transcripts". However, as shown in Figure 5F, the highest level of Eggpl::GFP appeared in the cell that contains a spectrosome, suggesting that is either a GSC or a CB obtaining highest Eggpl::GFP (KI) expression. Furthermore, as shown in Figure 4E, similar level of Eggpl protein expression indicated by immunostaining was found in GSCs and CBs, indicating a nice coupling between the mRNA and protein level of eggpl found in GSCs/CBs. Therefore, the authors need to simultaneously perform in situ hybridization and immunostaining of eggpl to visualize the expression of eggpl transcripts and protein within the same germarium and to directly determine whether the statement that "protein level of Eggpl was actually highest in cells that have no detectable eggpl transcripts" holds true.
- The genetic experiments done by the authors show that disruption of eggpl led to an increase in the average number of Brdu+ cysts, the size of the ovary and ultimately the number of eggs produced. Interestingly, such phenotypes were reminiscent to the effects caused by rich dietary intakes. The authors interpret these results by proposing that eggpl negatively regulates GSC proliferation in regulating oogenesis. However, as shown in Figure 5J, the number of GSCs was not significantly altered upon eggpl RNAi but was increased when over-expressing Eggpl::GFP, indicating that eggpl promotes the proliferation of GSCs. To be able to determine the function of eggpl in regulating GSC proliferation during oogenesis and to support the stamen that "We also found that cell cycle in germ cell cysts was accelerated" (in introduction), the authors need to perform clonal analyses to monitor the progression rate of eggpl mutant cells (of both germline clones and follicle cell clones) compared to control cells. An accelerated rate of germline development shall be predicted by increased GSC proliferation. Moreover, to rule out the possibility that changes in the dynamics of germline apoptosis can affect the number of Brdu+ cysts observed upon eggpl loss, the authors should also perform experiments assaying the number of apoptotic cells in the germarium when manipulating the expression level of eggpl.
- The authors examined the expression level of Eggpl::GFP (KI) in different feeding conditions and found a reverse correlation between Eggpl::GFP (KI) protein level and the quality of dietary condition. Moreover, the enlarged ovary seen upon eggpl loss was recapitulated when over-expressing Timp and Mmp1/2. Interestingly, the authors also found that overexpression of Timp and Mmp1/2 led to lower protein expression of Eggpl::GFP (KI). The authors interpret these results by proposing that eggpl mediates the GSC differentiation via MMP-dependent Timp regulation pathway. However, the authors should perform experiments to directly examine if eggpl and Timp (and Mmp1/2) interact genetically to regulate GSC proliferation/egg production while monitoring the dietary status.
Minor points:
- Please clarify what you mean in this sentence in the introduction as the message is not clear: "Mei-P26 suppress transcripts that promote differentiation in CB by antagonizing miRNA pathway
- In Figure 3B, for the genes that were listed for more than once (CG42250, CG10295, CG6904 and CG15845), please note that those are different RNAi lines.
- In Figure S2A, for the genes that were listed for more than once, please note that those are different RNAi lines.
- If applicable, more than one RNAi line of each candidate gene should be examined to validate the specific effects caused by gene KD.
- As shown in Figure S2B, knocking down CG31666 and CG12743 led to impaired oogenesis. Please add their gene name (CG31666 (chinmo) and CG12743 (otu)) for acknowledging their well-documented role in regulating oogenesis.
- How was the RNAi generally performed. Please add into material and method.
- In Figure 7A, it is difficult to tell how many of the BrdU+ cysts was shown in Eggpl[1] mutant germarium and whether it is qualitatively/quantitively different from control.
- The authors compared their scRNA-Seq dataset with other 4 public available Drosophila ovary scRNA-Seq datasets and concluded that comparable features were found among these datasets by UMAP analysis. The authors should also comment on what are the potential differences among these adult ovary scRNA-Seq datasets in the discussion section. Providing such information will not only highlight the importance of this dataset and will also be beneficial to the fly community for future research.
Referees cross-commenting
I also agree with the comments from both reviewers 1 and 3.
Significance
In summary, the characterization of single-cell atlas of adult fly ovary from this study adds additional datasets for future investigation on cell-specific differentiation in vivo. Meanwhile, the identification of a novel molecular link between nutritional status and germline differentiation is of broad interest to readers in the field of dietary/metabolic control on stem cell function. However, there are specific concerns about how the results were presented and interpreted while some important controls are missing. I believe this article would be much improved if the points mentioned can be effectively addressed.
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Referee #1
Evidence, reproducibility and clarity
In this manuscript, Sun et al. conduct single-cell RNAseq on germline stem cells in the Drosophila ovary to better understand the transcriptional profile of this small, but important, population of stem cells. This resulted in the identification of a subset of genes and gene networks proposed to be involved in early germ cell development and differentiation. A subsequent RNAi screen of these candidate genes indicated that an uncharacterized one (renamed eggplant) was expressed at the transcriptional level in GSCs and at the protein level in the germarium. RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of eggplant resulted in an accelerated cell cycle in germ cell cysts and flies with overall larger ovaries. These results were not observed, however, when flies were fed a rich yeast-based diet. The authors also draw a connection between the MMP-Timp pathway and the regulation of eggplant in ovarian germ cells. Collectively, the findings presented in this manuscript detail novel modulators of germ cell differentiation and proliferation that are regulated by nutrient availability. While the data presented are intriguing (especially in the first part of the manuscript), there are several major and minor issues that should be addressed prior to publication.
Major issues:
- The bioinformatic analysis of the scRNA-seq data is elegant and well done. However, the data from the follow-up studies on eggplant are mostly observational, correlative, and not convincing. There is little to no mechanism described regarding the function of eggplant. This may be beyond the scope of this paper, but it should at least be addressed in more detail in the Discussion section.
- Along the lines of point 1, what is the amino acid sequence of eggplant? What is its predicted molecular weight? Does it contain any predicted domains that may hint at its function in the germarium? The answers to these questions should be included in the manuscript.
- There is no validation of the novel eggplant antibody that was generated. This needs to be demonstrated in order to be confident that the antibody is working as expected to interpret the immunofluorescence data.
- In Figure 8, the connection between MMPs, TIMPs, and eggplant is very weak. It is unclear how the regulation is occurring. Is it direct or indirect? And by what mechanism? Also need to show representative images to go along with the graphs. But as is, this is the weakest data in the manuscript and needs more clarification.
Minor issues:
- It is unclear what it is being shown in Figure 5M.
- In Figure 7B, how was the egg-laying assay performed. This information was not included in the Materials and Methods section.
- There are some grammar issues in the Introduction section.
Referees cross-commenting
My comments align closely with those of Reviewer #3. I also agree that the specific points raised by Reviewer #2 should be addressed to strengthen the manuscript.
Significance
The significance of this work lies in the transcriptional profiling of the Drosophila ovarian germline stem cells. These cells are scarce and are notoriously difficult to isolate and study. This work makes headway in that regard, laying the foundation for future studies, and as mentioned, the bioinformatic analysis of the scRNA-seq data is the strength of the study.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity):
Summary<br /> Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.<br /> Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.<br /> Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.
Major comments:<br /> - Are the key conclusions convincing?<br /> Most conclusions are convincing; however, some issues are pointed out below.
- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.<br /> Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.
While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.
We will characterise the temporal identity, termination and cellular identity of Lola-Ri induced ectopic neuroblasts. If these parameters are disrupted, we will overexpress D to assess whether this can trigger the progression of the temporal series.
Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.
We will include that Dpn is a target of Notch when first mentioned.
Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.<br /> Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.
We will make a more conservative claim and clarify all of our conclusion are medulla neuron-specific.
Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.<br /> Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.<br /> Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.
We will assess the temporal identity of neuroblasts in nerfin-1 mutant clones.
Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.
Ey is shown in Fig-2 (D-D’’) already. Slp is used as a marker of mid tTFs as Ey is expressed also in neurons thus would also be present in deep sections of control clones, whereas Slp is not expressed in neurons. We therefore used Slp as a proxy for mid-temporal identity throughout our study. We will include this text in our revision.
In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.
In Figure 2H, we have shown in clones that overexpression of Dpn induced a significant increase of Slp. In Figure S3B-B’’, we have shown that Dpn overexpression causes an upregulation of Slp at 6 hr APF. We can think we have pretty convincingly shown that Dpn overexpression activates Slp.
For Dichaete, our existing data shows that Dpn overexpression did not significantly alter D expression. To assess if using a stronger driver might allow us to see some changes, we will induced dedifferentiation via Dpn overexpression using the Eyeless-Gal4 driver. In this experiment, we will quantify the amount of D upon Dpn overexpression. Depending on this result, we will revise our conclusion on whether Dpn overexpression represses D.
Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.<br /> Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.<br /> - Are the data and the methods presented in such a way that they can be reproduced?<br /> Yes
Are the experiments adequately replicated and statistical analysis adequate?<br /> Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.
We will repeat Notch experiments to increase the n number for these experiments.
Minor comments:<br /> - Specific experimental issues that are easily addressable.<br /> There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.
In the text we said: “We found that D overexpression did not significantly increase neuronal production, suggesting that it is likely that cell cycle progression lies upstream or in parallel to the temporal series, to promote the generation of neurons.”
In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).
We will add “medulla NBs” to the end of this sentence.
In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).<br /> Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.
We will expand in discussion to discuss the link between cell cycle/tTFs.
In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?<br /> In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.<br /> Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.
While Dpn is overexpressed, it progresses through the temporal series at a slower pace due to a delay in cell cycle progression, as well as delayed onset of D, these NBs still eventually reach the terminal temporal identity, and are thus about to undergo terminal differentiation. We will include an additional piece of data that shows NBs induced by Dpn overexpression do eventually turn on Tll.
Are prior studies referenced appropriately ?<br /> Yes, but in a few places, some references can be added.<br /> An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.14134
We beg to disagree with the reviewer in terms of Pros is not required for terminal differentiation of medulla neuroblasts. Li et al., 2013 shows that nuclear Pros is found in the oldest NBs. We do agree that differentiated state of medulla neurons is less stable, possibly owing to absence of Pros, and we will include that in our discussion.
In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.<br /> Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.
We will discuss the study looking at N targets in NB hyperplasia in the discussion of the revised manuscript.
We will mention that Dpn is a target of Notch in the results section.
Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.
We will comment on Brat in the discussion.
Are the text and figures clear and accurate?<br /> The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.<br /> There are 1 or 2 places where figure calling is wrong in the text.<br /> The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.
We thank the reviewers for identifying these mistakes, and will correct them.
Do you have suggestions that would help the authors improve the presentation of their data and conclusions?<br /> The presentation is okay, in my opinion.
Reviewer #1 (Significance):
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.
This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.<br /> However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.
- Place the work in the context of the existing literature (provide references, where appropriate).<br /> Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.<br /> Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).
The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.<br /> Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.
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State what audience might be interested in and influenced by the reported findings.<br /> The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.
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Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.<br /> Drosophila, central nervous system patterning and cell fate determination of neural stem cells.
Reviewer #2 (Evidence, reproducibility and clarity):
Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.<br /> Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.
This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:
- Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?
We will include an expression analysis of EyOK107-GAL4 and eyR16F10-GAL4. GMR31HI08-GAL4 expression analysis was previously published (Vissers et al., 2018). We will explain in the text the benefits of each driver.
- The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.
We will repeat this experiment and mark the progeny with the neuronal marker Elav, to demonstrate that they are neurons. Additionally, we will add the control to this figure.
- How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).<br /> Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?<br /> Additional explanations in the methods section is needed to assess the robustness of the data.
We will expand the methods section and mention that we used similar thresholding in antibody staining between control and uas dpn in all instances, so even if the antibody is weaker (eg hth) it is consistently quantified. Additionally, we can increase the intensity of Ey in Figure 2D-2D’, as it is expressed at low levels.
- This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'<br /> The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.
We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence.
- Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".
We will split figure 2 into two images. The first image including A-H and the control data. And the second including I-Q and the control data. This will increase the size of the images. Additionally, we will group I and J with corresponding data.
- Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.
Most NBs have terminated by this time point, we will explain this within the text.
- Figure 3K-M: Please explain how the Toy signal was measured and quantified.
We will expand the methods section and explain how Toy quantification is made.
- The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."<br /> More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.<br /> Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?
The DamID data do actually show that Dpn binds (i.e. there is a statistically significant peak at FDR<0.01) directly at these loci (see the TaDa supp fig A & B). Whether it’s doing anything functional or not, we can’t say, but our data shows that Dpn directly binds to slp1 and D. We will clarify the sentence to indicate this in our revision.
- This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.
Our data already shows that these NBs express Tll, the terminal temporal transcription factor (Figure 4F). In addition, we show that there is an increase in Tll+ and Repo+ progeny (Figure 4K, L). Together, this suggests that D overexpression promotes the progression of the temporal series. However, it is possible that Mira+ cells can disappear via cell death. We will assess this possibility by staining for cell death marker Dcp1 at 6hr APF.
Reviewer #2 (Significance):
These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.<br /> I think the findings will be of interest to cell, developmental cell and stem cell biologists.
My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.
Reviewer #3 (Evidence, reproducibility and clarity):
The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing.
Our data relate specifically to gene regulation by Dpn in a dedifferentiated context, and do not seek to understand Dpn regulation in wt neuroblasts. The reviewer is assuming our scope is greater here: we’re not trying to claim that we know what Dpn is doing in wt NBs, and it’s not surprising that ectopic effects in neurons may be different to wt NBs.
To assess whether the mechanisms described apply to more than Dpn overexpression, we will also assess whether the temporal series progression is affected in Lola RNAi and Nerfin-1 mutant.
Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.<br /> The author's state:<br /> "However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."<br /> at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.
We will add this to the abstract.
Results<br /> 1. The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.
We will include expression analysis of all three drivers in a supplementary figure and explain in the text the benefit of each driver.
- The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.
We have conducted lineage tracing (G-Trace) analysis of the medulla neuron driver GMR31H08-GAL4 which we utilise in our study, this driver is predominantly expressed within the medulla neurons (real time) except for a few GMCs present in the lineage. Therefore, the Mira positive cells induced via Dpn overexpression are most likely from dedifferentiation (We will include this data in a supplemental figure in our revised manuscript).
To further support this, we will use GMR31H08-GAL4 with a Gal80ts, to restrict the timing to dedifferentiation induction to 3rd instar, so that the driver is restricted to neurons. Similar strategy to induce dedifferentiation was utilised in DOI: 10.1242/dev.141341 and DOI: 10.1016/j.devcel.2014.01.030.
- What is a conclusion of fig 2C-H?
Fig 2C-H assess the expression of tTFs in UAS-dpn induced ectopic NBs. We will make these conclusions clearer in the text.
- "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.
We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence. Additionally, we will make the connection to the experiments which follow it clearer.
- The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.
We agree with the reviewer that the profile is not specific to medulla NBs. To assess Dpn binding profiles specifically in the medulla NBs, we will use the recently-published NanoDam technique (https://doi.org/10.1016/j.devcel.2022.04.008) for profiling GFP-fusion proteins, with a medulla specific driver (eyR16F10-GAL4) and Dpn-GFP (recombineered locus under endogenous control). This should inform us whether the target genes we have identified are relevant in the medulla.
We will include the tracks of the other transcription factors.
- Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.
We observed that high levels of Slp-1 is correlated with low levels of D. This suggest to us that the initial stochastic differences accounts for where Slp-1 is high is where D is low, and vice versa.
- The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.
We will discuss these models in the Discussion, and make our claims more conservative, as we do not have direct experimental evidence to prove or disprove the model that Dpn is acting as an activator in this context.
- Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.
We will make our claims more conservative, and discuss this point further in the Discussion.
- I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?
The data points towards that Dpn overexpression has two separate roles in regulating the cell cycle. Ofcourse dedifferentiation requires a commitment of neurons into the cell cycle (this we think is still happening), however, we think once these cells have turned on NB markers, they have limited ability to progress through the cell cycle. We will discuss this point in the Discussion.
- Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.
A direct target does not necessarily infer the same phenotype. To assess whether the mechanisms apply to other dedifferentiation models, we will add Lola-RNAi and Nerfin-1 data to our revised manuscript.
Minor comments:<br /> 1. Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"<br /> 2. In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include<br /> 3. Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"
We will correct these typos.
Reviewer #3 (Significance):
Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.
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Referee #3
Evidence, reproducibility and clarity
The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing. Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.
The author's state:
"However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."<br /> at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.
Results
- The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.
- The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.
- What is a conclusion of fig 2C-H?
- "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.
- The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.
- Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.
- The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.
- Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.
- I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?
- Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.
Minor comments:
- Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"
- In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include
- Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"
Significance
Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.
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Referee #2
Evidence, reproducibility and clarity
Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.<br /> Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.
This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:
- Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?
- The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.
- How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).<br /> Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?<br /> Additional explanations in the methods section is needed to assess the robustness of the data.
- This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'<br /> The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.
- Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".
- Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.
- Figure 3K-M: Please explain how the Toy signal was measured and quantified.
- The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."<br /> More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.<br /> Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?
- This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.
Significance
These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.<br /> I think the findings will be of interest to cell, developmental cell and stem cell biologists.
My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.
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Referee #1
Evidence, reproducibility and clarity
Summary
Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.<br /> Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.<br /> Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.
Major comments:
- Are the key conclusions convincing?
Most conclusions are convincing; however, some issues are pointed out below.<br /> - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.<br /> Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.
While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.<br /> Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.
Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.<br /> Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.<br /> - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.<br /> Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.<br /> Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.<br /> In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.<br /> - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.<br /> - Are the data and the methods presented in such a way that they can be reproduced?
Yes<br /> - Are the experiments adequately replicated and statistical analysis adequate?
Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.
Minor comments:
- Specific experimental issues that are easily addressable.
There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.
In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).
In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).<br /> Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.
In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?<br /> In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.<br /> Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.<br /> - Are prior studies referenced appropriately ?
Yes, but in a few places, some references can be added.<br /> An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.141341).<br /> In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.<br /> Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.<br /> Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.<br /> - Are the text and figures clear and accurate?
The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.<br /> There are 1 or 2 places where figure calling is wrong in the text.<br /> The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.<br /> - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
The presentation is okay, in my opinion.
Significance
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.
This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.<br /> However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.<br /> - Place the work in the context of the existing literature (provide references, where appropriate).
Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.<br /> Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).
The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.<br /> Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.<br /> - State what audience might be interested in and influenced by the reported findings.
The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.<br /> - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
Drosophila, central nervous system patterning and cell fate determination of neural stem cells.
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Reply to the reviewers
We propose three revisions, that have not yet been included in the current manuscript:
- All three reviewers comment on the data in figure 7, in which the application of the sensor is shown. We agree that the number of cells is low, and we plan to repeat this experiment to increase the number of cells, and better demonstrate the usefulness of the new probe. We note that the improved Cdc42 sensor is used in a recent preprint (see figures 7 and 9 of: https://www.biorxiv.org/content/10.1101/2022.06.22.497207v2.full), clearly showing the potential of the probe for detection of Cdc42 and increasing our confidence that we can generate higher quality data.
- The ratio of expression of the different components was not quantified. We have these data and we will (re)analyze it and present the results (related to Reviewer #3, point2).
- We will reanalyze the images to ensure that representative images are depicted in the manuscript (related to Reviewer #2, point 3).
Reviewer #1
1) It is not clear why RhoA data were included in this manuscript (Fig. 1), since they seem irrelevant to the primary topic addressed.
We have cell-based data from our previous (published) work that we can use to check whether these results align with the mass-spec data. To make this point clearer we add “We looked first into GBDs for Rho, to compare the results of the mass spectrometry screen with the results of our cell-based assays”.
2) It is not clear what cell type was used when screening for p67phox. The expression of this component of the NADPH oxidase is restricted to a few specific cell types.
That’s a relevant point and therefore observation that p67phox is not detected is perhaps not surprising. We removed this statement.
3) There is precious little quantitation of the colocalization or translocation of the probes throughout the manuscript. It is difficult to assess the validity of the conclusions in the absence of analysis of the statistical significance of the colocalization.
In figure 2, which is an initial screen, there is only a qualitative assessment. However, for the promising candidates, there is a quantitative assessment in Figures 3B and 4 B as to which extent the candidates colocalize with the nuclear localized target. From the rank order and individual datapoints the best performing binder can be inferred.
4) It is not clear why translocation to mitochondria was used in some experiments and translocation to the nucleus in others.
To clarify, we have added text: ”We have previously used nuclear localized, constitutive active Rho GTPases, but these are not accessible for larger proteins that cannot enter the nucleus”
5) In the S1P experiments, it is difficult to ascertain whether increased fluorescence resulted from membrane folding/ruffling or is actually a consequence of localized activation of receptors. Why does the fluorescence decrease progressively over 1500 seconds? Isn't maximal receptor activation accomplished much sooner?
This experiment suffered from bleaching. We will redo the experiment to get higher number of cells and to improve the data.
Reviewer #2
Major comments
- Statistical tests are missing in most of the figures. If the principal purpose of this work is to compare the performance of candidate peptides, the quantitative comparison is essential. If the purpose is just to report another relocation probe, then, more application data may be necessary.
We will improve the quality of the application data. As for statistics, we have added the effect size to figures 5C-F and figure 6A. To explain this (not so common) statistic we add to the materials and methods: “The effect size that quantifies the difference and its distribution was calculated with the web tool ‘PlotsofDifferences’”.
- The criteria for selecting the best peptide should be clearly described. Is it just by inspection or based on any quantitative data? We know that quantification of colocalization is a difficult task. Therefore, it depends on the aim of this work whether the authors are asked to show quantitative data or not. If a strict comparison of peptides is aimed at, the expression level of each target peptide should be at a comparable level. It will be also required whether the design of each probe guarantees the proper folding to bind to GTPases.
There are two stages for the selection. First, we did a qualitative analysis of colocalization (shown in figure 2). Based on the results (“Candidates colocalizing with the mitochondrial tagged Rho GTPase were further tested for their potential as localization-based sensors”), we generated smaller biosensor candidates of which binding to a nuclear target was quantitatively analyze (figures 3B and 4B). As the expression level is an important factor, we ascertained potential candidates were expressed at roughly the same level in the nuclear accumulation assay.
- About the images of cells: When a fluorescent image is presented, we assume it represents all other cells. Please check all images whether they are truly representing the data. For example, in Fig. S3 the nuclei of ABI1-expressing cells look weird, and the nucleus of CYRI-A is very large. If this is true, the reason why ABI1 and CYRI-A should be excluded from the candidate is not the relocation efficiency but the undesired effect on cell physiology. For the screening of the peptides, this information is also very important. With that, this paper becomes more valuable for scientists.
We agree that this is an important point. We will reanalyze the data as indicated in the ‘planned revisions’.
- Please examine the order of panels. For example, the result of mScarlet is on the top in Fig3, but at the bottom in Fig4. Such inconsistency would disturb readers.
We thank the reviewer for this suggestion and we changed figure 4.
- The label should be consistent throughout the paper. For example, in Fig. 5A, Lck-FRB-mTurquoise2 is labeled as Lck-FRB (without the fluorescent protein's name). WASp(CRIB)-mScarlet-I-WASp(CRIB) is labeled as WASp(CRIB)-mScar-WASp(CRIB) (with fluorescent protein's name). Moreover, the same peptide is labeled as mSca-1xWASp(CRIB) in Panel B. Such inconsistency is confusing.
We agree, we have updated figure 5A by adding the abbreviations of the fluorescent proteins. Please note that WASp(CRIB)-mSca-WASp(CRIB), mSca-1xWASp(CRIB) and mSca-2xWASp(CRIB) are three different constructs. In the first one the CRIB domains are sandwiching the fluorescent protein and in the third one they are in tandem downstream of the fluorescent protein.
- Quantitative insight would improve this work. For example, in Fig. 7, the reason why the authors believe that the probe worked is the accumulation of probe at the tip of lamellipodia and the decrease in cytoplasmic intensity. This reviewer does not think the accumulation of the probe in the small area of the lamellipodia explains the massive decrease of cytoplasmic signals. Probably, a substantial amount of the probe is relocated to the plasma membrane, not limited to the lamellipodia.
Minor comments
We propose to repeat the experiment shown in figure 7 and to improve the quality of the data.
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Introduction, "FRET signal is typically measured with a wide field microscope.": This reviewer does not agree with this statement. Confocal and two-photon microscopes have also been used widely.
Fair point. We changed the text to “when the FRET signal is measured with a wide field microscope”
Introduction, "G-protein activating proteins (GAP)": It should read as "GTPase-activating proteins (GAPs)"
Thanks, corrected.
TRIF should read as TIRF.
All instances have been corrected.
Fig.1: To the best of this reviewer's knowledge, PKN1 was first used as the RhoA target peptide by Yoshizaki et al in 2003. J Cell Biol 162, 223-232. They also examined mDia, Rhoteki, and Rhophilin as the target peptides. Pak1 was first used as the Rac1 probe by Kraynov et al. Science 290, 333-337, 2000. Use of Pak1 as the Cdc42 probe was reported by Itoh et al. Mol Cell Biol 22, 6582-659, 2002. This reviewer believes that the priority of the first report should be respected.
We changed part of the introduction to:
High scoring proteins for interacting with constitutively active RhoA(Q63L) included ANLN part of the AniRBD Rho location sensor (Piekny and Glotzer, 2000), PKN1 part of aRho FRET sensor (Yoshizaki et al., 2003) and RTKN part of the rGBD Rho location sensor (Benink and Bement, 2005; Mahlandt et al., 2021) (Fig. 1A,B). This suggested that proteins with a high score in the mass spectrometry screen are potentially suitable as Rho GTPase activity biosensor. Indeed, the GBDs used for Cdc42 location sensors from, PAK1 used in the PBD location sensor (Itoh et al., 2002; Petrie et al., 2012) and N-WASP similar to WASp used in the wGBD location sensor (Benink and Bement, 2005) showed a high score in the screen (Fig. 1A,B).
Discussion:
Another challenge is the Rho GTPase specificity of the relocation-based sensor. For example, Pak1(CRIB) was first used in a Rac1 FRET sensor (Kraynov et al., 2000)____. ThenPak1(CRIB) has been utilized in Cdc42 FRET sensors and in an intensiometric Cdc42 sensor (Hanna et al., 2014; Itoh et al., 2002; Kim et al., 2019). However, Pak1(CRIB), also named PBD sensor, has then been reintroduced by Weiner and colleagues as a Rac1 specific location-based sensor and is often used in neutrophil HL60 cells (Brunetti et al., 2022; Graziano et al., 2019; Le et al., 2021; Weiner et al., 2007).
We also updated the tables in Figure 1.
Fig. 1: Why do the authors omit other promising candidates shown in panel 1B? Please describe the reason for the choice.
We took into account the availability of plasmid DNA, as also explained in the manuscript: “candidate GBDs were selected from top 30 scores of the mass spectrometry screen, that were specific for one Rho GTPase and their DNA was available on addgene”
Fig. 1B: Be consistent to use either "Name" or "Uni Prot name" in Panel A.
We updated figure 1.
Fig. 2: Please include information on TOMM20. The readers may not read the paper by Gillingham et al.
We added an explanation: “To this end, a fusion with TOMM20 was used for mitochondrial localization.”
Fig3 and 4: The authors should show the images of control H2A.
We provide the data for control H2A in figures 3B and 4B.
In Fig3B and 4B, "Cdc42/Rac1 affinity" would be misleading, because the control dots represent their authentic localization rather than "Cdc42/Rac1 affinity".
We agree, we have updated figure 3B and 4B.
Fig. 4: More explanation of this figure is required.
We added text: “Hence, the sensor candidate can freely partition between Rac and Cdc42 binding.”
Fig. 5: More explanation about the FKBP-FRB system will be helpful.
We changed the text to: “The system used rapamycin induced heterodimerization of the two domains FRB and FKBP to recruit the DHPH domain of the Cdc42 specific GEF ITSN1 to the plasma membrane, where it induces activity of the endogenous Cdc42”
Fig. 6: It is rather surprising to see that control-mScarlet also responds to Rac1 activation. What is the explanation for this observation?
We agree and have no explanation.
Fig. 7: A single champion data may not be convincing to prove the usefulness of this probe.
We agree and propose to repeat the experiment.
Reviewer #3
1) The discussion comparing different types of biosensors missed important points. Although the advantages of localization biosensors listed by the authors are correct, they gave the impression that these should simply be an improved replacement for FRET biosensors. There are times when FRET biosensors provide clear advantages. Unlike other proteins, Rho GTPases are well suited for localization sensors because the activated conformation, and only the activated conformation, localizes to the membrane. For diffuse or 3D localization FRET can provide better quantification. Subtle features such as gradients are not easily quantified over a background of unattached domain. The authors state that localization biosensors have enhanced spatial resolution, but this is not explained.
We agree that our introduction is biased towards a preference for relocation based biosensors. However, having used both approaches, we see that both strategies have pro’s and cons. Therefore, we removed the claim for higher resolution and we added: “Still, the ratiometric mode of imaging FRET sensors is beneficial for detection of gradients or activity in 3D imaging”.
2) Throughout the paper, the ratio between the GTPase and the domain, and the overall expression level of each, was not sufficiently examined. The results in many cases would be dependent on both these factors (was a large excess of domain used? Was there insufficient domain to bind the GTPase and provide a signal? Did this vary for different domains, and therefore produce the differences observed? A lack of apparent binding specificity could be produced by high domain expression.)
This is an important point. We will re-analyze the data and include a figure where we add the binding efficiency versus the expression level.
3) In the nuclear exclusion assay, some GTPases were excluded from the nucleus and others not. This was true even without expression of the domains. When GTPases were excluded from the nucleus, domains were eliminated from contention, even when this was true without domain. The authors could at least mention that these domains may be viable.
Correct, and we have added this text: “we cannot exclude that these would be viable Cdc42 sensor candidates”
4) In the multiplexing experiment, only two cells were imaged. In one cell RhoA activity was inversely correlated with Cdc42 activity. In the other cell it was not. It seems there is insufficient information to reach firm conclusions.
We agree and in the revision plan we indicate that we will repeat this experiment to increase the number of cells.
Minor points:
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There appear to be errors in naming mutants. Q60L is used for constitutively active Rac, but Q61L is likely meant. H2A-mTurquoise2-Rac1(G12V)-ΔCaaX is used when it likely should be H2A-mTurquoise2-Rac1(Q61L)-ΔCaaX. There are other examples -- a careful check of these names throughout the manuscript would be valuable.
Thanks for spotting this. Q60L is changed to Q61L. Note that the Rac1(G12V) is correct as it also is a constitutive active Rac1.
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Intro-Paragraph 1-line 5: change present to presence
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Intro-Paragraph 5- line 7: use them instead of theme.
Thanks, both corrected.
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Referee #3
Evidence, reproducibility and clarity
The manuscript was clearly written, but the introduction or discussion could provide a more balanced view of the significance. There are important experiments that are required to support the conclusions regarding selectivity and differences between the domains, particularly the role of expression level and the ratio of expressed proteins. Our review is summarized here:
The authors set out to improve localization-based biosensors for Rac1 and Cdc42 by identifying domains that bind selectively to the activated conformation of Rac1 and Cdc42. Using screens based on mitochondrial and nuclear relocalization, together with mass spec and proximity biotinylation, several potential candidates were identified. The work concludes with the identification of a WASp CRIB domain that can be used as a useful Cdc42 relocalization biosensor. It was applied in a well-executed proof of principle study demonstrating multiplexed imaging of RhoA and Cdc42 localization biosensors.
- The discussion comparing different types of biosensors missed important points. Although the advantages of localization biosensors listed by the authors are correct, they gave the impression that these should simply be an improved replacement for FRET biosensors. There are times when FRET biosensors provide clear advantages. Unlike other proteins, Rho GTPases are well suited for localization sensors because the activated conformation, and only the activated conformation, localizes to the membrane. For diffuse or 3D localization FRET can provide better quantification. Subtle features such as gradients are not easily quantified over a background of unattached domain. The authors state that localization biosensors have enhanced spatial resolution, but this is not explained.
- Throughout the paper, the ratio between the GTPase and the domain, and the overall expression level of each, was not sufficiently examined. The results in many cases would be dependent on both these factors (was a large excess of domain used? Was there insufficient domain to bind the GTPase and provide a signal? Did this vary for different domains, and therefore produce the differences observed? A lack of apparent binding specificity could be produced by high domain expression.)
- In the nuclear exclusion assay, some GTPases were excluded from the nucleus and others not. This was true even without expression of the domains. When GTPases were excluded from the nucleus, domains were eliminated from contention, even when this was true without domain. The authors could at least mention that these domains may be viable.
- In the multiplexing experiment, only two cells were imaged. In one cell RhoA activity was inversely correlated with Cdc42 activity. In the other cell it was not. It seems there is insufficient information to reach firm conclusions.
Minor points:
- There appear to be errors in naming mutants. Q60L is used for constitutively active Rac, but Q61L is likely meant. H2A-mTurquoise2-Rac1(G12V)-ΔCaaX is used when it likely should be H2A-mTurquoise2-Rac1(Q61L)-ΔCaaX. There are other examples -- a careful check of these names throughout the manuscript would be valuable.
- Intro-Paragraph 1-line 5: change present to presence
- Intro-Paragraph 5- line 7: use them instead of theme.
Significance
The assays and approach to finding domains for biosensors were novel and interesting. The end result was not as surprising as one might have hoped, but the approach alone made the paper worthwhile.
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Referee #2
Evidence, reproducibility and clarity
Mahlandt et al report Rho GTPase relocation sensors. First, the authors picked up candidate peptides based on the Mass-Spec data reported by Sean Munro's laboratory. The authors repeated the experiments to confirm the binding of peptides to mitochondria-targeted Cdc42 and Rac1 and narrowed down the candidate peptides by binding to nuclear Cdc42. The specificity of binding to Rac1 and Cdc42 was also tested. Eventually, they concluded that dimeric Tomato-WASp(CRIB) is the best sensor for Cdc42, which could detect S1P-induced Cdc42 activation in primary endothelial cells. The effort to improve the relocation sensors should be evaluated highly. This reviewer has some suggestions to improve this paper.
Major comments:
- Statistical tests are missing in most of the figures. If the principal purpose of this work is to compare the performance of candidate peptides, the quantitative comparison is essential. If the purpose is just to report another relocation probe, then, more application data may be necessary.
- The criteria for selecting the best peptide should be clearly described. Is it just by inspection or based on any quantitative data? We know that quantification of colocalization is a difficult task. Therefore, it depends on the aim of this work whether the authors are asked to show quantitative data or not. If a strict comparison of peptides is aimed at, the expression level of each target peptide should be at a comparable level. It will be also required whether the design of each probe guarantees the proper folding to bind to GTPases.
- About the images of cells: When a fluorescent image is presented, we assume it represents all other cells. Please check all images whether they are truly representing the data. For example, in Fig. S3 the nuclei of ABI1-expressing cells look weird, and the nucleus of CYRI-A is very large. If this is true, the reason why ABI1 and CYRI-A should be excluded from the candidate is not the relocation efficiency but the undesired effect on cell physiology. For the screening of the peptides, this information is also very important. With that, this paper becomes more valuable for scientists.
- Please examine the order of panels. For example, the result of mScarlet is on the top in Fig3, but at the bottom in Fig4. Such inconsistency would disturb readers.
- The label should be consistent throughout the paper. For example, in Fig. 5A, Lck-FRB-mTurquoise2 is labeled as Lck-FRB (without the fluorescent protein's name). WASp(CRIB)-mScarlet-I-WASp(CRIB) is labeled as WASp(CRIB)-mScar-WASp(CRIB) (with fluorescent protein's name). Moreover, the same peptide is labeled as mSca-1xWASp(CRIB) in Panel B. Such inconsistency is confusing.
- Quantitative insight would improve this work. For example, in Fig. 7, the reason why the authors believe that the probe worked is the accumulation of probe at the tip of lamellipodia and the decrease in cytoplasmic intensity. This reviewer does not think the accumulation of the probe in the small area of the lamellipodia explains the massive decrease of cytoplasmic signals. Probably, a substantial amount of the probe is relocated to the plasma membrane, not limited to the lamellipodia.
Minor comments:
- Introduction, "FRET signal is typically measured with a wide field microscope.": This reviewer does not agree with this statement. Confocal and two-photon microscopes have also been used widely.
- Introduction, "G-protein activating proteins (GAP)": It should read as "GTPase-activating proteins (GAPs)"
- TRIF should read as TIRF.
- Fig.1: To the best of this reviewer's knowledge, PKN1 was first used as the RhoA target peptide by Yoshizaki et al in 2003. J Cell Biol 162, 223-232. They also examined mDia, Rhoteki, and Rhophilin as the target peptides. Pak1 was first used as the Rac1 probe by Kraynov et al. Science 290, 333-337, 2000. Use of Pak1 as the Cdc42 probe was reported by Itoh et al. Mol Cell Biol 22, 6582-659, 2002. This reviewer believes that the priority of the first report should be respected.
- Fig. 1: Why do the authors omit other promising candidates shown in panel 1B? Please describe the reason for the choice.
- Fig. 1B: Be consistent to use either "Name" or "Uni Prot name" in Panel A.
- Fig. 2: Please include information on TOMM20. The readers may not read the paper by Gillingham et al.
- Fig3 and 4: The authors should show the images of control H2A.
- In Fig3B and 4B, "Cdc42/Rac1 affinity" would be misleading, because the control dots represent their authentic localization rather than "Cdc42/Rac1 affinity".
- Fig. 4: More explanation of this figure is required.
- Fig. 5: More explanation about the FKBP-FRB system will be helpful.
- Fig. 6: It is rather surprising to see that control-mScarlet also responds to Rac1 activation. What is the explanation for this observation?
- Fig. 7: A single champion data may not be convincing to prove the usefulness of this probe.
Significance
- The authors have screened many peptides, which may serve as the relocation sensor for Rho-family GTPases.
- There are precedent relocation sensors, a part of which is listed in Fig. 1A. This work discloses an improved relocation biosensor.
- Cell biologists who is working on Cdc42 will be interested in this probe.
- Expertise of this reviewer: Signal transduction, Fluorescence microscopy.
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Referee #1
Evidence, reproducibility and clarity
In this manuscript Mahlandt et al. report efforts to generate sensitive fluorescent biosensors to monitor the activation of Rac and Cdc42. While biosensors for these GTPases have been designed and used by others, some have poor specificity while others require the measurement of FRET, which is technically more complex and usually yields low signal to noise ratios. The efforts of Mahlandt et al. are therefore well justified and commendable, and follow on the heels of their successful development of a Rho-selective probe (J. Cell Sci., 2021).
The authors used existing data of interacting proteins to identify candidate domains that could be adapted for use as Rac or Cdc42 activation indicators. They proceeded to select the most promising candidates, assessed their specificity and tried to improve their efficiency by generating tandem constructs that are expected to have higher avidity. They identify CYRA-I as a positive Rac interactor, but the affinity and selectivity of this construct were not deemed to be sufficient and this line of enquiry was not pursued further. In contrast, the WASp-CRIB was found to be sufficiently Cdc42-specific and its avidity was improved by generating a construct tagged with dimeric Tomato fluorescent protein. Unfortunately, while data using overexpression of active Cdc42 are convincing and clear, the results obtained under physiological conditions -stimulating cells with S1P- show very modest recruitment. The general usefulness of the probe is therefore questionable.
There are also a number of specific issues:
- It is not clear why RhoA data were included in this manuscript (Fig. 1), since they seem irrelevant to the primary topic addressed.
- It is not clear what cell type was used when screening for p67phox. The expression of this component of the NADPH oxidase is restricted to a few specific cell types.
- There is precious little quantitation of the colocalization or translocation of the probes throughout the manuscript. It is difficult to assess the validity of the conclusions in the absence of analysis of the statistical significance of the colocalization.
- It is not clear why translocation to mitochondria was used in some experiments and translocation to the nucleus in others.
- In the S1P experiments, it is difficult to ascertain whether increased fluorescence resulted from membrane folding/ruffling or is actually a consequence of localized activation of receptors. Why does the fluorescence decrease progressively over 1500 seconds? Isn't maximal receptor activation accomplished much sooner?
Significance
While the purpose and intent of the study are commendable, the results are far from convincing and the probes designed do not represent a sufficient improvement over existing biosensors. The lack of quantitative and statistical analyses is problematic, as is it is difficult to assess the significance of the results.
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Reply to the reviewers
1. General Statements [optional]
We thank the reviewers for fair and constructive comments, and for good suggestions for how to further improve the manuscript. We also appreciate the comments on novelty and potential importance for future clinical approaches for CADASIL. Below we provide our point-by-point responses to the reviewers’ comments. We start with a description of planned revisions, followed by describing changes already carried out in the transferred manuscript.
2. Description of the planned revisions
We plan to experimentally address the following points raised by the reviewers.
First, we will address the ASMA staining in the arterioles by isolating brain microvessels from the TgN3R182C150 mouse and WT mouse and strain for ASMA and perlecan/PDGFRb (stain pericytes and VSMC) to in more detail visualize that ASMA is only expressed in the arterioles and not in the pericytes in the capillaries (reviewer 1, point 4).
Second, we will assess the safety and toxicity of the active immunotherapy (reviewer 2, point 4). Specifically, we plan to analyse the structure and morphology of the kidneys from sham and vaccinated mice for renal damage by histology, H/E staining as well as creatinine levels in the serum. If the histology shows signs of damage (such as necrosis, apoptosis or granules in the tubules), we will stain the tissue with KIM1 for acute renal damage and caspase 3 for apoptosis. Moreover, we will analyse the inflammation C-Reactive Protein (CRP) marker in the serum by western blot analysis and/or ELISA and finally Neurofilament Light chain protein in the serum by SIMOA analysis to monitor if the vaccination is causing any neurodegeneration.
Third, we will improve the image quality for Figure 5A (reviewer 1, point 5).
Reviewer #1 (Evidence, reproducibility and clarity):
Summary:
This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.
We thank the reviewer for the positive comments and for finding it to be a potential novel and safe therapy for CADASIL.
Major comments:
The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.
We thank the reviewers for the positive words on the manuscript and understand the concerns over the lack of effects in the arterioles. We still believe that observing NOTCH3 ECD reductions around capillaries and in the blood is a significant step forward and supports further endeavors in the area of active immunization, especially considering that the treatment is well tolerated with no overt NOTCH3-related toxicity effects. With this said, we of course realize that there would still be a long way towards clinical use, but all therapy developments have to start somewhere, and future studies (from us and others) can build on the data presented here. There are a number of possible explanations as to why there was not a significant reduction of NOTCH3 around the arterioles. It might be due to that the TgN3R182C150 model is a mild disease model which has a NOTCH3 accumulation onset around 6-7 months of age that starts in the capillaries and at later ages spreads to the arterioles, and that a different time axis or longer immunization period would also lead to a reduction of NOTCH3 in the arterioles, which can be addressed in future experiments. We however believe it was worthwhile to first explore an early immunization protocol, as it may pave the way to a treatment strategy that can be used early on the disease progress, maybe already at the pre-symptomatic phase.
Minor comments:
- Figure EV1 is redundant as Figure 2C has the same information.
We agree the figures are redundant and have deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively.
- P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?
The reviewer raises an interesting point. While we do not strictly know why a mixing of mutated and wildtype fragments promote aggregation, our observations are in line with previous reports. Duering et al. Hum. Mol. Genet, 2011 used a single-particle approach SIFT (scanning for intensely fluorescent targets) to study the co-aggregation of WT and mutant NOTCH3 EGF1-5 proteins and reported that mixtures of WT and mutant proteins showed dual colour high-intensity signals, representing de novo aggregates. They also observed WT/R133C multimer formation over three days as compared to five days used in our study. It is also of note that most CADASIL cases are heterozygous for the NOTCH3 mutation, which suggests that wildtypeand mutant NOTCH3 proteins co-exist in vivo, which also have been confirmed by co-immunoprecipitation experiments from cell lysates by Opherk et al. Hum. Mol. Genet. 2009. In the light of these previous reports, we performed a variety of ratio between WT and R133C NOTCH3 EGF1-5 proteins and opted for a 1:1 mixture since it showed the highest amount of multimers in relation to monomers after incubation.
- How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?
The dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay. The protein was then mixed with the adjuvant or PBS to the desired concentration. This is now more clearly described in the Materials and Methods.
- P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?
We have used a direct fluorescent conjugated ASMA antibody for this mouse strain and another mouse strain with same mutation but higher NOTCH3 expression (described in Rutten et al, Acta Neuropathol Commun, 2015) as well as for the wildtype mice. In all cases, we find that pericytes have no or very low ASMA expression. This is in line with a report from Ghezali et al. Ann Neurol 2018, in which they used perlecan as a marker for pericytes in the rat Notch3 R169C mutation model, since ASMA did not stain the capillaries. The lack of ASMA staining in pericytes is in agreement with our previous large-scale single cell RNA-seq study of the mouse brain vasculature, where we find very little ASMA (ACTA2) gene expression in pericytes, while it is abundant in the vascular smooth muscle cells (Vanlandewijck et al., Nature 2018).
With this said, we will in the revised version include images on isolated brain microvessels from the N3TgR182C150 mouse model and WT that are stained with ASMA and perlecan/PDGFRb to clearly show that ASMA only stains the arterioles and not the capillaries, see Point 2 “Description of planned revisions” part above.
- In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).
We agree that Figure 5A is not clearly showing the differences between vaccinated and sham although highly significant. We have included more representative images and we have also improved the description of the quantification in the method part (page 18-19).
- In Figure 8B, what does the "% of microglia area" mean and how was it calculated?
The % of microglia area represents the percentage that the CD68 staining occupies per microglia area (stained by Iba1), meaning that the vaccinated mice contain more activated microglia compared to sham. This was calculated by measuring the area of the CD68 staining that colocalizes with the microglia. In the transferred version, we have improved the description of this in the text (page 18-19).
- Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?
Thank you for the comments and we understand the concern, given the highly complex morphology and dynamics of microglia, which makes it difficult to describe potential differences in a straightforward manner. In the first graph in Figure 9B we show that the vaccinated mice had a higher percentage of microglia with NOTCH3 ECD deposits. However, these deposits did not occupy more area of the microglia (% microglia area, second graph), or showed a significant increase in the number of deposits per microglia area (number/1000 µm2 microglia, third graph). The average size of these deposits did not increase in a significant way per microglia area (average size per microglia µm2, fourth graph) in comparison to sham and non-vaccinated mice. We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).
Reviewer #1 (Significance):
CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.
This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.
My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.
We thank the reviewer for these comments and that the manuscript provides proof-of-principle for a novel strategy towards a safe and tolerable therapy for future treatment of CADASIL patients. As such, we believe the study will be a useful platform and resource for future studies from us and others.
Reviewer #2 (Evidence, reproducibility and clarity):
The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.<br /> The study is well written, very clear and the results are very promising.
We thank the reviewer for positive comments on our work and pleased that you find the manuscript well and clearly written and containing promising data.
Same minor comments need to be clarified before publication.<br /> 1. In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).<br /> References should be updated with recent clinical studies about the efficacy of Aducanumab.
This is a valid point. We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021).
- Schedule used to induce active immunization should be justified or referenced.
We appreciate this comment and realize that we did not explain the rationale for the immunization scheme in the original version. We used a similar immunization schedule as used by Kontsekova et al. Alzheimer's Research & Therapy 2014 in their study of active immunization of a tau peptide in a transgenic rat Alzheimer disease model. This is now mentioned in the text on page 6. The slight modifications of the Kontsekova et al., scheme are also described in the Materials and Methods, page 15 (we made some slight modifications in order to be compatible to mouse and also immunized with an aggregated protein of 25kD instead of a 2 kD tau peptide).
- CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.
Thank you for pointing this out. We have previously reported MRI data on a more severe CADASIL mouse model (TgN3R182C350), which harbors the same transgene but expressed at a 2.3 times higher level compared to the mouse model used in this study (Rutten et al. Acta Neuropathol. Commun. 2015 and Gravesteijn et al. Transl. Stroke Res. 2020). We did not observe any consistent differences on MRI or behavior between the TgN3R182C350 model and controls at 20 months of age. From this data we do not expect to see any differences on MRI in our milder model at 7 months of age. We have however included a short description of the previous MRI data in the transferred version on page 12.
- It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker.
Thank you for pointing out this important issue regarding the safety aspects for our active immunotherapy. We will address this by additional experiments of the renal damage by histology and inflammation markers and neuronal damage markers in the serum with western blotting/ELISA and SIMOA, which we have written in more detail in Point 2 “Description of planned revisions” part above.
The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similarly long time axis.
Reviewer #2 (Significance):
The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.
We thank the reviewer for these positive comments. We have updated the manuscript with a new reference regarding the effect of Aducanumab. For the safety and tox analysis we will do additional experimental analysis which we have addressed above in “Description of planned revisions”.
3. Description of the revisions that have already been incorporated in the transferred manuscript
Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.
We deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively. (Reviewer 1, point 1).
We provide an improved description of how the dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay (Reviewer 1, point 3) (Materials and Methods, page 15).
We have improved the description of the quantification of the immunohistochemistry in the method part (Reviewer 1, point 5) (Materials and Methods, page 18-19).
We have improved the description of microglia in the quantification in the method part (Reviewer 1, point 6 and 7) (Materials and Methods, page 18-19).
We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).
We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021). (Reviewer 2, point 1).
We have provided a rationale and a more detailed description of the immunization protocol. (Reviewer 2, point 2) (Materials and Methods, page 15).
We have included a short description of the previous MRI data in the transferred version. (Reviewer 2, point 3), (Discussion, page 12)
We have included the following new references (Vanlandewijck et al., Nature 2018, Knopman et al. Alzheimers Dement. 2021, Kontsekova et al. Alzheimer's Research & Therapy 2014, Gravesteijn et al. Transl. Stroke Res. 2020).
4. Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similar time axis.
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Referee #2
Evidence, reproducibility and clarity
The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.<br /> The study is well written, very clear and the results are very promising.
Same minor comments need to be clarified before publication.
- In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).<br /> References should be updated with recent clinical studies about the efficacy of Aducanumab.
- Schedule used to induce active immunization should be justified or referenced.
- CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.
- It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker, ....
Significance
The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.
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Referee #1
Evidence, reproducibility and clarity
Summary:
This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.
Major comments:
The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.
Minor comments:
- Figure EV1 is redundant as Figure 2C has the same information.
- P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?
- How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?
- P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?
- In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).
- In Figure 8B, what does the "% of microglia area" mean and how was it calculated?
- Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?
Significance
CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.
This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.
My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.
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Reply to the reviewers
Author response (Tane at al: RC-2022-01646)
Reviewer #1 (Evidence, reproducibility and clarity (Required)): * Comments The work described in this manuscript starts with an in-silico analysis of the primary amino-acid sequence of CAP-H proteins that reveals the presence in vertebrate orthologs of an N-terminal extension of ~80 amino acids in length which contains 19 serine or threonine residues and also, in its centre, a stretch of conserved basic amino acids predicted to form a helix. These features suggest a regulatory module. Using xenopus egg extracts depleted of endogenous condensins and supplemented with recombinant condensin I holocomplexes, either wildtype or mutants, the authors show that deleting the N-terminal tail of CAP-H, or just the central helix (CH), increases the association condensin I with chromatin in mitotic egg extracts and accelerates the formation of mitotic chromosomes. Interestingly, they also show that deleting the N-tail enables a substantial amount of condensin I to associate with chromatin in interphase extracts and to form chromosome-like structures, while WT condensin I cannot. Using in vitro assays and naked DNA as substrate, the authors further show that removing the N-terminal tail of CAP-H improves both the topological (salt-resistant) association of condensin I with DNA and it loop extrusion activity. These experiments appear to me as are clear and robust. They convincingly reveal that N-tail of human CAP-H hinders the binding of condensin I to DNA and both its loop-extrusion and chromosome-shaping activities. However, the mechanism through which such hindrance is achieved remains elusive (see major comments 1-3). A complementary part of the work tackles the important question of the cell cycle control of such counteracting effect. Using newly-designed antibodies against two phospho-serine residues within the tail, the authors provide evidence that the tail is phosphorylated in mitosis-specific manner. This points towards phosphorylation as a biological mean to modulate the effect of the tail on condensin's binding during the cell cycle. In support to this idea, using non-phosphorylatable or phosphomimic substitutions of all the serine and threonine residues within the tail (n =19), including one substitution within the CH domain (Ser 70), the authors show that non-phosphorylatable mutations (H-N19A) or phosphomimic mutations (H-N19D) respectively reduce or improve condensin I binding to chromatin in mitotic egg extracts. This suggests that the phosphorylation of the N-terminal tail in mitosis might relieve its negative effect on condensin I binding to chromatin. The weaknesses I see in this part of the study concern (1) the validation of the phospho-antibodies, which appears to me as insufficiently described (major comment 4), (2) the possibility the bulk changes in amino acids (n=19 out of 80) could impact the folding of the central helix (minor comment X) and (3) that some substitutions could impact the binding of condensin I by different mechanisms (minor comment X).
Major comments:
- On the model. The authors propose that the N-tail could stabilise an interaction between the N-terminal part of CAP-H and SMC2's neck, which would restrain the transient opening of a DNA entry gate within the ring, necessary for the topological engagement of DNA and loop formation. Although the model is sound, I see no direct data that support it in the manuscript. Such model predicts that a CAP-H protein containing or not the N-terminal tail (or the central helix) should exhibit different binding strengths to SMC2 in vitro. It seems to me that the authors could easily test this prediction using the recombinant proteins they produced in the context of this study. *
Response
We thank the reviewer for pointing out this important issue. To test whether the CAP-H N-tail indeed contributes to the stabilization of the SMC2-kleisin gate, we set up a highly sophisticated functional assay described by Hassler et al (2019). The authors used this assay to demonstrate that an N-terminal fragment of kleisin (engineered to be cleaved by TEV protease) is released from the rest of the condensin complex in an ATP-dependent (i.e., head-head engagement-dependent) manner. We reasoned that this assay is most powerful to prove our hypothesis in a mechanistically relevant context. We envisioned that the CAP-H fragment lacking its N-tail can readily be released whereas the CAP-H fragment retaining its N-tail is more difficult to be released (because of the postulated stabilization of the SMC2-CAP-H interaction). Despite substantial efforts in making TEV-cleavable constructs and in testing various releasing conditions, we have not been able to recapitulate the ATP-dependent release even with the holo(H-dN) construct. Thus, unfortunately, this trial enabled us to neither prove nor disprove our hypothesis.
We are fully aware that the full reconstitution of ATP-dependent and phosphorylation-stimulated gate-opening reaction in vitro is a very important direction in the future. It is beyond the scope of the current study, however.
2. On ATP-hydrolysis. Given the importance of ATP hydrolysis for the engagement of condensin into a topological mode of association with DNA and for its loop extrusion activity, I suggest that the authors measure the impact of the N-tail and of the CH domain on the rate of ATP hydrolysis by condensin I holocomplexes. I suppose that it can be relatively easily done (PMID: 9288743) using the recombinant WT and mutant versions they purified in the course of this study.
Response
We appreciate this constructive comment. In fact, we did a preliminary experiment and found that ATPase activities (either in the absence or presence of DNA) were not significantly different between holo(WT) and holo(H-dN). We were not surprised with this result because our previous study on condensin II indicated that enhanced ATP hydrolysis by a class of mutant complexes is not directly coupled to their enhanced association with chromosomes (Yoshida et al., 2022, eLife). We consider that other functional assays, such as the topological loading assay and the loop extrusion assay shown in the current manuscript, are more informative assays to address ATP-dependent activities of the condensin complexes.
3. A conundrum with previous work? In Kimura et al. Science 1998 (PMID: 9774278), the lab of Tatsuya Hirano has shown that xenopus condensin I purified from mitotic egg extracts induces the supercoiling of plasmid DNA in vitro, but fails to do so when it is purified from interphase egg extracts. This echoes the inhibitory effect of the N-tail of the topological binding of condensin I described in the current manuscript. However, using a gel shift assay, Kimura et al. 1998 also provide evidence that interphase and mitotic condensin I bind plasmid DNA in vitro with similar efficiencies. At first sight, this prior observation seems to contradict the idea that the N-tail of CAP-H restrains DNA binding unless it is phosphorylated in mitosis. Is it possible that the in vitro binding assays used in Kimura et al. 1998 and in this work might assess different modes of binding? I suggest that this apparent conundrum should to be discussed.
Response
We thank the reviewer for following our early studies. As discussed below, we are confident that our conclusion reported in the current study by no means contradicts our previous observations.
We reason that the confusion expressed by the reviewer stems from intrinsic, technical limitations of the gel-shift assay. Such limitations become apparent especially when it is applied to the functional analyses of complicated protein machines such as condensins. For instance, the DNA-binding activity of condensin I detected by the gel-shift assay is neither ATP-dependent nor phosphorylation-dependent (Kimura and Hirano, 1997; Kimura et al., 1998). It is fundamentally different from the ATP-dependent activities detected by the topological loading and loop extrusion assays reported in the current study (It remains unknown whether the two activities are stimulated by mitotic phosphorylation). Thus, the DNA-binding activity detected by the gel-shift assay does not reflect “productive” DNA interactions that depend on ATP hydrolysis in vitro. We therefore consider that gel-shift analyses of holo(WT) and holo(H-dN) would not produce any useful information.
*Related to that, could it be possible for the authors to assess the impact of the N-tail on the salt-sensitive binding of condensin to DNA, i.e. by reproducing the topological binding assay but omitting the high salt washes? I guess this information could be useful to fully apprehend the impact of the N-tail on the binding of condensin. *
Response
When we set up the topological loading assay, we actually tested a low-salt wash condition that the reviewer suggests here. Although a much higher level of DNA recovery was observed with the low-salt condition than with the high-salt wash condition, no nucleotide dependency was detectable with the low-salt condition. Moreover, no difference in DNA recovery between holo(WT) and holo(H-dN) was observed. Thus, the low-condition condition allowed us to detect the “bulk” DNA-binding activity that is equivalent to that detected by the gel-shift assay. These results were fully consistent with the discussion above.
4. Validation of phospho-antibodies and by extension showing the phosphorylation of the tail. The newly-designed phospho-serine antibodies used in this study to show that the N-tail is phosphorylated at serine 17 and serine 76 in mitosis (Fig. EV3) are, in my view, not characterized enough. This piece of data is key to substantiate the idea that the tail is phosphorylated in mitosis. Yet, showing that these antibodies detect epitopes on WT condensin I from mitotic egg extracts but not on the H-N19A counterpart, nor on WT condensin I from interphase extracts, does not demonstrate the phospho-specificity of such antibodies. I suggest that a PPase treatment should be conducted to assess the phospho-specificity of these antibodies. Moreover, since the lab of Tatsuya Hirano has the know-how to deplete Cdc2/CDK1 from xenopus egg extract, such strategy could/should be used to further validate the antibodies and assess to which extent the N-tail is phosphorylated in a Cdc2-dependent manner.
Response
We have performed two sets of experiments to confirm the specificity of the phosphoepitopes recognized by anti-hHP1 and anti-hHP2. In the first set, we performed a phosphatase treatment assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated using an antibody against hCAP-G, treated with l protein phosphatase in the presence or absence of phosphatase inhibitors, and analyzed by immunoblotting using anti-hHP1 and anti-hHP2. The results (now shown in Supplementary Fig 3C) demonstrated that the epitopes recognized by anti-hHP1 and anti-hHP2 are sensitive to phosphatase treatment. In the second set, we performed a phosphopeptide competition assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated and subjected to immunoblotting. The membranes were triplicated and probed with anti-hHP1 in the presence of no competing peptide, hHP1 or hHP2. Similarly, another set of triplicated membranes was probed with anti-hHP2 in the presence of no competing peptide, hHP1 or hHP2. We found that the signal recognized by anti-hHP1 competed with hHP1, but not with hHP2, and that the signal recognized by anti-hHP2 competed with hHP2, but not with hHP1. The results (now shown in Supplementary Fig 3D) demonstrated the sequence specificity of the phosphoepitopes recognized by the two antibodies. The procedures for these experiments have been described in Materials and Methods.
Because Cdk1 depletion from M-HSS creates an HSS equivalent to I-HSS, we do not consider that the suggested experiment will provide additional information.
*Minor comments:
- The impact of the 19 mutations, A or D, introduced within the tail on the folding of the central helix? The idea that the negative effect of the N-tail is relieved by phosphorylation is based on the chromatin binding phenotypes exhibited by the H-N19D or H-N19A mutant holocomplexes, in which 19 amino-acids out of 80 have been modified, include one in the central helix. The authors also provide evidence that the central helix (CH) located within the tail plays a key role in the negative regulation of condensin I binding. Thus, I wonder to which extent the folding of the central helix could be impacted by the mutations introduced in the tail and notably the one within the central helix itself. Could the author assess the structure of mutated tails using Alpho-fold and/or discuss this point? *
Response
According to the reviewer’s suggestion, we performed structure predictions using Alphafold2, and found that neither the N19A nor N19D mutations alter the original prediction of helix formation that was made for the wild-type CH sequence. A conventional secondary structure prediction using Jpred4 reached the same conclusion.
2. Phosphorylation of serine 70 in the central helix by Aurora-B kinase? A prior study by Tada et al. (PMID: 21633354) has shown (1) that serine 70 of the N-tail of hCAP-H is phosphorylated by Aurora-B kinase, (2) that the mutation S70A reduces the binding of condensin I to chromatin in HeLa cells and (3) that hCAP-H interacts with histone H2A in an Aurora-B dependent manner. This draws a picture in which the phosphorylation of Ser70 by Aurora-B would improve condensin I binding to chromatin by promoting an interaction between hCAP-H and histone H2A/nucleosomes. Intriguingly, Ser 70 in Tada et al. correspond to the serine residue located within the conserved central helix analysed in this study, and this Ser70 residue is mutated in the H-N19D or H-N19A holocomplexes that show reduced chromatin binding in this study. This raises the question as what could be the contribution of the S70A or S70D substitution to the chromatin binding phenotypes shown by the H-N19D or H-N19A holocomplexes. This is not discussed in the manuscript, and the authors do not cite this earlier work (PMID: 21633354) in their manuscript. Is there any reason for that? I suggest it should be cited and discussed.
Response
We thank the reviewer for bringing up this issue. In many respects, we do not trust the data reported by Tada et al (2011) and the resultant model they proposed. Previous and emerging lines of evidence reported from our own and other laboratories indicate that histones compete with condensins for DNA binding, strongly arguing against the possibility that histone H2A acts as a “chromatin receptor” for condensins. We formally discussed and criticized the Tada 2011 model in our previous publications, which included Shintomi et al (2015) NCB, Shintomi et al (2017) Science, Hirano (2016) Cell and Kinoshita/Hirano (2017) COCB. We thought that those were enough. That said, we also consider that the reviewer is right. The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional line of evidence that argues against the Tada model. A critical comparison between the Tada model and our current model would benefit the readers. In the revised manuscript, we have added the following discussion:
In terms of the regulatory role of the CAP-H N-tail, it would be worthy to discuss the model previously proposed by Tada et al (2011). According to their model, aurora B-mediated phosphorylation of the CAP-H N-tail allows its direct interaction with the histone H2A N-tail, which in turn triggers condensin I loading onto chromatin. Accumulating lines of evidence, however, strongly argue against this model: (i) aurora B is not essential for single chromatid assembly in Xenopus egg extracts (MacCallum et al., 2002) or in a reconstitution assay (Shintomi et al., 2015); (ii) the H2A N-tail is dispensable for condensin I-dependent chromatid assembly in the reconstitution assay (Shintomi et al., 2015); (iii) even whole nucleosomes are not essential for condensin I-mediated assembly of chromatid-like structures (Shintomi et al., 2017). The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional piece of evidence against the model proposed by Tada et al (2011).
3. Other minor comments - Please provide a microscope image of DNA loop in Fig. 4D.
Response
In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.
*- The authors do not compare the kleisin of condensin I with the one of condensin II with respect to the features tackled in this work. Given the different behaviours condensin I and II, such comparison could be informative for the readers. *
Response
We thank the reviewer for this constructive comment. In the revised manuscript, we have added the following statement:
It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II.
*- The authors do not reference the work of Robellet et al. Genes & Dev (2015) (PMID: 25691469) on the regulation of condensin binding in budding yeast by an SMC4 phospho-tail. I suggest that the analogy should be discussed. *
Response
According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.
Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015; Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.
- In the introduction section, lane 5, the sentence "Many if not all eukaryotic species have two different condensin complexes" appears inappropriate since budding and fission yeast cells possess a single condensin complexes, similar to condensin I in term of primary amino-acid sequence.
Response
We thought that the original wording “Many if not all” was good enough to imply that some species, which include budding yeast and fission yeast, have only a single condensin complex. To make it clear, however, we have modified the sentence in the revised manuscript as follows:
Many eukaryotic species have two different condensin complexes although some species including fungi have only condensin I.
*- page 4; typo: motif I and V bind to the SMC neck and the SMC4 cap regions, respectively. Should read SMC2 neck. *
Response
The reviewer is right. It should read the SMC2 neck. Corrected.
*- Are the data and the methods presented in such a way that they can be reproduced? YES - Are the experiments adequately replicated and statistical analysis adequate? YES - Are prior studies referenced appropriately? Not all of them (see above) - Are the text and figures clear and accurate? YES
CROSS-CONSULTATION COMMENTS I consider the comments from all reviewers as helpful for the authors.
Reviewer #1 (Significance (Required)):
Summary Condensins are genome organisers of the family of SMC ATPase complexes and are best characterized as the drivers of mitotic chromosome assembly (condensation). It is acknowledged that condensins shape mitotic chromosomes by massively associating with DNA upon mitotic entry (loading step) and by folding chromatin fibres into arrays of loops, most likely through an ATP-dependent extrusion of DNA into loops, as seen in vitro. What remains unclear, however, are the mechanisms by which condensins load onto DNA and fold it into arrays of loops in vivo, and how these reactions are coupled with the cell cycle, i.e. restricted mostly to mitosis. Condensins are ring shaped pentamers that change conformation upon ATP-hydrolysis. In vitro studies suggest that condensins bind DNA via ATP-hydrolysis-independent, direct electrostatic contacts between condensin subunits and DNA. Such electrostatic contacts are salt-sensitive in in-vitro assays. Upon ATP-hydrolysis, condensins engage into an additional mode of binding that is resistant to high salt concentration and likely to be topological in nature. Both modes of association are necessary to form DNA loops. Vertebrates possess two types of condensin complexes, condensin I and II, each composed of a same SMC2-SMC4 ATPase core but associated with two different sets of three non-SMC subunits; a kleisin and two HEAT-repeat proteins. Condensin II is nuclear during interphase and stably binds chromatin upon mitotic entry, while condensin I is located in the cytoplasm during interphase and binds chromatin in a dynamic manner upon nuclear envelope breakdown. How the spatiotemporal control of condensin I and II is achieved remains poorly understood. Previous studies have shown that the phosphorylation of condensin I by mitotic kinases, such as CDK1, Aurora-B and Polo, play a positive role in its binding to chromatin and/or its functioning, but the underlying mechanisms remain to be characterised. In this manuscript, Shoji Tane and colleagues provide good evidence that the N-terminal tail of the human kleisin subunit of condensin I, hCAP-H, serves as a regulatory module for the cell-cycle control of condensin I binding to chromatin and chromosome shaping activity. The authors clearly show that the N-tail of CAP-H hinders the binding of condensin I to chromatin in xenopus egg extracts and, using in vitro assays, that the N-tail also hinders the topological association of condensin I with DNA and its loop extrusion activity. The authors provide additional data suggesting that the phosphorylation of the N-tail of CAP-H, in mitosis, relieves its inhibitory effect on condensin I binding. Based on their findings, Tane et al. propose a model suggesting that the N-terminal tail of CAP-H constitutes a gate keeper that maintains condensin-rings in a closed conformation that is unfavourable for topological binding to DNA, and whose locking effect is relieved in mitosis by phosphorylation.
Taken as a whole, this work has the potential to reveal a molecular basis for the cell cycle regulation of condensin I in vertebrate cells and as such to significantly improve our understanding of the integrated functioning condensin I. The characterisation of the inhibitory effect of the N-tail on condensin binding to chromatin and to naked DNA in vitro is well described, the data are clear and robust and the results convincing. On the other hand, some of the data on the phospho-regulation appear to me as are more debatable and I think that some of the results described here deserve to be discussed in the context of previous works. Finally, I see no data in the manuscript that directly supports the mechanistic model proposed by the authors, while it seems to me that such model could have been easily tested exprimentally. Thus, I suggest that Tane and colleagues should perform a couple of relatively easy experiments to strengthen their claims and that a few omitted prior studies on the topic should be referenced and discussed. *
Reviewer #2 (Evidence, reproducibility and clarity (Required)): * The manuscript reveals that the N-terminal region of CAPH could play a role in regulating condensin I activity, using a range of in vitro methods. They propose that the N-terminal region of CAPH inhibits complex activity, and this is turned off upon deletion or phosphorylation, by using truncations, phospho-mimics or phospho-deficient mutations. While the results are interesting to the field, and helps to address the question as to how condensin complexes are controlled in a cell cycle dependent manner, some key data and controls are necessary to ensure the conclusion is robust.
Main comments
• What is meant by "unperturbed I-HSS" on page 7, ie membrane containing versus membrane free or condensin depleted? *
Response
We apologize for having created unnecessary confusion. We meant that the “unperturbed I-HSS” is the “undepleted I-HSS”. As far as the issue of membrane-containing vs membrane-free is concerned, we explicitly mentioned that “we used membrane-free I-HSS in the following experiments” several lines above. In the revised manuscript, we have revised the statement accordingly.
In many of the protein gels eg figure 4B, the bands for SMC2 and 4 are more intense that the non-SMC components. The method for protein purification also does not include a size exclusion step to ensure sample homogeneity. Authors should perform some sort of quality control checks on samples such as analytical gel filtration or mass photometry to ensure stoichiometry/homogeneity. This is particularly important for samples eg the N19A, where activity is reduced compared to wild-type as poor protein behaviour could create false negative results.
Response
As the reviewer is fully aware, the reconstitution and purification of multiprotein complexes, such as condensins, is by no means an easy task. We notice that many groups in the field share common concerns about sample homogeneity and subunit stoichiometry, and that these concerns cannot completely be eliminated even after size exclusion chromatography. Because the current study handles a large number of mutant complexes, we consider that the purification by two-step column chromatography is the most practical approach. We do not notice any abnormal behaviors of holo(H-N19A) in the processes of expression and purification. It is also important to emphasize that the H-N19D mutations cause the completely opposite phenotype. Taken all together, we are confident of our current conclusions.
That said, in the revised manuscript, we have added the following statement in Results:
Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….
- Loop extrusion assays in figure 4D-G shows no example data i.e. no pictures or videos of loops being formed. These should also be included.*
Response
In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.
- Given the mutant holo(H-dN) has higher activity than wild-type, a negative control such as holo(H-dN) without ATP or holo(H-dN) ATPase deficient mutant should also be measured in loop extrusion assays, to ensure the activity is derived from the ATPase activity.*
Response
In the revised manuscript, we have added loop formation data for both holo(WT) and holo(H-dN) in the absence or presence of ATP (Supplementary Fig 5). We are confident that both complexes support loop extrusion strictly in an ATP-dependent manner.
- According to the methods, this work performs the same loop extrusion assay as described in Kinoshita et al, 2022, however, in Kinoshita et al, wild type condensin I makes loops in 30-50% of DNA molecules, where in this study the percentage is less than half that. Can the author please explain the discrepancy given the same method was used?*
Response
First of all, we wish to remind the reviewer that the holo(WT) constructs used in the two studies are not identical: CAP-H was N-terminally HaloTagged in all constructs used in Kinoshita et al (2022), whereas the same subunit was C-terminally HaloTagged in the pair of constructs used in the current study. Because we wanted to compare the activities between the full-length CAP-H and N-terminally deleted version of CAP-H (H-dN), we reasoned that it would be inappropriate to put the HaloTag to the N-terminus of CAP-H. The difference in the constructs could explain the observed discrepancy, even if it might not be the sole reason.
The design of the constructs was accurately described in each manuscript, but the statements were not very explicit about the positions of the HaloTag. To clarify this issue, we have added the following sentences in the revised manuscript:
Note that the HaloTag was fused to the C-terminus of CAP-H in the current study because we wanted to investigate the effect of the N-terminal deletion of CAP-H. We used N-terminally HaloTagged CAP-H constructs in our previous study (Kinoshita et al., 2022).
- In the concluding statement the author suggests "Upon mitotic entry, multisite phosphorylation of the N-tail relieves the stabilization, allowing the opening of the DNA entry gate, hence, the loading of condensin I onto chromosomes." This seems unlikely as fusion the N-terminus of the of the kleisin to the C-terminus of SMC2 is able to function for yeast (Shaltiel et al 2022) and condensin II (Houlard et al 2021), and equivalently in cohesin (Davidson et al 2019).*
Response
We appreciate the reviewer’s concern. In our view, however, the issue of the “DNA-entry gate” remains under debate in the SMC field (e.g., Higashi et al [2020] Mol Cell; Taschner and Gruber [2022] bioRxiv). For instance, Shaltiel et al (2022) demonstrated that neck-gate fusion constructs can support in vitro activities including topological loading under certain conditions, but also showed that such constructs greatly reduce the cell viability, leaving the possibility that the gate opening is required for some physiological functions.
That said, it is true that the data reported in the current manuscript do not exclude the possibility that the SMC2 neck-kleisin interface is not used as a DNA entry gate for condensin I loading. In the revised manuscript, we have added the following statement in Discussion:
Although our model predicts that the SMC2 neck-kleisin interface is used as a DNA entry gate, we are aware that several studies reported evidence arguing against this possibility (e.g., Houlard et al [2021]; Shaltiel et al [2022]). Our current data do not exclude other models.
*Reviewer #2 (Significance (Required)):
This is an interesting story that reveals new insights about condensin regulation.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This paper reveals a role of an N-terminal extension of CAP-H in the regulation of condensin-I activity in Xenopus extracts using biochemical reconstitution experiments. The authors demonstrate that a motif in the N-terminal tail that is conserved in vertebrates acts as an inhibitor of condensin I activity. Using several mutant constructs, it is shown that the inhibition by this motif is in turn counteracted by the phosphorylation of neighbouring serine and threonine residues in mitosis, presumably at least in part by CdK. Mutants that have lost this inhibition are able to condense chromatin into chromatid-like structures more efficiently and to some degree even in interphase extracts. Moreover, one such mutant is characterized in detail by biochemical and biophysical experiments and shown to have increased capacity in salt-stable DNA loading and in DNA loop extrusion.
Major comments: This is a beautiful and thorough study that is presented in a clear and concise manner. The main conclusions are well justified. No additional experiments are needed to support them. Replication and statistical analysis appear adequate. The final model is however largely speculative. Recent work has indicated that loading of yeast condensin does not require gate opening. The authors may thus want to include alternative scenarios or remain more vague. *
Response
This comment is related to the last comment of Reviewer#2. See above for our response.
*The H-N19A mutant has a loss of function phenotype (possibly due to folding problem caused by 19 point mutations rather than lack of phosphorylation), the authors could consider to rescue the phenotype by also including the CH motif mutations in this construct (or make an explanatory statement in the text). *
Response
We understand the reviewer’s logic here, but overlaying additional mutations into the H-N19A mutations could cause an unforeseeable effect, potentially making the interpretation of the outcome complicated.
We also wish to point out that it may be inappropriate to regard the phenotype exhibited by holo(H-N19A) as a simple loss-of-function phenotype. This is because the opposite, accelerated loading phenotype exhibited by holo(H-dN) can be regarded as a consequence of loss of negative regulation. Like holo(H-dN), the phosphomimetic mutant complex holo(H-N19D) displayed an accelerated loading phenotype, fully supporting our conclusion. In the revised manuscript, we have added the following statement in Results:
Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….
*Albeit not necessary for the main conclusions, the authors could possibly significantly strengthen their study by testing for binding partners of the N-tail and the CH motif by running AlphaFold predictions against the condensin I subunits. *
Response
We appreciate this constructive comment. We attempted to predict possible interactions between SMC2 and a CAP-H fragment containing its N-tail and motif I using
ColabFold (Mirdita et al., 2022, Nat. Methods). The algorism excellently predicted the proper folding of the CAP-H motif I and its interaction with the SMC2 neck. Under this condition of predictions, however, the N-tail remained largely disordered (except for the CH), and no interaction with any part of SMC2 was predicted. The same was true when the N19D mutations were introduced in the N-tail sequence. Thus, this trial did not provide much information about the potential interaction target(s) of the CAP-H N-tail.
*The efficiency of depletion of condensin subunits from I-HSS extracts is not documented (in contrast to M-HSS extracts - figure EV1C). While any condensin remaining in these extracts might not be active (or interfering), the authors may want to at least comment on this in the text. *
Response
We check the efficiency of immunodepletion every time by immunoblotting and confirm that a high level of depletion is achieved from both M-HSS and I-HSS. According to the reviewer’s comment, the following statement was placed in Materials and Methods:
The efficiency of immunodepletion was checked every time by immunoblotting. An example of immunodepletion from M-HSS was shown in Supplemental figure 1C. We also confirmed that a similar efficiency of immunodepletion was achieved from I-HSS.
*The authors should include information on the quantification of chromatid morphology. Is the analysis based on chromatids taken from the same images/imaging session, from technical replicates, biological replicates? *
Response
In the revised manuscript, we have added statements on image presentation and experimental repeats in the appropriate figure legends and methods section. During the revision process, we repeated the experiments shown in Supplementary Fig 2, and obtained the same results. In the revised manuscript, the original set of data has been replaced with the new set of data along with panel C (Quantification of the intensity of mSMC4 per DNA area).
Minor comment: The colour scheme in Figure 5A is confusing. Use less colour? The orange and red colours are moreover quite similar.
Response
According to the reviewer’s comment, we have modified Figure 5A.
*Reviewer #3 (Significance (Required)):
The findings provide new insights into how condensin-I activity is restricted outside of mitosis. It was previously assumed that this regulation was (largely) due to the exclusion of condensin I from the nucleus prior to nuclear envelope breakdown. This study shows that another pathway is contributing to the regulation and implies that controlling condensin I activity is more important than previously appreciated. Whether all residual nuclear condensin I is inactivated, remains to be determined. The physiological impact of loss of autoinhibition on chromosome segregation and cell cycle progression also remains to be uncovered. The observed effects are robust and appear significant. Future research on condensin I using reconstitution will likely benefit from being able to control or eliminate the self-inhibition.
This reviewer has expertise on the biochemistry and structural biology of SMC protein complexes.
Reviewer #4 (Evidence, reproducibility and clarity (Required)):
Mitotic chromosome formation is a cell cycle-regulated process that is crucial for eukaryotic genome stability. The chromosomal condensin complex promotes chromosome condensation, but the temporal control over condensin function is only scantly understood. In this impressive manuscript, "Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit", Tane and colleagues provide important new insight into the cell cycle-regulation of condensin. The authors identify a kleisin N-tail that acts as a negative regulator of condensin's DNA interactions. Removal of this N-tail, or its cell cycle-dependent phosphorylation, relieves inhibition and activates condensin. This is a simple, yet very important story, that advances our molecular understanding of chromosome formation. The experiments are performed to a very high technical standard and support the authors conclusions. This manuscript is highly suitable for publication in any molecular biology journal, once the authors have considered the following points.
- Introduction. a) The authors could better explain their own prior work (Kimura et al. 1998), which has identified the condensin XCAP-D2 and XCAP-H as the targets of phosphoregulation. The current manuscript explains the role of XCAP-H phosphorylation. *
Response
According to the reviewer’s comment, we have added the following sentence in Introduction:
The major targets of mitotic phosphorylation identified in these studies included the CAP-D2 and CAP-H subunits.
1. b) Given the limited knowledge about condensin cell cycle regulation, it seems prudent to provide a brief summary of what is known. Fission yeast Smc4 phosphorylation regulates condensin nuclear import (Sutani et al. 1999), while budding yeast Smc4 phosphorylation slows down the dynamic turnover of the condensin complex on chromosomes (Robellet et al. 2015 and Thadani et al. 2018).
Response
We appreciate this constructive comment. According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.
Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015 and Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.
2. Extracts were mixed with mouse sperm nuclei. If there is a reason why mouse rather than Xenopus sperm nuclei were used, this would be interesting to know.
Response
The original motivation for introducing mouse sperm nuclei into Xenopus egg extracts was to test the functional contribution of nucleosomes to mitotic chromosome assembly. When mouse sperm nuclei are incubated with an extract depleted of the histone chaperone Asf1, the assembly of octasomes can be suppressed almost completely. Remarkably, we found that even under this “nucleosome-depleted” condition, mitotic chromosome-like structures can be assembled in a manner dependent on condensins (Shintomi et al., 2017, Science). Xenopus sperm nuclei cannot be used in this type of experiment because they endogenously retain histones H3 and H4 and are therefore competent in assembling octasomes even in the Asf1-depleted extract. During this study, we realized that the use of mouse sperm nuclei in Xenopus egg extracts provides additional and deep insights into the basic mechanisms of mitotic chromosome assembly. For instance, the functional contribution of condensin II to chromosome assembly could be observed more prominently when mouse sperm nuclei are used as a substrate than when Xenopus sperm nuclei are used (Shintomi et al., 2017, Science). We suspected that the slow kinetics of nucleosome assembly on the mouse sperm substrate creates an environment in favor of condensin II’s action. For these reasons, our laboratory now extensively uses mouse sperm nuclei for the functional analyses of condensin II (Yoshida et al., 2022. eLife) and other purposes (Kinoshita et al., 2022, JCB). Yoshida et al (2022) used experimental approaches analogous to the current study, and found that the deletion of the CAP-D3 C-tail, causes accelerated loading of condensin II. One of the long-term goals in our laboratory is to critically compare and contrast the actions of condensin I and condensin II in mitotic chromosome assembly. Thus, the use of the same substrate in the two complementary studies can be fully justified.
During the preparation of this response, we realized that the readers would benefit from a brief statement about the comparison between condensin I and condensin II. In the revised manuscript, we have added the following statement in Discussion:
It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II. Interestingly, however, a recent study from our laboratory has shown that the deletion of the CAP-D3 C-tail causes accelerated loading of condensin II onto chromatin (Yoshida et al., 2022). It is therefore possible that condensins I and II utilize similar IDR-mediated regulatory mechanisms, but they do so in different ways.
3. Page 5. "we next focused on the conserved helix (CH) [...], that is enriched with basic amino acids." Based on the provided sequence alignment, the helix contains an equal number of both basic and acidic residues. Is it correct to characterize this helix as positively charged?
Response
The reviewer is right. In the revised manuscript, we have used a more neutral expression as follows:
we next focused on the conserved helix (CH) [...], that contains conserved basic amino acids.
4. To prevent N-tail phosphorylation, the authors create a (H-N19A) allele, referring to Cdk promiscuity. Cdk cooperation with other mitotic kinases can also be expected. Nevertheless, in case the authors created a variant with only the 4 Cdk consensus sites mutated, it would be interesting to know its consequences.
Response
We consider that this is a reasonable question. In our early experiments, we noticed that introduction of multiple SP/TP sites in the non-SMC subunits of condensin I including CAP-H caused a relatively mild phenotype in mitotic chromosome assembly in Xenopus egg extracts. Then we found that the deletion of the CAP-H N-tail caused a very clear, accelerated loading phenotype, prompting us to focus on the regulatory function of the CAP-H N-tail. As the reviewer correctly points out, the current study does not pinpoint the number and position of target sites involved in the proposed phosphoregulation by the CAP-H N-tail. We wish to address this important issue in the near future, along with reconstitution of the phosphoregulation using purified components.
5. Fig EV3A, a second region of mitotic condensin phosphorylation is XCAP-D2. The authors state that XCAP-D2 phosphorylation does not impact on condensin function in their assays. This is very relevant to the current paper, so it would be good to see the Yoshida et al. 2022 Elife publication (in press) as an accompanying manuscript.
Response
We thank the reviewer for pointing out this issue, but it is not necessarily clear to us what the reviewer requests. In the original manuscript, we cited Yoshida et al (2022) in Discussion as follows:
Recent studies from our laboratory showed that the deletion of the CAP-D2 C-tail, which also contains multiple SP/TP sites (Supplementary Figure 3A), has little impact on condensin I function as judged by the same and related add-back assays using Xenopus egg extracts (Kinoshita et al, 2022; Yoshida et al, 2022).
We believe that the current statement is good enough.
6. One of the authors' most striking results is chromosome formation in interphase egg extracts using condensin (H-dN). At the same time, condensin (H-dN) is unable to support DNA supercoiling or chromosome reconstitution with recombinant components. More emphasis might be placed on this important piece of information, and possible reasons should be discussed. Can Cdk-treatment restore condensin (H-dN) biochemical activity? If not, then condensin (H-dN) might have lost more than just an inhibitory N-tail. The cohesin N-tail is thought to fulfil a positive role during DNA loading (Higashi et al. 2020). Could it be that the condensin N-tail encompasses both positive and negative roles?
Response
We were also surprised to find that holo(H-dN) gains the ability to assemble mitotic chromosome-like structures in interphase extracts. It should be stressed, however, that the formation of mitotic chromosome-like structures in I-HSS requires a much higher concentration (150 nM) than the standard concentration used in M-HSS (35 nM). Thus, the deletion of the CAP-H N-tail alone cannot make the condensin I complex fully active in I-HSS. We think that the negative regulation by the CAP-H N-tail is not the sole mechanism responsible for the very tight cell cycle regulation of condensin I function. We emphasize this important point by mentioning that “our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes” in Summary.
At the end of Discussion, we describe the limitations of the current study: “we have so far been unsuccessful in using these recombinant complexes to recapitulate positive DNA supercoiling or chromatid reconstitution, both of which require proper Cdk1 phosphorylation in vitro”. We are fully aware that full reconstitution of phosphorylation-dependent activation of condensin I in vitro is one of the most important directions in the future.
Although we currently do not have any evidence to suggest that the H N-tail has a positive role, we do not exclude such a possibility.
7. Here comes my main question for the authors (though I should stress that I do not expect an answer for publication in a Review Commons journal). The authors now have a unique opportunity to gain key mechanistic insight into condensin by answering the question, 'how does the kleisin N-tail inhibit condensin'? The authors allude to a model in which the N-tail interacts with Smc2 to close/obstruct the kleisin N-gate, through which the DNA likely enters the condensin ring. Can the authors biochemically recapitulate an interaction between an isolated N-tail (or N-terminal section of XCAP-H) and Smc2? Does Cdk phosphorylation alter this interaction?
Response
This comment is related to Comment #1 of Reviewer#1. See above for our response.
*Minor points. 8. The condensin loop extrusion results would benefit from a supplementary movie or time-series, to illustrate the comparison. Details of how loop rate, duration and sizes were assessed should be added to the methods section. *
Response
In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E. We have also added the following explanations for how the parameters of loop extrusion reactions were assessed in Materials and Methods:
To determine the loop size, the fluorescence intensity of the looped DNA was divided by that of the entire DNA molecule for each image, and multiplied by the length of the entire DNA molecule (48.5 kb). The loop rate was obtained by averaging the increase in looped DNA size per second. The loop duration was calculated by measuring the time from the start of DNA loop formation until the DNA loop became unidentifiable.
9. Figure EV3A legend, "hHP4" should probably read "hHP2".
Response
The reviewer is right. It should read hHP2. Corrected.
*Reviewer #4 (Significance (Required)):
see above *
-
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Referee #4
Evidence, reproducibility and clarity
Mitotic chromosome formation is a cell cycle-regulated process that is crucial for eukaryotic genome stability. The chromosomal condensin complex promotes chromosome condensation, but the temporal control over condensin function is only scantly understood. In this impressive manuscript, "Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit", Tane and colleagues provide important new insight into the cell cycle-regulation of condensin. The authors identify a kleisin N-tail that acts as a negative regulator of condensin's DNA interactions. Removal of this N-tail, or its cell cycle-dependent phosphorylation, relieves inhibition and activates condensin. This is a simple, yet very important story, that advances our molecular understanding of chromosome formation. The experiments are performed to a very high technical standard and support the authors conclusions. This manuscript is highly suitable for publication in any molecular biology journal, once the authors have considered the following points.
-
Introduction.
- a) The authors could better explain their own prior work (Kimura et al. 1998), which has identified the condensin XCAP-D2 and XCAP-H as the targets of phosphoregulation. The current manuscript explains the role of XCAP-H phosphorylation.
- b) Given the limited knowledge about condensin cell cycle regulation, it seems prudent to provide a brief summary of what is known. Fission yeast Smc4 phosphorylation regulates condensin nuclear import (Sutani et al. 1999), while budding yeast Smc4 phosphorylation slows down the dynamic turnover of the condensin complex on chromosomes (Robellet et al. 2015 and Thadani et al. 2018).
- Extracts were mixed with mouse sperm nuclei. If there is a reason why mouse rather than Xenopus sperm nuclei were used, this would be interesting to know.
- Page 5. "we next focused on the conserved helix (CH) [...], that is enriched with basic amino acids." Based on the provided sequence alignment, the helix contains an equal number of both basic and acidic residues. Is it correct to characterize this helix as positively charged?
- To prevent N-tail phosphorylation, the authors create a (H-N19A) allele, referring to Cdk promiscuity. Cdk cooperation with other mitotic kinases can also be expected. Nevertheless, in case the authors created a variant with only the 4 Cdk consensus sites mutated, it would be interesting to know its consequences.
- Fig EV3A, a second region of mitotic condensin phosphorylation is XCAP-D2. The authors state that XCAP-D2 phosphorylation does not impact on condensin function in their assays. This is very relevant to the current paper, so it would be good to see the Yoshida et al. 2022 Elife publication (in press) as an accompanying manuscript.
- One of the authors' most striking results is chromosome formation in interphase egg extracts using condensin (H-dN). At the same time, condensin (H-dN) is unable to support DNA supercoiling or chromosome reconstitution with recombinant components. More emphasis might be placed on this important piece of information, and possible reasons should be discussed. Can Cdk-treatment restore condensin (H-dN) biochemical activity? If not, then condensin (H-dN) might have lost more than just an inhibitory N-tail. The cohesin N-tail is thought to fulfil a positive role during DNA loading (Higashi et al. 2020). Could it be that the condensin N-tail encompasses both positive and negative roles?
- Here comes my main question for the authors (though I should stress that I do not expect an answer for publication in a Review Commons journal). The authors now have a unique opportunity to gain key mechanistic insight into condensin by answering the question, 'how does the kleisin N-tail inhibit condensin'? The authors allude to a model in which the N-tail interacts with Smc2 to close/obstruct the kleisin N-gate, through which the DNA likely enters the condensin ring. Can the authors biochemically recapitulate an interaction between an isolated N-tail (or N-terminal section of XCAP-H) and Smc2? Does Cdk phosphorylation alter this interaction?
Minor points.
- The condensin loop extrusion results would benefit from a supplementary movie or time-series, to illustrate the comparison. Details of how loop rate, duration and sizes were assessed should be added to the methods section.
- Figure EV3A legend, "hHP4" should probably read "hHP2".
Significance
see above
-
-
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Referee #3
Evidence, reproducibility and clarity
This paper reveals a role of an N-terminal extension of CAP-H in the regulation of condensin-I activity in Xenopus extracts using biochemical reconstitution experiments. The authors demonstrate that a motif in the N-terminal tail that is conserved in vertebrates acts as an inhibitor of condensin I activity. Using several mutant constructs, it is shown that the inhibition by this motif is in turn counteracted by the phosphorylation of neighbouring serine and threonine residues in mitosis, presumably at least in part by CdK. Mutants that have lost this inhibition are able to condense chromatin into chromatid-like structures more efficiently and to some degree even in interphase extracts. Moreover, one such mutant is characterized in detail by biochemical and biophysical experiments and shown to have increased capacity in salt-stable DNA loading and in DNA loop extrusion.
Major comments:
This is a beautiful and thorough study that is presented in a clear and concise manner. The main conclusions are well justified. No additional experiments are needed to support them. Replication and statistical analysis appear adequate. The final model is however largely speculative. Recent work has indicated that loading of yeast condensin does not require gate opening. The authors may thus want to include alternative scenarios or remain more vague.
The H-N19A mutant has a loss of function phenotype (possibly due to folding problem caused by 19 point mutations rather than lack of phosphorylation), the authors could consider to rescue the phenotype by also including the CH motif mutations in this construct (or make an explanatory statement in the text).
Albeit not necessary for the main conclusions, the authors could possibly significantly strengthen their study by testing for binding partners of the N-tail and the CH motif by running AlphaFold predictions against the condensin I subunits.
The efficiency of depletion of condensin subunits from I-HSS extracts is not documented (in contrast to M-HSS extracts - figure EV1C). While any condensin remaining in these extracts might not be active (or interfering), the authors may want to at least comment on this in the text.
The authors should include information on the quantification of chromatid morphology. Is the analysis based on chromatids taken from the same images/imaging session, from technical replicates, biological replicates?
Minor comment:
The colour scheme in Figure 5A is confusing. Use less colour? The orange and red colours are moreover quite similar.
Significance
The findings provide new insights into how condensin-I activity is restricted outside of mitosis. It was previously assumed that this regulation was (largely) due to the exclusion of condensin I from the nucleus prior to nuclear envelope breakdown. This study shows that another pathway is contributing to the regulation and implies that controlling condensin I activity is more important than previously appreciated. Whether all residual nuclear condensin I is inactivated, remains to be determined. The physiological impact of loss of autoinhibition on chromosome segregation and cell cycle progression also remains to be uncovered. The observed effects are robust and appear significant. Future research on condensin I using reconstitution will likely benefit from being able to control or eliminate the self-inhibition.
This reviewer has expertise on the biochemistry and structural biology of SMC protein complexes.
-
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Referee #2
Evidence, reproducibility and clarity
The manuscript reveals that the N-terminal region of CAPH could play a role in regulating condensin I activity, using a range of in vitro methods. They propose that the N-terminal region of CAPH inhibits complex activity, and this is turned off upon deletion or phosphorylation, by using truncations, phospho-mimics or phospho-deficient mutations.
While the results are interesting to the field, and helps to address the question as to how condensin complexes are controlled in a cell cycle dependent manner, some key data and controls are necessary to ensure the conclusion is robust.
Main comments
- What is meant by "unperturbed I-HSS" on page 7, ie membrane containing versus membrane free or condensin depleted?
- In many of the protein gels eg figure 4B, the bands for SMC2 and 4 are more intense that the non-SMC components. The method for protein purification also does not include a size exclusion step to ensure sample homogeneity. Authors should perform some sort of quality control checks on samples such as analytical gel filtration or mass photometry to ensure stoichiometry/homogeneity. This is particularly important for samples eg the N19A, where activity is reduced compared to wild-type as poor protein behaviour could create false negative results.
- Loop extrusion assays in figure 4D-G shows no example data i.e. no pictures or videos of loops being formed. These should also be included.
- Given the mutant holo(H-dN) has higher activity than wild-type, a negative control such as holo(H-dN) without ATP or holo(H-dN) ATPase deficient mutant should also be measured in loop extrusion assays, to ensure the activity is derived from the ATPase activity.
- According to the methods, this work performs the same loop extrusion assay as described in Kinoshita et al, 2022, however, in Kinoshita et al, wild type condensin I makes loops in 30-50% of DNA molecules, where in this study the percentage is less than half that. Can the author please explain the discrepancy given the same method was used?
- In the concluding statement the author suggests "Upon mitotic entry, multisite phosphorylation of the N-tail relieves the stabilization, allowing the opening of the DNA entry gate, hence, the loading of condensin I onto chromosomes." This seems unlikely as fusion the N-terminus of the of the kleisin to the C-terminus of SMC2 is able to function for yeast (Shaltiel et al 2022) and condensin II (Houlard et al 2021), and equivalently in cohesin (Davidson et al 2019)
Significance
This is an interesting story that reveals new insights about condensin regulation.
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Referee #1
Evidence, reproducibility and clarity
Comments
The work described in this manuscript starts with an in-silico analysis of the primary amino-acid sequence of CAP-H proteins that reveals the presence in vertebrate orthologs of an N-terminal extension of ~80 amino acids in length which contains 19 serine or threonine residues and also, in its centre, a stretch of conserved basic amino acids predicted to form a helix. These features suggest a regulatory module. Using xenopus egg extracts depleted of endogenous condensins and supplemented with recombinant condensin I holocomplexes, either wildtype or mutants, the authors show that deleting the N-terminal tail of CAP-H, or just the central helix (CH), increases the association condensin I with chromatin in mitotic egg extracts and accelerates the formation of mitotic chromosomes. Interestingly, they also show that deleting the N-tail enables a substantial amount of condensin I to associate with chromatin in interphase extracts and to form chromosome-like structures, while WT condensin I cannot. Using in vitro assays and naked DNA as substrate, the authors further show that removing the N-terminal tail of CAP-H improves both the topological (salt-resistant) association of condensin I with DNA and it loop extrusion activity. These experiments appear to me as are clear and robust. They convincingly reveal that N-tail of human CAP-H hinders the binding of condensin I to DNA and both its loop-extrusion and chromosome-shaping activities. However, the mechanism through which such hindrance is achieved remains elusive (see major comments 1-3).
A complementary part of the work tackles the important question of the cell cycle control of such counteracting effect. Using newly-designed antibodies against two phospho-serine residues within the tail, the authors provide evidence that the tail is phosphorylated in mitosis-specific manner. This points towards phosphorylation as a biological mean to modulate the effect of the tail on condensin's binding during the cell cycle. In support to this idea, using non-phosphorylatable or phosphomimic substitutions of all the serine and threonine residues within the tail (n =19), including one substitution within the CH domain (Ser 70), the authors show that non-phosphorylatable mutations (H-N19A) or phosphomimic mutations (H-N19D) respectively reduce or improve condensin I binding to chromatin in mitotic egg extracts. This suggests that the phosphorylation of the N-terminal tail in mitosis might relieve its negative effect on condensin I binding to chromatin. The weaknesses I see in this part of the study concern (1) the validation of the phospho-antibodies, which appears to me as insufficiently described (major comment 4), (2) the possibility the bulk changes in amino acids (n=19 out of 80) could impact the folding of the central helix (minor comment X) and (3) that some substitutions could impact the binding of condensin I by different mechanisms (minor comment X).
Major comments:
- On the model. The authors propose that the N-tail could stabilise an interaction between the N-terminal part of CAP-H and SMC2's neck, which would restrain the transient opening of a DNA entry gate within the ring, necessary for the topological engagement of DNA and loop formation. Although the model is sound, I see no direct data that support it in the manuscript. Such model predicts that a CAP-H protein containing or not the N-terminal tail (or the central helix) should exhibit different binding strengths to SMC2 in vitro. It seems to me that the authors could easily test this prediction using the recombinant proteins they produced in the context of this study.
- On ATP-hydrolysis. Given the importance of ATP hydrolysis for the engagement of condensin into a topological mode of association with DNA and for its loop extrusion activity, I suggest that the authors measure the impact of the N-tail and of the CH domain on the rate of ATP hydrolysis by condensin I holocomplexes. I suppose that it can be relatively easily done (PMID: 9288743) using the recombinant WT and mutant versions they purified in the course of this study.
- A conundrum with previous work? In Kimura et al. Science 1998 (PMID: 9774278), the lab of Tatsuya Hirano has shown that xenopus condensin I purified from mitotic egg extracts induces the supercoiling of plasmid DNA in vitro, but fails to do so when it is purified from interphase egg extracts. This echoes the inhibitory effect of the N-tail of the topological binding of condensin I described in the current manuscript. However, using a gel shift assay, Kimura et al. 1998 also provide evidence that interphase and mitotic condensin I bind plasmid DNA in vitro with similar efficiencies. At first sight, this prior observation seems to contradict the idea that the N-tail of CAP-H restrains DNA binding unless it is phosphorylated in mitosis. Is it possible that the in vitro binding assays used in Kimura et al. 1998 and in this work might assess different modes of binding? I suggest that this apparent conundrum should to be discussed. Related to that, could it be possible for the authors to assess the impact of the N-tail on the salt-sensitive binding of condensin to DNA, i.e. by reproducing the topological binding assay but omitting the high salt washes? I guess this information could be useful to fully apprehend the impact of the N-tail on the binding of condensin.
- Validation of phospho-antibodies and by extension showing the phosphorylation of the tail. The newly-designed phospho-serine antibodies used in this study to show that the N-tail is phosphorylated at serine 17 and serine 76 in mitosis (Fig. EV3) are, in my view, not characterized enough. This piece of data is key to substantiate the idea that the tail is phosphorylated in mitosis. Yet, showing that these antibodies detect epitopes on WT condensin I from mitotic egg extracts but not on the H-N19A counterpart, nor on WT condensin I from interphase extracts, does not demonstrate the phospho-specificity of such antibodies. I suggest that a PPase treatment should be conducted to assess the phospho-specificity of these antibodies. Moreover, since the lab of Tatsuya Hirano has the know-how to deplete Cdc2/CDK1 from xenopus egg extract, such strategy could/should be used to further validate the antibodies and assess to which extent the N-tail is phosphorylated in a Cdc2-dependent manner.
Minor comments:
- The impact of the 19 mutations, A or D, introduced within the tail on the folding of the central helix? The idea that the negative effect of the N-tail is relieved by phosphorylation is based on the chromatin binding phenotypes exhibited by the H-N19D or H-N19A mutant holocomplexes, in which 19 amino-acids out of 80 have been modified, include one in the central helix. The authors also provide evidence that the central helix (CH) located within the tail plays a key role in the negative regulation of condensin I binding. Thus, I wonder to which extent the folding of the central helix could be impacted by the mutations introduced in the tail and notably the one within the central helix itself. Could the author assess the structure of mutated tails using Alpho-fold and/or discuss this point?
- Phosphorylation of serine 70 in the central helix by Aurora-B kinase? A prior study by Tada et al. (PMID: 21633354) has shown (1) that serine 70 of the N-tail of hCAP-H is phosphorylated by Aurora-B kinase, (2) that the mutation S70A reduces the binding of condensin I to chromatin in HeLa cells and (3) that hCAP-H interacts with histone H2A in an Aurora-B dependent manner. This draws a picture in which the phosphorylation of Ser70 by Aurora-B would improve condensin I binding to chromatin by promoting an interaction between hCAP-H and histone H2A/nucleosomes. Intriguingly, Ser 70 in Tada et al. correspond to the serine residue located within the conserved central helix analysed in this study, and this Ser70 residue is mutated in the H-N19D or H-N19A holocomplexes that show reduced chromatin binding in this study. This raises the question as what could be the contribution of the S70A or S70D substitution to the chromatin binding phenotypes shown by the H-N19D or H-N19A holocomplexes. This is not discussed in the manuscript, and the authors do not cite this earlier work (PMID: 21633354) in their manuscript. Is there any reason for that? I suggest it should be cited and discussed.
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Other minor comments
- Please provide a microscope image of DNA loop in Fig. 4D
- The authors do not compare the kleisin of condensin I with the one of condensin II with respect to the features tackled in this work. Given the different behaviours condensin I and II, such comparison could be informative for the readers.
- The authors do not reference the work of Robellet et al. Genes & Dev (2015) (PMID: 25691469) on the regulation of condensin binding in budding yeast by an SMC4 phospho-tail. I suggest that the analogy should be discussed.
- In the introduction section, lane 5, the sentence "Many if not all eukaryotic species have two different condensin complexes" appears inappropriate since budding and fission yeast cells possess a single condensin complexes, similar to condensin I in term of primary amino-acid sequence.
- page 4; typo: motif I and V bind to the SMC neck and the SMC4 cap regions, respectively. Should read SMC2 neck.
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Are the data and the methods presented in such a way that they can be reproduced? YES
- Are the experiments adequately replicated and statistical analysis adequate? YES
- Are prior studies referenced appropriately? Not all of them (see above)
- Are the text and figures clear and accurate? YES
Referees cross-commenting
I consider the comments from all reviewers as helpful for the authors.
Significance
Summary
Condensins are genome organisers of the family of SMC ATPase complexes and are best characterized as the drivers of mitotic chromosome assembly (condensation). It is acknowledged that condensins shape mitotic chromosomes by massively associating with DNA upon mitotic entry (loading step) and by folding chromatin fibres into arrays of loops, most likely through an ATP-dependent extrusion of DNA into loops, as seen in vitro. What remains unclear, however, are the mechanisms by which condensins load onto DNA and fold it into arrays of loops in vivo, and how these reactions are coupled with the cell cycle, i.e. restricted mostly to mitosis. Condensins are ring shaped pentamers that change conformation upon ATP-hydrolysis. In vitro studies suggest that condensins bind DNA via ATP-hydrolysis-independent, direct electrostatic contacts between condensin subunits and DNA. Such electrostatic contacts are salt-sensitive in in-vitro assays. Upon ATP-hydrolysis, condensins engage into an additional mode of binding that is resistant to high salt concentration and likely to be topological in nature. Both modes of association are necessary to form DNA loops. Vertebrates possess two types of condensin complexes, condensin I and II, each composed of a same SMC2-SMC4 ATPase core but associated with two different sets of three non-SMC subunits; a kleisin and two HEAT-repeat proteins. Condensin II is nuclear during interphase and stably binds chromatin upon mitotic entry, while condensin I is located in the cytoplasm during interphase and binds chromatin in a dynamic manner upon nuclear envelope breakdown. How the spatiotemporal control of condensin I and II is achieved remains poorly understood. Previous studies have shown that the phosphorylation of condensin I by mitotic kinases, such as CDK1, Aurora-B and Polo, play a positive role in its binding to chromatin and/or its functioning, but the underlying mechanisms remain to be characterised. In this manuscript, Shoji Tane and colleagues provide good evidence that the N-terminal tail of the human kleisin subunit of condensin I, hCAP-H, serves as a regulatory module for the cell-cycle control of condensin I binding to chromatin and chromosome shaping activity. The authors clearly show that the N-tail of CAP-H hinders the binding of condensin I to chromatin in xenopus egg extracts and, using in vitro assays, that the N-tail also hinders the topological association of condensin I with DNA and its loop extrusion activity. The authors provide additional data suggesting that the phosphorylation of the N-tail of CAP-H, in mitosis, relieves its inhibitory effect on condensin I binding. Based on their findings, Tane et al. propose a model suggesting that the N-terminal tail of CAP-H constitutes a gate keeper that maintains condensin-rings in a closed conformation that is unfavourable for topological binding to DNA, and whose locking effect is relieved in mitosis by phosphorylation.
Taken as a whole, this work has the potential to reveal a molecular basis for the cell cycle regulation of condensin I in vertebrate cells and as such to significantly improve our understanding of the integrated functioning condensin I. The characterisation of the inhibitory effect of the N-tail on condensin binding to chromatin and to naked DNA in vitro is well described, the data are clear and robust and the results convincing. On the other hand, some of the data on the phospho-regulation appear to me as are more debatable and I think that some of the results described here deserve to be discussed in the context of previous works. Finally, I see no data in the manuscript that directly supports the mechanistic model proposed by the authors, while it seems to me that such model could have been easily tested exprimentally. Thus, I suggest that Tane and colleagues should perform a couple of relatively easy experiments to strengthen their claims and that a few omitted prior studies on the topic should be referenced and discussed.
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Reply to the reviewers
We really appreciate the reviewers’ insightful comments, which help improve the quality of this work. We have responded to the reviewers’ questions/comments point by point in the following text and made the corresponding changes in the revised manuscript. Lastly, we added one more figure (Fig. 7) with lineage tracing experiments demonstrating the conversion of id2a+ liver ductal cells to hepatocytes in extreme hepatocyte loss condition.
Reviewer #1 (Evidence, reproducibility and clarity):
Mi and Andersson describe a method for creating efficient 3' knock-ins in zebrafish using a combination of end-modified dsDNA and Cas9/gRNA RNPs. They tested their method on four genetic loci where they introduced Cre recombinase endogenously, and obtained high F0 mosaicism and germline transmission. The authors included fluorescent proteins with self-cleaving peptides to determine that endogenous expression patterns are observed. By crossing their knock-in Cre lines with lineage tracing reporter lines, the authors temporally traced lineage divergences in zebrafish liver and pancreas.
The authors should clarify the following points before I can recommend publication:
Overall, I suggest that the authors consider paring down their figures. Throughout the paper, multiple figure panels convey the same point but for different genes. Furthermore, many construct configurations are shown that are not used in the subsequent panels. For example, the mNeonGreen only (no Cre) constructs and the EGFP constructs are largely not used in downstream experiments. The authors could pick the important constructs and show the relevant data, and summarize all their other constructs in one supplementary figure. The authors also jump around in different parts of the paper with regards to using iCre or CreERT2 and ubi:Switch or ubi:CSHm. It's not clear to me why they're doing that? It makes the paper hard to follow. For example, why use iCre - it's not temporal if I understand correctly (and I'm not sure what improved Cre is - could they reference a paper and include a small explanation) so CreERT2 seems suitable especially for their temporal lineage tracing experiments. Why not limit the description to CreERT2 in the main text/figures? Also, isn't ubi:Switch and ubi:CSHm pretty similar except the latter is nuclear mCherry due to H2B? Why not only focus on ubi:CSHm experiments? I found the paper to be unnecessarily long and think it would benefit from editing to describe the most important concepts and experiments.
Response: Thank you for your constructive and helpful comments. We do agree that sometimes the schematic constructs seem redundant. This is because the krt4, nkx6.1, and id2a genes have similar gRNA targeting sites (all spanning over the stop codon). However, we prefer to keep these schematic constructs as we have all the statistical results showing the knock-in efficiency in the subsequent figure panels. Such layout can allow readers to make comparisons and better understand the efficacy of this method. However, combined with the comments from the second reviewer, we indeed need to add more detailed information, including the sequence and the length of the short left and right homologous arms in the schematics, to enable the readers to follow this strategy more easily. Meanwhile, we added a new supplementary figure with the sequences of the long left and right homologous arms, as well as the genetic cassettes/point mutations for krt92 knock-in (Figure EV1).
As for the color switch lines we used, we appreciate your comments and replaced Fig. 5E-G with new fluorescent images using zebrafish larvae carrying the ubb:CSHm transgene. For most of the lineage tracing experiments in this study, we used Tg(ubb:CSHm) as the H2BmCherry is more stable, located in the nucleus, and the fluorescence intensity is stronger than in Tg(ubb:Switch). However, for the lineage tracing experiments in the liver injury model, we believe that Tg(ubb:switch) is a better option than Tg(ubb:CSHm). In the absence of a hepatocyte specific far-red reporter line, we can distinguish the hepatocytes derived from the id2a+ origin using the Tg(ubb:Switch) line, as the cells with Cre recombination express mCherry in the cytoplasm; i.e. we can tell the cell types based on the cell morphology in combination with the ductal anti-vasnb staining. This strategy was previously used by Dr. Donghun Shin’s group in their 2014 Gastroenterology paper (Figure 4B, DOI: 10.1053/j.gastro.2013.10.019). Therefore, we still kept the ubb:switch in the Fig. 1F schematic, and we have elaborated on why we chose Tg(ubb:switch) line for the id2a+ cell conversion experiments in Fig. 7 and Figure EV14.
The iCre we used is a codon-improved Cre (iCre). The original cDNA sequence was from pDIRE (Addgene plasmid #26745; provided by Dr. Rolf Zeller, University of Basel) (Osterwalder et al., 2010).
At the beginning of this project, we actually didn’t know whether there were any differences between iCre and CreERT2 in labelling of the cells of interest. Here, using both the iCre and CreERT2 lines, we for the first time, formally show the developmental lineage path of nkx6.1-expressing cells in the zebrafish pancreas. Our data suggested that the early nkx6.1-expressing cells are multipotent pancreatic progenitors giving rise to all three major cell types in the pancreas (endocrine, ductal and acinar cells, shown by nkx6.1 knock-in iCre) and gradually the nkx6.1-expressing cells become restricted in the ductal/endocrine lineages (shown by the nkx6.1 knock-in CreERT2 treated with 4-OHT at different timepoints). In addition, we also aim to use these knock-in lines for multiple studies in which we need to perform many quantitative experiments. As expected, we are unable to reach 100% labeling using the knock-in CreERT2 lines, even if we treated the larvae with very high concentration of 4-OHT over a long period of time. This means that the CreERT2 induced recombination will introduce more variation for quantitative experiments (for instance, the number of regenerated beta-cells from the ductal origin). As we were quite confident with the efficiency of this knock-in strategy, we decided to make both iCre and CreERT2 lines in krt4, nkx6.1, and id2a locus and just observe how they performed. We often use iCre knock-in lines for lineage tracing experiments, because the iCre lines reach near 100% labeling efficiency. Such iCre lines are particularly useful if they only label terminally differentiated cell types. Thus, the near 100% labeling efficiency in iCre lines can be of great help for initial experiments, which later can be confirmed by temporal labeling using CreERT2 lines.
- Could the authors describe the purpose of the 5'AmC6 modification earlier in the paper? I didn't see much text about it until the discussion. It seems that the speculation is that it provides end protection and prevents degradation (based on in vitro studies in human). This should be inserted into the introduction as a reader might be wondering about this and won't find an answer until near the end. Also, is this the first in vivo use of this modification for knock-ins? If so, that should be highlighted in the text.
Response: This is a helpful comment. In the revised manuscript, we elaborate more on why we chose 5'AmC6 modification in our donors. To our knowledge, this is the first time this 5’ modification is used in vivo, however, bulky 5’modification (5'Biotin - 5x phosphorothioate bonds) has been used in medaka (DOI: https://doi.org/10.7554/eLife.39468.001, 2018 Elife, as we previously referenced). The cell division rate is much faster in zebrafish embryos compared with medaka embryos during early development, so we speculate that such modification might be of more importance in zebrafish to achieve early integration. Another advantage is that the 5'AmC6 modification is commercially available, allowing researchers to prepare the donor dsDNA in a handy fashion. We have now expanded on these details and advantages in the introduction.
- The authors do not show any sequencing data confirming that their insert was knocked-in as designed with no disruption to the immediate upstream and downstream endogenous sequences. Can they sequence the loci to confirm?
Response: This is indeed a question we frequently get – thank you for making us relay this information more clearly! We have put the raw Sanger sequencing data in a public repository (mentioned in the Data Availability section), and included the sequencing primers in the method paragraph. Now we also refer to this data in the discussion section in conjunction to highlighting that the integrations were correctly placed in the loci. If you think there are better ways to show the sequencing results, please let us know.
- I found the descriptions of the long and short HA to be confusing when describing the results, especially since the first tested gene krt92 only has long and all subsequent ones are short. The discussion made it more clear that short HA is more efficient and applicable when gRNAs span the stop codon. Perhaps that wasn't possible with krt92, but the authors could prevent the confusion by clearly stating the design requirements of long and short HA and that they wanted to test which is more efficient before starting to describe the data. I also didn't see a description of what the length difference between long and short HA is? How short is short HA?
Response: This is a great question that is well worth discussing. In the revised manuscript, we changed the order in which the parts are described, with nkx6.1 knock-in in front of krt4 knock-in. Here we explain why we would like to do that:
At the beginning of this project, we did not know if the 5’ modified dsDNA could be an effective donor. To test our hypothesis, we chose the krt92 gene as our first target, as this is a keratin protein and expressed in the epithelial cells. We can easily detect the fluorescence in the epithelial cells (most notably in the skin), which allow us to sort the F0 mosaic embryos with high percentage of integration. Notably, from our experience, the most difficult part of the knock-in method is the sorting step (usually performed during 1-3 dpf). This is because the fluorescence signal is highly dependent on the endogenous gene expression level and is usually dimmer with an overall integration efficiency that is lower compared to canonical transgenesis. Therefore, we thought that targeting an epithelial cell marker would be informative and help us to evaluate the validity and reproducibility of the method. If it worked, then we could move on targeting genes expressing in more restricted tissues or cell types. For krt92 gene, the gRNA targets the region upstream of the stop codon. To prevent the cleavage of the donor template, we had to introduce several point mutations and at the same time keep the amino acid sequence intact. However, such mutations can restrict the knock-in and lower the integration efficiency when using shorter arms (due to the sequence mismatch).
After we managed to make the krt92 knock-in, our next question was, what about using a gRNA spanning over the stop codon region? In this way, we don’t need to introduce point mutations on neither the left nor the right homologous arm. Also, for the purpose of our biological study, the nkx6.1 were on top of our gene list for lineage tracing experiments and we luckily identified that there is very good gRNA targeting this locus. After we successfully made the nkx6.1 knock-in, we were thinking that we could simplify the protocol even further, i.e. switching to short homologous arms so that we can prepare the donor by a one-step PCR instead of making complicated constructs. We tested that hypothesis in nkx6.1, krt4, and id2a sites and obtained very promising efficiency. Also, we did some further testing with dsDNA without the 5’ modifications and showed that the 5’ modifications indeed greatly increased integration efficiency. Therefore, although the short homologous arm method is a highlight here, we also point out that it was not planned from the beginning. In the revised manuscripts, we want to convey our method in a logical way and show how we modify the method in a step-by-step fashion.
Moreover, with regards to the comments from the second reviewer, we now added the length of the homologous arms as well as the mutation site on the schematics. We chose short homologous arm because in previous literature it was suggested that short homologous arms (36-48 bp, which we now write out in both the results and the methods) can promote microhomology-mediated end joining (doi: 10.1096/fj.201800077RR). We also noticed that the recent Geneweld method (DOI: 10.7554/eLife.53968) also adheres to a similar length for homology mediated integration. In this study, HAs even shorter than 36 bp also perform well.
- The authors state that they could not use in situs to confirm krt92 endogenous and knock-in expression overlap, but rather say that they match based on data from an intestine scRNA-seq dataset. Can they elaborate on this? Which clusters/cell types show overlap? Furthermore, is there any krt92:GFP transgenic line that can be used as a reference for expression as well? This point is also applicable for krt4 described in Fig.2
Response: We appreciated this point. In the beginning, we contacted Molecular Instruments to synthesize krt92 HCR3.0 in situ hybridization probes. However, the technical staff there told us that they are unable to make specific probes due to high sequence similarity to other keratin protein families. We can see that the sequence similarity mostly occurs in the middle of krt92 genes, and the HCR3.0 probes rely on a probe set (preferably 20-30 probes with different sequences) to target the mRNA.
The scRNA-seq data that we referenced are from 10X platform, which is based on a 3’enrichment methodology. The reads mapping to krt92 genes are mostly located on the 3’ end. This is good as there is much less similarity to other cytoskeleton genes in the 3’ end of the gene. Unfortunately, there is no krt92 transgenic lines available, so we relied on the single-cell data to correlate expression patterns in this case.
There are two zebrafish intestine single-cell data sets available, with the following links:
We can see that krt92 is widely expressed in different types of intestinal epithelial cells (absorptive enterocytes, secretory enteroendocrine/goblet cells and ionocyte).
For the krt4 gene, we now added the HCR3.0 in situ hybridization and immunofluorescence for both krt4 knock-in EGFP-t2a-CreERT2 lines and the Tg(krt4:EGFP-rpl10a) transgenic line (a construct from Anna Huttenlocher, https://www.addgene.org/128839/, which has been widely used to label skin cells). The results are shown in Figure EV9. We show that krt4 has very high expression in the intestinal bulb and hindgut based on the HCR3.0 in situ. The Immunofluorescence of the krt4 knock-in fully recapitulate the krt4 expression pattern in the intestine, while there is almost no fluorescence signal in Tg(krt4:EGFP-Mmu.Rpl10a). We believe this is another advantage of using the knock-in method, over transgenics, for cellular labeling and lineage tracing. Classical transgenics often rely on short promoters of the proximal/enhancer region upstream of ATG with various length (arbitrarily or based on clues from motif analysis/DNA methylation sites). However, different tissues/cell types tend to use different cis-_regulatory elements and the chromatin structure/enhancer-promoter loops might differ dramatically among different cell types. It is hard to predict the exact region of the regulatory sequences that is sufficient for driving the gene expression in a certain cell type. Thus, such reasoning consolidates with that our knock-in lines recapitulate the endogenous _krt4 gene expression. Therefore, we believe that the knock-in based genetic lineage tracing will become the standard in the zebrafish field, as theoretically it avoids both the lack of relevant expression and leakage problems of transgenics.
- I think Figure 2A needs the dotted lines on the last construct to be fixed (points to p2A)
Response: Thank you for noticing! This was due to a bug in the IBS software, and we changed it manually using Adobe Illustrator in the revised manuscript.
- There are a few instances where the authors describe performing 4-OHT treatment for long period (e.g. over a 20 hour or 24 hour period). Is fresh 4-OHT added after a certain amount of time or is it a one-time addition? Is such long periods of 4-OHT required or has maximal recombination already occurred within a few hours after addition of 4-OHT?
Response: For 4-OHT treatment, we referred to the method described by Dr. Christian Mosimann (DOI: 10.1371/journal.pone.0152989). We actually tried different conditions (dosage, duration, refresh or not). This is particularly important for the knock-in CreERT lines because the level of CreERT2 is highly dependent upon the endogenous gene expression level. In our case, the nkx6.1 and id2a are transcriptional regulators and relatively lowly expressed compared with structural proteins. We maximized the labeling efficiency by using the highest concentration and longest duration suggested for 4-OHT treatment. The 4-OHT was stored in -20 ℃ and it would become less effective after 30 days of storage. Therefore, we first incubated the 4-OHT in 65 ℃ for 10 min (as recommended by Dr. Christian Mosimann) in order to convert it to a bioactive form. Next, we treated the zebrafish embryos with 4-OHT using a final concentration of 20 μM for 24 hours. We didn’t refresh the 4-OHT since there was no significant difference compared with a one-time addition. Moreover, using higher dosage or longer treatment time can lead to less survival and increased deformity rate. 20 μM 4-OHT treatment for shorter time periods (6 or 12 hours) can cause high labeling variability (some larvae have good labeling while others not). In the end, after several rounds of experiments, we settled on 20 μM 4-OHT treatment for 24 hours as it can reach the highest labeling efficiency, lower variability, and good survival.
- For Figures 4-6 where confocal images of lineage tracing experiments are shown, there is no indication of how many times the experiments were repeated, how many sections were images, how many animals used, how many cells counted. All of this information should be included in the figure legends and plots should be added showing quantification and statistical analysis (where appropriate).
Response: The reviewer makes a good point and we have now added the number of larvae used and statistical results for the quantitative experiments. The quantification of experiments in Figure 3E-H (originally Figure 4E-H) are shown in Figure EV6D using box/dotplot. We randomly selected 3 secondary islets of different sizes (large, middle, and small) from each juvenile fish (n=5) and pooled the number of mCherry/ins double positive cells and ins positive cells together. The quantification of the lineage-tracing efficiency in the experiments in Figure 6 are shown in Figure EV13.
- Figure 4 C, C' - I'm not sure what to look for. Is the message that there is no Cherry positive cells that are vasnb negative when labelling is done at 8 somite? But the vasnb positive cells that are also Cherry positive remain? The vasnb staining seems much weaker/harder to see in C C' compared to B, B'. As mentioned above, these data should be quantified and statistical significance indicated.
Response: Thank you for pointing this out; the second reviewer made a similar point. We redid the experiments using zebrafish larvae carrying the ptf1α:EGFP transgene to indicate the acinar cells (Figure 3B-D, Figure EV4G). We also quantified the results and performed statistical testing.
- I recommend the authors include a short section in the discussion comparing the efficiency of their method to other knock-in strategies used in zebrafish. This is an important claim of the paper yet it is not clear how much better it is (if at all) in terms of frequency of F0 mosaicism and identification of founders relative to other methods. I do appreciate the relative simplicity of the molecular steps of construct design/generation.
Response: This is indeed important. It is also tricky since we are unable to make head-to-head comparisons between different methods as we are targeting different genetic loci and do not have the other methods up and running in our lab. However, the general comparison is based on the statistics shown in the hallmark papers describing these other methods, regardless of which genes were selected for targeting. In the discussion, we added a list of points that are novel/improved with our method versus previous ones, including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product. We believe this is critical for the field of developmental biology, regenerative medicine, and disease modeling in zebrafish – and perhaps a similar 3’ knock-in based lineage-tracing method can become commonly used to delineate the cell differentiation and plasticity during homeostatic and diseased conditions in additional organisms.
Reviewer #1 (Significance):
Overall, the study contributes a new knock-in strategy in zebrafish that appears to be more user-friendly and results in high germline transmission. The authors also identify nkx6.1+ ductal cells as progenitors of endocrine cells in the pancreas highlighting the biological applications of their method. I think this study represents an important advancement in zebrafish genetics and will have future impact in lineage tracing during development, regeneration, and disease.
Reviewer #2 (Evidence, reproducibility and clarity):
Summary:
Here, the authors present a strategy where they performed knock-in at the level of the STOP codon, taking care of not perturbing the coding region. They integrate cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides.
The cassettes are done by PCR with primers with 5' AmC6 modifications and they test short (36 to 46 bp) or long homologous arms (~950bp). For nkx6.1 gene, they observed a dramatic increase of recombination efficiency when injecting the donors with short Homology arms compared to long arms suggesting that short arms could be used. Indeed, short arms used with krt4 and id2a allow them to obtain K.I lines.
The techniques described here look promising. Indeed, even if the proportion of F0 showing adequate reporter expression is low (usually about 2%), the percentages of founders among these mosaic F0 were quite high (between 50% and 100%). And this is the most important aspect as it is usually the most time-consuming aspect of the work.
Major comment:
The authors claim that the knock-in lines can precisely reflect the endogenous gene expression, as visualized by optional fluorescent proteins. But are the authors sure that the integration of the cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides, will not affect the level of expression of the targeted genes . Indeed, it has been shown that sometimes self-cleavable peptides could affect the expression of the genes of the cassette like for example in this reference ([https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034980]. Therefore it is important that the authors check whether the cassette affect the level of expression of the targeted gene if they want to claim that the knock-in lines precisely reflect the endogenous gene expression.
Response: Thank you for your insightful comments. With regards to the endogenous gene expression, we now use qPCR for further validation. We added the qPCR results to the supplement material (Figure EV15) in the revised manuscript. In brief, we pooled 4 larvae in one tube per biological replicate and have 4 biological replicates for each knock-in line. We didn’t see a significant change in the endogenous expression for any gene. In addition, we have grown up homozygous knock-in lines to adulthood and they are fertile without any overt phenotype.
The highlighted reference is dealing with a cardiomyocyte specific transgenic line, and we assume figure 3-Supplementary figure 1 is what the reviewer is referring to. The altered level of erbb2 expression might be due to the experimental conditions (no treatment or 3 days post treatment). Also, it is possible multiple transgenic insertions occur, as well as gene silencing at some insertion sites. However, such issues would not present, or very limited, with knock-in methods.
Minor comments:
General points:
I believed that the authors should improve the presentation of their data. Indeed, based on what they present, it would be impossible for me to reproduce their technique. Indeed, it is not clear at all how they design the short and long arm, where they are exactly located, which mutations they have done (for fig1), where is located the guide RNA compared to the STOP codon and the HA arms. Graphics that exactly place all these sequences are absolutely required to understand the strategy used and should be placed in figure 1, 2, 3 and 4.
Response: Thank you for these comments. In the revised version, we added the sequence information of the short homologous arms in each of the schematics. As for the krt92 gene, we added the sequence information in the first supplement results (Figure EV1) with the genetic cassettes and point mutation information. We list all the primer information in the methods. Also, we have uploaded our vector templates in the public repository (as listed in the Data availability section). Lastly, we added a key resource table in the supplement file with all the detailed information of reagents for the ease of reproducibility (including all the primers sequences used). We are also willing to share our constructs with the scientific community upon request.
Specific points:
Introduction:
"In zebrafish, the NHEJ-mediated methods have been intensively investigated in 5'knock-in upstream of ATG using donor plasmid containing in vivo linearization site flanking the insertion sequences (11,12,17-20). The 3' knock-in method has also been examined using circular plasmid as the donor with either long or short homologous arms (HAs) flanked by in vivo linearization sites (14, 21-23). Recently, intron-based and exon-based knock-in approaches have remarkably expanded the knock-in toolbox by targeting genetic loci beyond the 5' or 3' end (8-10,13,24-26)."<br /> This part should be explained better in order that the readers could really understand the differences between these old studies and this new one. And really insist on what is the novelty of their technique.
Response: Good points. In the revised version, we elaborated more on the previous discoveries, the major challenges, the knowledge gap in zebrafish knock-in methodology, and what is novel and improved with our new technique. Please, see clarifications and the expanded text in both the introduction and discussion.
Results:
Page 4: To my opinion, the first paragraph should be removed and the technique directly explained based on krt92 strategy as this paragraph does not allow to understand the technique. As indicated above, figure 1 should indicate more clearly the location of the long arms and which mutations they have done and where is located the guide RNA.
Figure 1G: The expression in the skin is far from obvious and the image should be improved (for example with some inset).
Response: Thank you for the comments. We added a new supplementary figure (Figure EV1) and show the sequences of left and right homologous arms, the genetic cassettes, as well as the point mutations with different background color highlight. We added the insets to show the magnified regions of interest. Also, we added the images from the fluorescent microscope used for sorting, to show the EGFP signals in live zebrafish embryos (Figure EV2D and Figure EV8D).
Figure 3E: The authors say that "cells expressing nkx6.1 (displayed by the green fluorescence) were located on the ventral side of the spinal cord whereas H2BmCherry positive cells, which include all the progenies of nkx6.1+ cells after the iCre recombination, resided in both the ventral and dorsal parts of spinal cord". This differential expression in the spinal cord is not obvious and a more closer view should be provided.
Response: Thank you for the comment. First, we changed the order and now describe all nkx6.1 content in Figure 2 and 3 and the krt4 content in Figure 4. We added insets to show the magnified regions and better display the expression pattern of the two fluorescence proteins in Figure 2E-G. One can now clearly see from the magnified insets that the green signals driven by the endogenous nkx6.1 gene are present in the ventral part of the spinal cord, while the red signals are present in both the ventral and the dorsal side of the spinal cord.
Fig S4H: The authors say that" using lineage tracing, we could trace back all three major cell types in the pancreas (acinar, ductal and endocrine cells) to nkx6.1 lineage (Figure 3H-H',Supplementary Figure S4G, H)". While this is obvious for endocrine, the colocalisation with ela3l:GFP is not obvious and the figure should be improved.
Response: This is a very good point, and the first reviewer gave similar suggestions. In the revised version (shown in Figure EV4H and I), we added the insets to show the magnified regions to better display the expression pattern of two fluorescence proteins. The ela3l reporter line is using a short promoter to drive the expression of H2B-EGFP (doi: 10.1242/dmm.026633). However, this short promoter cannot reach 100% labeling of acinar cells, so we also use the ptf1α:EGFP transgene for further validation (new Figure EV4G). Both transgenic reporter lines showed many EGFP and mCherry double-positive cells, indicating that these acinar cells are derived from a nkx6.1-expressing origin. Here we did not use the anti-GFP antibody, as our color switch lines contains CFP and anti-GFP antibody can also recognize CFP. However, the GFP signal is strong enough to show the expression. We hope the additional experiments and insets clarifies this point.
Page 8: the authors say that "The immunostaining at 6 dpf showed that both intrapancreatic ductal cells and a portion of acinar cells can be lineage traced when the 4-OHT treatment started at the 6 somite stage (Figure 4B and B'). The identification of the acinar cells has been done based on the absence of the ductal marker vasnb. To trace efficiently the acinar cells, this should be done with an acinar marker.
Response: Another good point also mentioned by reviewer one. We redid the analyses using zebrafish larvae containing the ptf1α:EGFP transgene to indicate the acinar cells and the co-expression pattern with the lineage-tracing (the data is shown in new Figure 3B-D).
Reviewer #2 (Significance):
I do not have enough expertise in the KI field to evaluate whether this strategy is really novel and as mentioned above, the authors should better explain what is really the novelty of their strategy.
Response: In our answers to the comments of the first reviewer, we elaborated more on the points that are novel/improved with our method vs previous methods, as reiterated here:
“…including that: 1) we simplify the knock-in methodology circumventing complicated molecular cloning; 2) we have very high germline transmission rate, which means that one morning of injection is often enough to get a founder; and the expression of fluorescence proteins avoids tedious work in identifying founders, which also saves a lot of space in the fish facility; 3) our lines can be applied for multiple utilities; 4) the method does not disrupt the endogenous gene product.”
Moreover, the first reviewer asked about the difference between the krt4 knock-in and krt4 transgenics, and based on the in situ data, we showed that our krt4 knock-in can fully recapitulate the endogenous gene expression, while the krt4 transgenics can hardly label the intestinal bulb and hindgut. This might be due to that different tissues/cell types may depend on different _cis-_regulatory elements to drive the gene expression. The chromatin structure and the enhancer/promoter loop might also differ dramatically among different tissues. Therefore, the transgenics might be useful for one type of cells, while they might be not useful at all for other cell types. In the future, we believe that, similar to the mouse field, the 3’ knock-in based lineage tracing methods might become the standard method in the zebrafish field, to delineate cellular differentiation and plasticity during homeostatic and diseased conditions.
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Referee #2
Evidence, reproducibility and clarity
Summary:
Here, the authors present a strategy where they performed knock-in at the level of the STOP codon, taking care of not perturbing the coding region. They integrate cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides.<br /> The cassettes are done by PCR with primers with 5' AmC6 modifications and they test short (36 to 46 bp) or long homologous arms (~950bp). For nkx6.1 gene, they observed a dramatic increase of recombination efficiency when injecting the donors with short Homology arms compared to long arms suggesting that short arms could be used. Indeed, short arms used with krt4 and id2a allow them to obtain K.I lines.<br /> The techniques described here look promising. Indeed, even if the proportion of F0 showing adequate reporter expression is low (usually about 2%), the percentages of founders among these mosaic F0 were quite high ( between 50% and 100%). And this is the most important aspect as it is usually the most time-consuming aspect of the work
Major comment:
The authors claim that the knock-in lines can precisely reflect the endogenous gene expression, as visualized by optional fluorescent proteins. But are the authors sure that the integration of the cassettes coding for fluorescence protein and Cre recombinase, which are separated from the endogenous gene and each other by two self-cleavable peptides, will not affect the level of expression of the targeted genes . Indeed, it has been shown that sometimes self-cleavable peptides could affect the expression of the genes of the cassette like for example in this reference (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034980/) . Therefore it is important that the authors check whether the cassette affect the level of expression of the targeted gene if they want to to claim that the knock-in lines precisely reflect the endogenous gene expression
Minor comments:
General points :
I believed that the authors should improve the presentation of their data. Indeed, based on what they present, it would be impossible for me to reproduce their technique. Indeed, it is not clear at all how they design the short and long arm, where they are exactly located, which mutations they have done (for fig1), where is located the guide RNA compared to the STOP codon and the HA arms. Graphics that exactly place all these sequences are absolutely required to understand the strategy used and should be placed in figure 1, 2, 3 and 4.
Specific points :
Introduction :
"In zebrafish, the NHEJ-mediated methods have been intensively investigated in 5'knock-in upstream of ATG using donor plasmid containing in vivo linearization site flanking the insertion sequences (11,12,17-20). The 3' knock-in method has also been examined using circular plasmid as the donor with either long or short homologous arms (HAs) flanked by in vivo linearization sites (14, 21-23). Recently, intron-based and exon-based knock-in approaches have remarkably expanded the knock-in toolbox by targeting genetic loci beyond the 5' or 3' end (8-10,13,24-26)."<br /> This part should be explained better in order that the readers could really understand the differences between these old studies and this new one. And really insist on what is the novelty of their technique.
Results :
Page 4 : To my opinion, the first paragraph should be removed and the technique directly explained based on krt92 strategy as this paragraph does not allow to understand the technique. As indicated above, figure 1 should indicate more clearly the location of the long arms and which mutations they have done and where is located the guide RNA.
Figure 1G : The expression in the skin is far from obvious and the image should be improved (for example with some inset) .
Figure 3E : The authors say that "cells expressing nkx6.1 (displayed by the green fluorescence) were located on the ventral side of the spinal cord whereas H2BmCherry positive cells, which include all the progenies of nkx6.1+ cells after the iCre recombination, resided in both the ventral and dorsal parts of spinal cord". This differential expression in the spinal cord is not obvious and a more closer view should be provided.
Fig S4H: The authors say that" using lineage tracing, we could trace back all three major cell types in the pancreas (acinar, ductal and endocrine cells) to nkx6.1 lineage (Figure 3H-H',Supplementary Figure S4G, H)". While this is obvious for endocrine, the colocalisation with ela3l:GFP is not obvious and the figure should be improved.
Page 8: the authors say that "The immunostaining at 6 dpf showed that both intrapancreatic ductal cells and a portion of acinar cells can be lineage traced when the 4-OHT treatment started at the 6 somite stage (Figure 4B and B'). The identification of the acinar cells has been done based on the absence of the ductal marker vasnb. To trace efficiently the acinar cells, this should be done with an acinar marker.
Significance
I do not have enough expertise in the KI field to evaluate whether this strategy is really novel and as mentioned above, the authors should better explain what is really the novelty of their strategy.
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Referee #1
Evidence, reproducibility and clarity
Mi and Andersson describe a method for creating efficient 3' knock-ins in zebrafish using a combination of end-modified dsDNA and Cas9/gRNA RNPs. They tested their method on four genetic loci where they introduced Cre recombinase endogenously, and obtained high F0 mosaicism and germline transmission. The authors included fluorescent proteins with self-cleaving peptides to determine that endogenous expression patterns are observed. By crossing their knock-in Cre lines with lineage tracing reporter lines, the authors temporally traced lineage divergences in zebrafish liver and pancreas.
The authors should clarify the following points before I can recommend publication:
- Overall, I suggest that the authors consider paring down their figures. Throughout the paper, multiple figure panels convey the same point but for different genes. Furthermore, many construct configurations are shown that are not used in the subsequent panels. For example, the mNeonGreen only (no Cre) constructs and the EGFP constructs are largely not used in downstream experiments. The authors could pick the important constructs and show the relevant data, and summarize all their other constructs in one supplementary figure. The authors also jump around in different parts of the paper with regards to using iCre or CreERT2 and ubi:Switch or ubi:CSHm. It's not clear to me why they're doing that? It makes the paper hard to follow. For example, why use iCre - it's not temporal if I understand correctly (and I'm not sure what improved Cre is - could they reference a paper and include a small explanation) so CreERT2 seems suitable especially for their temporal lineage tracing experiments. Why not limit the description to CreERT2 in the main text/figures? Also, isn't ubi:Switch and ubi:CSHm pretty similar except the latter is nuclear mCherry due to H2B? Why not only focus on ubi:CSHm experiments? I found the paper to be unnecessarily long and think it would benefit from editing to describe the most important concepts and experiments.
- Could the authors describe the purpose of the 5'AmC6 modification earlier in the paper? I didn't see much text about it until the discussion. It seems that the speculation is that it provides end protection and prevents degradation (based on in vitro studies in human). This should be inserted into the introduction as a reader might be wondering about this and won't find an answer until near the end. Also, is this the first in vivo use of this modification for knock-ins? If so, that should be highlighted in the text.
- The authors do not show any sequencing data confirming that their insert was knocked-in as designed with no disruption to the immediate upstream and downstream endogenous sequences. Can they sequence the loci to confirm?
- I found the descriptions of the long and short HA to be confusing when describing the results, especially since the first tested gene krt92 only has long and all subsequent ones are short. The discussion made it more clear that short HA is more efficient and applicable when gRNAs span the stop codon. Perhaps that wasn't possible with krt92, but the authors could prevent the confusion by clearly stating the design requirements of long and short HA and that they wanted to test which is more efficient before starting to describe the data. I also didn't see a description of what the length difference between long and short HA is? How short is short HA?
- The authors state that they could not use in situs to confirm krt92 endogenous and knock-in expression overlap, but rather say that they match based on data from an intestine scRNA-seq dataset. Can they elaborate on this? Which clusters/cell types show overlap? Furthermore, is there any krt92:GFP transgenic line that can be used as a reference for expression as well? This point is also applicable for krt4 described in Fig.2
- I think Figure 2A needs the dotted lines on the last construct to be fixed (points to p2A)
- There are a few instances where the authors describe performing 4-OHT treatment for long period (e.g. over a 20 hour or 24 hour period). Is fresh 4-OHT added after a certain amount of time or is it a one-time addition? Is such long periods of 4-OHT required or has maximal recombination already occurred within a few hours after addition of 4-OHT?
- For Figures 4-6 where confocal images of lineage tracing experiments are shown, there is no indication of how many times the experiments were repeated, how many sections were images, how many animals used, how many cells counted. All of this information should be included in the figure legends and plots should be added showing quantification and statistical analysis (where appropriate).
- Figure 4 C, C' - I'm not sure what to look for. Is the message that there is no Cherry positive cells that are vasnb negative when labelling is done at 8 somite? But the vasnb positive cells that are also Cherry positive remain? The vasnb staining seems much weaker/harder to see in C C' compared to B, B'. As mentioned above, these data should be quantified and statistical significance indicated.
- I recommend the authors include a short section in the discussion comparing the efficiency of their method to other knock-in strategies used in zebrafish. This is an important claim of the paper yet it is not clear how much better it is (if at all) in terms of frequency of F0 mosaicism and identification of founders relative to other methods. I do appreciate the relative simplicity of the molecular steps of construct design/generation.
Significance
Overall, the study contributes a new knock-in strategy in zebrafish that appears to be more user-friendly and results in high germline transmission. The authors also identify nkx6.1+ ductal cells as progenitors of endocrine cells in the pancreas highlighting the biological applications of their method. I think this study represents an important advancement in zebrafish genetics and will have future impact in lineage tracing during development, regeneration and disease.
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Reply to the reviewers
We thank the Reviewers for their comments. Below we have the Reviewers’ comments and our responses.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this work, the authors claim that their machine learning approach can be combined with a biophysical model to predictably engineer sensors. The concept is interesting, but there are many issues that must be addressed before considering its publication.
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It is surprising that their citations are too biased. They keep citing nonrelevant papers from several groups while omitting many key papers regarding genetic sensors and circuits in the field. Some can be justified (e.g., Voigt lab's reports), but others (e.g., reports on dynamic controllers too often) would not be relevant.
There are hundreds (possibly thousands) of papers that have been published on genetic sensors. Most of those papers report only qualitative results (e.g., genetic sensor implemented in a new host organism or demonstrated to sense a ligand of interest).
The purpose of this manuscript is to demonstrate methods for quantitative engineering of genetic sensors. Specifically, the manuscript is focused on quantitative tuning of the genetic sensor dose-response curve. So, in deciding which previous papers to cite, we chose several review articles (to cover the many, many qualitative results), any previous papers we could find that reported strategies for tuning the dose-response curve of genetic sensors (the Voigt lab’s reports and others), and any papers we could find that discussed reasons/applications for quantitative tuning of a genetic sensor dose-response curve (e.g., dynamic controllers).
We added a new paragraph to the beginning of the Results section to explain this focus on quantitative tuning (and to clearly state which statistic we use for assessing accuracy – see response to next comment; lines 72-83 in the revised manuscript).
We would also like to add more relevant citations as suggested by the reviewer, but that is difficult based on the reviewer’s comment, which just indicates that we have omitted many “key” papers. For the central focus of this manuscript, we think the “key” papers are those that describe methods to tune the dose-response curve of genetic sensors, and we have done our best to cite all of those that we could find. So, we ask the reviewer to please suggest some specific papers that they consider to be “key” that we should cite, or at least some more specific definition of what they think constitutes a “key” paper that should be cited.
It is very unclear which statistical analysis has been done for their work.
The main statistical metric used in the manuscript is the fold-accuracy. The fold-accuracy was defined in the previous version of the manuscript, but we agree that it could have been stated more clearly. So, we have moved the definition of fold-accuracy to the (new) first paragraph of the Results section, and identified it as “…the primary statistic we will use to assess different methods.” (line 77 of the revised manuscript)
There are many practical sensors for real applications, but their work focuses on IPTG-responsive sensors or circuits. I was wondering whether this work would have significant impacts on the field or the advancement of knowledge.
Similarly, it is questionable that their approach is generalizable.
Currently, there is only one published dataset that can be used for the methods described in this manuscript, for IPTG-responsive LacI variants.
However, previous work (cited in our manuscript) has shown that directed evolution can be used to qualitatively “improve” a wide range of genetic sensors beyond LacI. Furthermore, some of those previous studies used a single round of mutagenesis and libraries with diversity similar to the size of the LacI dataset (104 to 105 variants). Based on that, we think it is highly likely that our in silico selection approach will generalize to other sensor proteins.
With regard to the ML methods used in our manuscript, we showed in the initial publication describing the LANTERN method that the approach is generalizable to different types of proteins and protein functions (LacI sensor protein, GFP fluorescence protein, SARS Cov-2 spike-binding protein). So, we don’t see any reason to question the generalizability of that approach to other sensor proteins.
We have edited the Discussion section of the manuscript to include these points regarding the generalizability of our approach (lines 340-350 in the revised manuscript).
Due to the biased literature review, it is unclear to me whether this work is novel.
The majority of relevant literature on genetic sensor engineering is qualitative in nature and is not particularly comparable to the work here. We have tried to emphasize this in the introduction and discussion. We have searched the relevant literature extensively, and we have only found a small number of papers that describe quantitative methods to tune the dose-response of genetic sensors. Furthermore, there are only a few that contain any kind of quantitative assessment of that tuning. We have cited all of those papers and included specific discussions and comparisons between them and our results.
If the reviewer knows of any specific papers that we missed we would be happy to include them in our literature review.
I am unsure whether their correlation is sufficiently high.
This comment is too vague to address.
Again, we ask the reviewer for more specific information: What “correlation” are you referring to? And what is “sufficiently high”?
We have provided statistics on the accuracy of our methods, as discussed above.
Is EC50 the only important parameter? Or is it really relevant for real applications where the expression levels would change due to RBS changes, context effects, metabolic burdens, circuit topologies, etc.?
EC50 is not the only important parameter. That is why we also demonstrate the ability to quantitatively tune other aspects of the dose-response (e.g., G∞).
In any real application of genetic sensors, the EC50 will have to be engineered to have a quantitatively specified value (within some tolerance). So, yes, it really is relevant.
There is an important question about the effect of context however, and perhaps that is what the reviewer is really asking: If we engineer a genetic sensor that has a given EC50 in the context used for the large-scale measurement, will we be able to use that genetic sensor in a different context where, because of the change in context, its EC50 may be different?
This is one of the outstanding challenges in the field, to be able to predict the effect of a change in context. But for genetic sensors, there are several previous publications that demonstrate promising routes to quantitatively predict the effect of context on genetic sensor function.
So, we have added a paragraph to the Discussion section addressing this point and citing the relevant previous publications (lines 315-339 in the revised manuscript).
There are many reports on mutations or part-variants and their impacts on circuit behaviors. Those papers have not been cited. This is another omission.
As discussed in response to Comment 1, above, there are many hundreds of such papers. It would not be practical or appropriate for us to cite all of them. However, there are only a few that contain any kind of quantitative assessment of the predictability of mutational effects or of efforts to use mutations to engineer sensors to meet a quantitative specification. We have done our best to cite and discuss all of those. Again, if the reviewer knows of any specific additional papers that we should cite, please tell us.
CROSS-CONSULTATION COMMENTS
In general, I agree with the other reviewer. Its significance would be too incremental.
Reviewer #1 (Significance (Required)):
See above.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This paper proposes two approaches for forward-design of genetically encoded biosensors. Both methods rely on a large scale dataset published earlier by the authors in Mol Syst Biol, containing ~65k lacI sequences and their measured dose response curves. One approach, termed 'in silico selection', is proposed as a way to find variants of interest according to phenotypic traits such as the dynamic range and IC50 of the biosensor dose-response curve. The second approach uses machine learning to regress the dynamic range, IC50 and others from the lacI sequences themselves - the ML regressor can then be used to predict phenotypes of new variants not present in the original dataset. The ML algorithm has been published by the same authors in a recent PNAS paper.
The manuscript has serious flaws and seems too preliminary/incremental:
1) The 'in silico selection' method corresponds to a simple lookup table. This is a perfectly acceptable method for sequence design, but the attempt to portray this as a new method or 'multiobjective optimization' is highly misleading. Also, the analogy between 'in silico selection' and darwinian evolution or directed evolution are inappropriate, because both latter approaches rely on iterative selection through fitness optimization and randomization of variants. The 'in silico selection' approach in contrast is one-shot and does not use randomization.
We agree with some of the reviewer’s points here. In making the analogy to directed evolution, we wanted to give the reader a connection to something familiar, but the reviewer is correct that the analogy is imperfect. The “lookup table” description is much better, and probably a familiar idea to most readers. So, we edited the relevant paragraph to describe in vitro selection as the use of the large-scale dataset as a lookup table instead of making the analogy to directed evolution. We thank the reviewer for this suggestion.
However, we disagree with the reviewer with regard to “multi-objective optimization.” We clearly demonstrate in Figures 3 and 4 that we can simultaneously tune multiple aspects the dose-response curve to meet quantitative specifications. If the reviewer is aware of any previous publications that they think provide a better demonstration of multi-objective engineering of biological function, please let us know; we would like to cite those papers appropriately.
Also, the reviewer is incorrect in stating that our in silico selection approach does not use randomization. The randomization occurs as part of the large-scale measurement. This is clearly stated in the second paragraph of the Results section.
2) The ML approach is a minor extension to what they already published in PNAS 2022. One could imagine an extra figure in that paper would be able to contain all ML results in this new manuscript. A couple of comments about the actual method: a) it seems unlikely to work on sequences of lengths relevant to applications, because it relies on gaussian processes that are known to scale poorly in high dimensions. b) The notion of 'interpretable ML' is misleading and quite different to what people in interpretable AI understand. Moreover, the connection between the three latent variables, which provide the 'interpretability', and biophysical models seems to come from their earlier PNAS work and this specific dataset, but there is no indication that such connection exists in other cases. Although this is somewhat acknowledged in L192-195, the text tends to portray the connection with biophysical models as something generalizable.
The ML results presented in this manuscript are specifically aimed to quantitatively assess the accuracy of the ML predictions for the parameters of a genetic sensor dose-response curve. So, we think those results belong in the current manuscript.
The reviewer’s comment on Gaussian processes and dimensionality is clearly contradicted by the results presented in this manuscript and in our previous publication describing the ML method: The ML method works quite well for “sequences of lengths relevant to applications,” including LacI (360 amino acids), the SARS-Cov2 receptor binding domain (200 amino acids), and GFP (250 amino acids). The reason for this is that the Gaussian process is only applied on the low-dimensional latent space learned by the ML method.
The reviewer’s comment on “interpretable ML” is not relevant to this manuscript but is instead a criticism aimed at our previous publication on the ML method.
The generalizability of this approach is an open question. The same could be said for most other publications describing new methods, since most of those publications include demonstrations with only a small number of specific systems. After re-reading the relevant portions of the manuscript, we disagree with the reviewer’s suggestion that we have exaggerated the potential generalizability of the approach. For example, in the last sentence of the Results paragraph, we state, “Although imperfect, this initial test of linking an interpretable, data-driven ML model to a biophysical model to engineer genetic sensors shows promise…” And, in the Discussion section, “The use of interpretable ML modeling in conjunction with a biophysical model also has the potential to become a useful engineering approach… But more rigorous methods would be needed…”
Other comments:
3) There are quite a few reduntant figures, eg Figure 1 contains too many heatmaps of the same variables. Fig 2B and C are redundant as the contain the same information. Altogether figures feel bloated and could have been compressed much more.
We disagree. The sub-panels of Figure 1 show different 2-D projections of the multi-dimensional data that are relevant to specific aspects of the results in Figs. 2-4.
Admittedly, Fig 2C shows the residuals from Fig 2B, which is in some sense the “same information.” But it is quite common, in papers focused on quantitative results, to have one sub-panel showing a comparison between predicted and actual and a second sub-panel showing the residuals.
4) Fig 2A and 3A have problems: the blue & orange lines (Fig 2A) and blue & green lines (Fig 3A) have a kink just before the second dot from the left. Such kinks cannot have been produced by a Hill function. This kind of errors cast doubt on the overall legitimacy and reproducibility of the results.
The kinks in the curves are a consequence of the use of the “symmetrical log” scale on the x-axis, which allows the zero-IPTG and non-zero-IPTG data to be shown on the same plot while showing the non-zero-IPTG data on a logarithmic scale. That symmetrical log axis uses a log scale for large x values, and a linear scale for smaller x values. The kink appears at the transition between the log and linear scales. We have re-plotted all of the figures showing dose-response curves to move the log-linear transition to overlap with the axis break.
CROSS-CONSULTATION COMMENTS
I agree with the other reviewer's comments, particularly on the lack of statistical analyses.
See our response to Reviewers #1, comment 2, above.
Reviewer #2 (Significance (Required)):
The work addresses a timely subject but is too incremental.
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Referee #2
Evidence, reproducibility and clarity
This paper proposes two approaches for forward-design of genetically encoded biosensors. Both methods rely on a large scale dataset published earlier by the authors in Mol Syst Biol, containing ~65k lacI sequences and their measured dose response curves. One approach, termed 'in silico selection', is proposed as a way to find variants of interest according to phenotypic traits such as the dynamic range and IC50 of the biosensor dose-response curve. The second approach uses machine learning to regress the dynamic range, IC50 and others from the lacI sequences themselves - the ML regressor can then be used to predict phenotypes of new variants not present in the original dataset. The ML algorithm has been published by the same authors in a recent PNAS paper.
The manuscript has serious flaws and seems too preliminary/incremental:
- The 'in silico selection' method corresponds to a simple lookup table. This is a perfectly acceptable method for sequence design, but the attempt to portray this as a new method or 'multiobjective optimization' is highly misleading. Also, the analogy between 'in silico selection' and darwinian evolution or directed evolution are inappropriate, because both latter approaches rely on iterative selection through fitness optimization and randomization of variants. The 'in silico selection' approach in contrast is one-shot and does not use randomization.
- The ML approach is a minor extension to what they already published in PNAS 2022. One could imagine an extra figure in that paper would be able to contain all ML results in this new manuscript. A couple of comments about the actual method: a) it seems unlikely to work on sequences of lengths relevant to applications, because it relies on gaussian processes that are known to scale poorly in high dimensions. b) The notion of 'interpretable ML' is misleading and quite different to what people in interpretable AI understand. Moreover, the connection between the three latent variables, which provide the 'interpretability', and biophysical models seems to come from their earlier PNAS work and this specific dataset, but there is no indication that such connection exists in other cases. Although this is somewhat acknowledged in L192-195, the text tends to portray the connection with biophysical models as something generalizable.
Other comments:
- There are quite a few reduntant figures, eg Figure 1 contains too many heatmaps of the same variables. Fig 2B and C are redundant as the contain the same information. Altogether figures feel bloated and could have been compressed much more.
- Fig 2A and 3A have problems: the blue & orange lines (Fig 2A) and blue & green lines (Fig 3A) have a kink just before the second dot from the left. Such kinks cannot have been produced by a Hill function. This kind of errors cast doubt on the overall legitimacy and reproducibility of the results.
Referees cross-commenting
I agree with the other reviewer's comments, particularly on the lack of statistical analyses.
Significance
The work addresses a timely subject but is too incremental.
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Referee #1
Evidence, reproducibility and clarity
In this work, the authors claim that their machine learning approach can be combined with a biophysical model to predictably engineer sensors. The concept is interesting, but there are many issues that must be addressed before considering its publication.
- It is surprising that their citations are too biased. They keep citing nonrelevant papers from several groups while omitting many key papers regarding genetic sensors and circuits in the field. Some can be justified (e.g., Voigt lab's reports), but others (e.g., reports on dynamic controllers too often) would not be relevant.
- It is very unclear which statistical analysis has been done for their work.
- There are many practical sensors for real applications, but their work focuses on IPTG-responsive sensors or circuits. I was wondering whether this work would have significant impacts on the field or the advancement of knowledge.
- Similarly, it is questionable that their approach is generalizable.
- Due to the biased literature review, it is unclear to me whether this work is novel.
- I am unsure whether their correlation is sufficiently high.
- Is EC50 the only important parameter? Or is it really relevant for real applications where the expression levels would change due to RBS changes, context effects, metabolic burdens, circuit topologies, etc.?
- There are many reports on mutations or part-variants and their impacts on circuit behaviors. Those papers have not been cited. This is another omission.
Referees cross-commenting
In general, I agree with the other reviewer. Its significance would be too incremental.
Significance
See above.
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Referee #3
Evidence, reproducibility and clarity
This study aims to gain a better understanding of PDLCs and their associated cementum and alveolar bone. The study provides a very clear results for differential expression of Plap-1 and IBSP the periodontal fibroblast and associated cementoblasts and osteoblasts.
The most infesting is the generation of reporter mice for identification of Plap-1+ cells. The generation of this mice lined allowed then to gain insight to the regeneration of periodontium as well as heterogeneity in of Plap-1+ cells.
Minor issues:
- Many abbreviation in the papers have to be better defined. (Spp1, Bgn, Sparc, Col1a. also DN and DP in legends to Figure 3.
- Legends to all figure can be written more clearly.
- Statement in the result (line 27 and 28) cement oblasts and osteoblasts were aligned ..... should be eliminated as the figure 1A does not allow appreciation of such features. Also, the statement does not add anything to the manuscript and its results.
- The statement on Page 5 (line 1, 2) the protein distribution of Plap-1 needs to be described.
- Line 14 and 15 on page 5. It should be noted that very few/if any cells are co-expressing Ibsp and td-tomato. The number is so few that brings questions to the conclusion.
Major/important issues to be addressed:
- The authors have very nicely and clearly shown that Ibsp is expressed by cementoblasts and osteoblasts but not by PDL fibroblasts. Therefore, the lineage tracing experiments after PDL injury should be followed by examination of Ibsp in cementoblasts and osteoblasts originating from the Plap-1+ cells.
- It is also important to know what is the percentage of Plap-1+/Ly6a+ cells.
- The author should include a stronger statement for the possible role of Plap-1+/Ly6a+ cells (not Plap-1+ alone) as a source pf progenitors for periodontium.
Significance
by providing new markers and new transgenic animal model, the paper makes an important and significant contribution to the field
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Referee #2
Evidence, reproducibility and clarity
- The authors describe in a richly illustrated manuscript periodontal ligament associated protein-1 Plap-1 as a periodontal fibroblast (PDLC) associated molecule that has the possibility to differentiate both into cementoblasts lining the tooth surface and into osteoblasts lining the alveolar bone.
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In the introduction, please not only refer to higher expression of Plap-1 in certain tissues, but also refer to the function, as revealed by Sakashita et al (also on the periodontium/susceptibility to periodontitis). Apart from the fact that it is a 43 kDa ECM protein, subtype of the leucine-rich etc., it is also important to briefly sum-up -if scientific data allow - the function of the protein.
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Page 5, line 2: have the authors investigated Plap-1 in tissues other than the periodontal ligament? These experiments seem essential to demonstrate the uniqueness of Plap-1, possibly as a confirmation of the Sakashita et al. paper of 2021. It is always a good habit to confirm previous work in a next study.
- Page 5 on lineage tracing with Tomato: please spend a few lines on the essence of the experiment, either on page 5 or in the legend of Fig. 2, or both. You will thus keep the readers involved who are not familiar with lineage tracing.
- Page 6 and 7: the description of the protocol is very valuable. It is also important the cell numbers of the various cell types were described in great detail (Fig 3). So, authors have now used cells derived from extracted teeth, which is world-wide a sample of convenience. However, after extraction, half of the PDLCs are likely attached to the alveolar bone of the tooth socket. Have the authors ever considered to harvest these cells? In principle, and biologically, PDLC cells at the site of the alveolar bone could be the more osteogenic cells. The PDLC that are attached to tooth could in principle be quite different, being anti-osteogenic and anti-osteoclastogenesis-stimulating.
- Page 6, line 8: indicate what CD51+ cells are. In corresponding figure 3, explain the abbreviations in the X-axis in the legend.
- Page 7 line 1: The proerythroblasts in the PDL are a surprise to me! I assumed that the bone marrow would be the natural niche. Authors are also encouraged to highlight plasma cell specific RNAs in their atlas, since these are quite abundant in periodontitis lesions.
- In figure 4, its seems to me that the stromal cells in 4C are scattered more or less in 3 domains. This idea is strengthened when interpreting 4E Plap-1 and lbsp. Could the authors specify these domains?
- On Page 7: again for the not-so-informed reader: briefly, in the first sentence, describe the phenomenon of RNA velocity.
- Page 7, line 20: delete "were".
- In figure 5A it could be helpful to put the numbers in the figure as well.
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In figure 6G, it seems like that some osteocytes are positive, which means that they were derived from the TomatoRed cells within 7 days. That is quite remarkable and should gain some attention. Probably use a white arrow and specific mentioning in the legend.
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In the discussion, I miss a clear link and comparison with human periodontal ligament. Is all this mouse specific or are some of these aspects also present in the human periodontal ligament? One study comes to mind that has actually studied gene expression of Plap-1 etc. in PDLC and in alveolar bone derived cells: Loo-Kirana R, et al., Frontiers in Cell and Developmental Biology, 2021: DOI: 10.3389/fcell.2021.709408. But there is bound to be other studies as well. A brief mirroring of these findings with other studies would be in place.
CROSS-CONSULTATION COMMENTS
I have read and seen the comments of the other reviewers. They are more or less in line with mine, and I have nothing to add.
Significance
Authors identify Flap-1 postive cells as key cells contributing to stem cell ness of the periodontium. With advanced techniques using GFP and Tomato Rd mice they are able to show a kind of hierarchy in cell differentiation. They also describe the presence of all kind of cells in the peridoontal ligament as well as the capacity of the Plap-1 positive cells to contribute to regeneration. It is a very valuable addition to existing literature.
Audience: those, basic scientist but also dentists in general for whom the biology of the periodontal ligament is crucial.
My expertise: periodontal ligament specialist, but more the human part. I use PDL to study osteogenesis and osteoclastogenesis, in presence of bacterial products, inflammatory and anti-inflammatory reagents.
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Referee #1
Evidence, reproducibility and clarity
Comments:
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In the presented study the authors attempt to perform an in vivo characterisation of the cellular hierarchies and cellular dynamics of the periodontal ligament (PDL), a largely unknown compartment of the periodontal tissue which function is to support mammalian teeth. Periodontal diseases are one of the major causes of adult tooth loss, hence understanding the cellular dynamics of this compartment is of particular interest. To do this, the authors develop a novel traceable mouse line based on the Plap-1 marker, which they identify specifically labels periodontal fibroblasts. The developed Plap-1-GFP-2A-CreER mice are then crossed onto an inducible Rosa26-tdTomato to enable in vivo tracing of Plap-1 PDL fibroblasts. This tool is of significant relevance as it allows for the first time to analyse the cellular fate of the elusive populations that constitute PDL under normal homeostatic and regenerative conditions. This mouse model also enables the authors to sort the cells of interest (as marked by GFP) to perform single-cell RNA sequencing analysis, providing further knowledge on the cellular heterogeneity of PDL cells.
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The work presented in this article is of interest for the periodontal stem cell field and more generally the mesenchymal stem cell field. In particular, through the development of new tools, including a novel lineage traceable mouse line amenable for lineage tracing studies, the authors provide knew knowledge advancing our understanding on the populations and hierarchies that constitute the PDL. Having said this, I find this study rather descriptive and, in certain cases, the significance of the results are somewhat overinterpreted. For instance, lineage tracing studies are rather vague... based on the colocalization of a widely induced traceable fluorophore and markers present in the relevant cell populations, or even just histological positioning of cells; something that entails numerous technical implications and potential artefacts. It would be more convincing to titrate down the tamoxifen levels used to induce Plap-1 traceable mice, in order to track how single-cell derived clones actually contribute to the formation of other PDL populations, and validate this using the relevant markers at critical time points. Quantification of clonal distribution would also provide a deeper understanding of the process.
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Another rather technical, but critical, aspect is the need for further validation of their new mouse model. Particularly, in order to interpret any prospective data on clonal dynamics, it is first important to know whether their new Cre system is tightly regulated, or whether there is leakage in the absence of Tamoxifen induction. Imaging of aged un-induced animals would help clarify this point.
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Finally, the scRNA-seq is rather superficial, a more in-depth analysis would be required to support the statements based on hierarchies and trajectories proposed by the authors.
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Despite all this, I believe the authors have the tools and data to address most of the aspects discussed below, which would make the study sound and result of advancement in the relevant field.
Significance
See above
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Reply to the reviewers
Manuscript number: RC-2022-01490
Corresponding author(s): Cariboni, Anna; Howard, Sasha R
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The current manuscript in question is well written and of general interest to the reproductive neuroendocrinology field. Overall it is a well written and substantiated.
Reply: We thank the reviewer for his/her positive and supportive comments on our manuscript.
The primary problem with the paper is the data derived from the microarray. While the experimental design included replicates (n = 3), although weak, the actual microarray data was based on a single data point. A major weakness. This experiment should be repeated using more up-to-date approaches such as RNA-seq or left out of the manuscript, because this data set is compromised due to the data collection procedure.
Reply: We thank the Reviewer for raising these points, which we wish to clarify. We respectfully disagree that the microarray data generated in this study is not valuable. The transcriptomic analysis of immortalized cells was performed on 3 biological replicates (specifically, RNA was extracted from n=3 samples, obtained from each cell line at 3 different passages) and run as 3 independent samples (for a total of 6, 3 for GN11 cells and 3 for GT1-7 cells). For the primary embryonic GFP-GnRH neurons, given the difficulty of isolating with FACS a sufficient number of GFP+ cells from each embryo due their very small number (around 1000 GnRH neurons/head), we had to pool sorted cells from 2-3 embryos for each time-point. Thus, although the primary cell microarrays were run on one sample for each time point, the RNA was not derived from one embryo only, but from at least 2/3 embryos.
Nevertheless, to overcome the issue of low number of replicates for the primary embryonic cells, we revised our manuscript by re-running our analyses, using as the starting dataset the analyses obtained from immortalized cells, which were based on a ‘true’ n=3 of biological replicates. In this context, we filtered DEGs from this microarray using logFC>2 and adj. p-value1) found in primary GFP-GnRH neurons. We believe that this revised analysis is statistically more powerful, as the core bioinformatic analyses were performed on triplicate samples, with a second filtering step to take advantage of biologically relevant data obtained from n=1 primary GFP-GnRH neurons to confirm in vivo the expression of selected genes. Whilst RNAseq offers wider coverage of the genome and has advantages over microarray, we do not believe that this renders unimportant the data generated from these unique experiments and the novel genomic discoveries it facilitated.
In line with this, our work may be considered as a proof-of-principle that transcriptomic profiles from rodent GnRH neurons can be exploited at different levels, including the possibility to identify novel GD candidate genes. Overall, our work also highlights the existence of similarities between two immortalized GnRH neuron cell lines with primary GnRH neurons, which was so far demonstrated by several functional studies, but not at molecular level.
The manuscript has been now edited as per the above amendments (see first and second paragraph of Results section, lines 86-135).
__CROSS-CONSULTATION COMMENTS __Notwithstanding the importance of neuroligin 3 during glutaminergic synaptogenesis, I agree with the reviewers on both points. Further screenings of the patient's family members should be done and the microarray data should be removed or potentially moved to a supplementary status.
Reply: we thank the reviewer for their comments and, accordingly with their suggestion, we revised the filtering strategy starting from immortalized cells microarray and therefore moved a substantial part of the microarray data from primary GFP+ neurons as supplementary data. We also unsuccessfully tried to collect information of the brother from case 2 and investigated datasets from both the DECIPHER and 100,000 genome projects, but have been limited to two cases for which we have familial consent to publish.
Reviewer #1 (Significance (Required)): The paper is of significance based on the neuroligin 3 data, which is indicative of abnormal synaptogenesis. However, these defects seem to only have a limited effect on the functionality of GnRH neuron system and do not seem to cause elimination of GnRH neurons themselves. Nevertheless these data do open end a new direction that may help explain some dysfunctions in reproductive health.
Reply: we thank the reviewer for their comments and agree that our findings have the potential to facilitate new avenues for the investigation of reproductive disorders.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): Oleari et al performed comparative transcriptome analysis on the different developmental stages of GnRH neurons, as well as two immortalized GnRH neuronal cells GT1-7 and GN11 which represent mature and immature GnRH neurons. As a results, they identified a panel of differentially expressed genes (DEG). They further used top DEGs as candidate disease-related genes for GnRH-deficiency (GD), a disorder characterized with absent of delayed puberty and infertility. To this end, they found two loss-of-function mutations in NLGN3 in patients with GD combined with autism. This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. I only have some minor concerns.
- According to the pedigree, both probands (case 1 and 2) inherited their NLGN3 mutations from their unaffected mother, consistent with an X-linked recessive inheritance. However, only "parent" was used in the manuscript, therefore, it is not clear if this "parent" is the probands' mother or father. __Reply: __Thank you for this comment. We were limited to the use of non-gendered terminology due to medRxiv policies. We have now amended the text and changed ‘parent’ to ‘mother’, lines 161, 173, 179, 185 and 730. We also integrated this sentence highlighting the X-linked pattern of inheritance: “Sanger sequencing of the probands’ mothers confirmed them to be the heterozygous carrier in each family, consistent with an X-linked recessive inheritance pattern.”, lines 185-186.
It is suggested to integrate Figure 2 as a panel in Figure 1.
__Reply: __We thank the reviewer for this suggestion. Due to our revision of first two Results paragraphs, we have now edited the Figures and the filtering flowchart has been added in Figure 2.
What is the meaning of Peak LH and Peak FSH, and how are they measured in Table 2?
Reply: This refers to peak value obtained after standard protocol GnRH stimulation testing with 100mcg GnRH (Gonadorelin) as an IV bolus and measurement of serum LH and FSH at 0, 20 and 60 minutes intervals. (e.g. Harrington et al., 2012, doi:10.1210/jc.2012-1598). This clarification has been added to the text in Table 2 legend (lines 681-683).
A genotyping for the elder brother of Case 2 will be a strong evidence to support NLGN3 as a GD-associated gene.
__Reply: __We thank the reviewer for this important point. In view of this issue, we have strived to collect DNA from this individual. Unfortunately, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.
We also identified a third case via a public database with central hypogonadism who carried a stop-gain variant in NLGN3, but unfortunately the family did not release their consent for publishing this case.
The authors claimed neither probands carried deleterious variants in known GD genes. It is suggested to indicate the exclusion criteria (which genes? How do they define a variant is deleterious?)
Reply: We thank this reviewer for raising this important point of clarification. Inclusion criteria for variants in known GD genes (updated gene list available in Supplemental Table 3) were as per Saengkaew et al., 2021 (doi: 10.1530/EJE-21-0387): “Only variants that met the ACMG criteria for pathogenicity, likely pathogenicity, or variants of uncertain significance (VUS) were retained in the analysis”. We have added this sentence in the manuscript, lines 150-151.
Please also include a sequence chromatogram for proband 2.
Reply: We thank the reviewer for their comment. We added the chromatograms for proband 2 and his heterozygous mother in revised Figure 3.
CROSS-CONSULTATION COMMENTS I agree with Reviewer 3, the genetics is not very strong, as NLGN3 mutations were only found in one GD case from their cohort and one pre-pubertal case from the literature. It will be nice to analyze the genotype and phenotype of Case 2's older brother. Further, it is important to screen NLGN3 rare sequencing variants in larger GD cohorts.
Reply: We thank the reviewer for their comment, but respectfully disagree with this assertion. The second case is not from the literature, but is a second case found thanks to GeneMatcher, an international tool that allows researchers to collaborate on novel gene discovery. We have also explored other cohorts that were available to us, including the DECIPHER and 100,000 genome project, but have been limited to two cases for which we have familial consent to publish. We anticipate that further international patient cohorts will be screened following the publication of this manuscript (added in Discussion section, lines 306-308). As described above, despite trying repeatedly to contact the family of proband 2, it has not been practically possible to collect these extra data from this family.
Reviewer #2 (Significance (Required)): This study provides a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. It may welcome by researchers and clinicians in the filed of neurodevelopment.
Reply: We thank the reviewer for their positive and supportive comments.
__Reviewer #3 (Evidence, reproducibility and clarity (Required)):
__Summary: Oleari et al used murine GnRH1, and immortalized GnRH cell lines (GT1-7, Gn11) to define genes of interest in GnRH development and used this list to filter exome sequencing data from patients with some evidence for GnRH Deficiency.
Title: I am concerned that the title of the paper overstates the results and conclusions.
Intro: use of "candidate causative genes" overstates the evidence presented.
__Reply: __We thank the reviewer for their comment and have revised the title to reflect the findings of the study. We have also edited the sentence in the abstract reporting "candidate causative genes" as follows: “Here, we combined bioinformatic analyses of primary embryonic and immortalized GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in GD pathogenesis”, lines 40-43.
Results: The transcriptomic profile of the developing human GnRH neuron has been published via in vitro differentiation protocols twice (Lund et al 2020, and Keen et al 2021). Gene set data is publicly available. This should be explicitly compared in results not relegated to discussion -- two or three examples it not enough to say mouse can be used instead of human.
__Reply: __We thank the reviewer for this comment. We apologize if our sentence in the Discussion was misleading, as we did not intend to make a conclusion on the similarities of the two datasets/cell types, neither to suggest the use of rodent instead of human.
Although we are aware that differences among species might exist, mouse/rodent models including immortalized cells have been instrumental to understand the molecular mechanisms of GnRH neuron development and to predict candidate genes. Indeed, our aim was to demonstrate that transcriptomic profiles of rodent GnRH neurons could be integrated with exome sequencing data from human patients to reveal novel candidate genes.
Therefore, the aim of our study was different to that of the Lund and Keen publications. Further, caution should be exercised in any deeper comparative analyses with our transcriptomes, for following reasons: first, the GnRH neurons generated from human iPSC and cultured for 20 and 27 days cannot be objectively defined for their ‘age’ in order to be then compared to immortalized or primary embryonic GnRH neurons; second, in these datasets a different and more extensive transcriptomic technique has been used (RNAseq vs microarrays).
There was no intention to relegate to the discussion the possible similarities with other transcriptomic datasets, but we felt that these comparative analyses were beyond the scope of our work.
However, following the Reviewer’s suggestion, we have tried to make comparative analyses with the publicly available datasets from Lund et al 2020 and Keen et al 2021, and with a paper just published (Wang et al 2022), as follows.
In Lund et al. paper, GnRH-like neurons were obtained from human iPSCs by dual SMAD inhibition and FGF8 treatment. We selected data obtained from cells treated with FGF8 and cultured for 20 days and 27 days for comparison with our early and late genes, respectively.
Because the authors of this paper did not publish the full list of differentially expressed genes (DEGs) from this specific comparison (20 vs 27days) and we were not able to retrieve it upon request, we used the normalized counts of these samples (available at ArrayExpress repository) to compare the two experimental groups with DESeq (Bioconductor release 3.15). To increase stringency of our analysis, we considered as differentially expressed those genes which displayed both an adjusted p-value of less than 0.05 and an absolute fold change of >2. The number of DEGs obtained was different and greater (5981) than from the published data, and this large number of genes may, by chance alone, contain a large fraction of any gene dataset (including the genes that we found with our analysis). For this reason, this particular comparison in this dataset cannot be informative or useful.
Next, we considered the dataset from Keen et al. In this paper, the authors have tested different differentiation protocols to obtain GnRH-like neurons from human wild-type or mCherry embryonic stem cells (hESC). They transcriptomically profiled hESC-mCherry-derived GnRH neurons at 8,15 and 25 days of culture.
Again, although we cannot precisely define the matching embryonic stage of cells cultured for 8, 15 or 25 days, we compared the lists of DEGs from immortalized GnRH neurons (GN11vsGT1-7) with the transcriptomic profiles of mCh-hESC at day 15 vs day 8 and mCh-hESC at day 25 vs day 15, respectively. We considered as differentially expressed the genes that displayed both an adjusted p-value of less than 0.05 and logFC>2. We found that the majority of the genes that were differentially expressed in one dataset were not in the other. However, the few genes that were differentially expressed in both datasets demonstrated a good correlation, i.e. the same expression trend. Although this latter approach was more fruitful, by suggesting a partial similarity between primary GFP-GnRH neurons and hESCs-derived GnRH neurons at day 25 vs day 15 time-point, we do not feel that we could draw significant and reliable conclusions.
Further, if we compare these two datasets obtained by RNAseq from hiPSC and hESC, even by taking into account the large amount of DEGs found in our re-analysis of Lund et al., 2021 raw data, a relatively small number of common DEGs were found. These data also suggest that there is transcriptomic heterogeneity even among human-derived GnRH neurons.
In addition to these two datasets, while our manuscript was under revision, a new paper was published, in which the authors dissected iPSC-derived GnRH neuron transcriptome with RNA-seq at single cell level (Wang et al., 2022, doi:10.1093/stmcls/sxac069). Again, although the same concerns may apply in comparing this dataset with ours and raw data of DEGs were not publicly available in this case, we compared the expression trends of our 29 candidates with gene expression trajectories identified in this work. As a result, 24/29 candidate genes, including NLGN3, were found to have an expression trend consistent with our dataset. The few remaining genes exhibited an opposite trend (2/29) or were not found in available data from this work (3/29). As this is a purely qualitative analysis, we do not feel it would be appropriate to include it in the Results section, but have included commentary on these comparative dataset analyses in the Discussion section (lines 247-257). A future study could be designed to mine the raw data from all the available transcriptomic profiles of developing GnRH neurons, but this is beyond the scope of our current manuscript.
The authors need to comment on other GnRH1 expression in the brain of developing rodent and if they think the GnRH1 sorted neurons are just "GnRH Neurons" associated with reproduction (Parhar et al 2005) due to microdissection.
__Reply: __We thanks the reviewer for raising this point of clarification. We have carefully selected by microdissection nasal areas from E14, nasal and basal forebrain areas from E17 and basal forebrain from E20 rat embryos (see revised Methods, lines 325-327). We are therefore confident that what we have obtained is RNA from ‘reproductive’ GnRH neurons only.
Questions about Cases/Missing Phenotypic Information: 1) Case 1: the patient underwent increased testicular volume on testosterone therapy -- testosterone therapy does not increase testicular volume. Has this patient undergone or been assessed for reversal of his hypogonadism?
__Reply: __We thank the reviewer for their comment. The patient had minimal testicular development on testosterone (from 10ml to 12ml) but did not increase testes volume beyond 12mls, consistent with a partial HH phenotype. He has had two trial periods of 3-4 months off testosterone treatment and during these periods had both low serum testosterone concentrations and symptoms of hypogonadism (tiredness, low energy and reduced muscle strength).
2) Case 2: Is too young to be classified as having a pubertal defect. Microphallus is mentioned but what size, was this diagnosed at birth and treated? I think the case for GD is overstated in the results and discussion (especially with the discussion of small testes).
Reply: We thank the reviewer for requesting these clarifications. The patient has not received any treatment for his microphallus (2.5 cm length in mid-childhood). We agree that this case is too young to be classified as having a pubertal defect, but the presence of microphallus and small testes volume in infancy and early childhood, in association with low gonadotrophins and absent erections, are well recognized as red flag signs for hypogonadotropic hypogonadism (Swee & Quinton, 2019, doi:10.3389/fendo.2019.00097). We added this information to the Results section, lines 175-177.
Genetic Information: Since this was a candidate gene search -- what other candidate genes were uncovered in these probands?
Reply: The revised list of 29 candidate genes were screened in the two probands from our study using the whole exome sequencing datasets for these individuals, and only the variants of interest in NLGN3 described in the manuscript were found.
By searching for mutations of the revised list of candidate genes in our GD cohort, we identified nonsense variants only in NLGN3 and no splice variants. We also found few rare and predicted damaging missense variants in this gene list identified. Indeed, two rare (MAF 25) missense variants were identified in the genes PLXNC1 and CLSTN2 in two further probands (now summarized in Supplemental table 4). We have not identified further probands with PLXNC1 or CLSTN2 variants of interest from additional cohorts and thus at present we have not yet taken these gene variants further for molecular characterization, but we will examine the relevance of this gene variant in future work.
Do the probands have a clear explanation for their developmental disability other than the gene noted?
__Reply: __We thank the reviewer for raising this point. Proband exomes were also screened for genes related to developmental delay and no other causal gene variant were identified. We added this information in the text, lines 183-185.
I would encourage the authors to update Table 3: they are missing IHH/KS genes such as GLI3, SEMA7A, SOX2, STUB1, TCF12. I suggest they update the Table and analyses.
Reply: we thank the reviewer for highlighting this point. Since we performed a new analysis, we also performed a new candidate gene prioritization using a more up-to-date gene list to instruct ToppGene (please see revised Supplemental table 3).
CROSS-CONSULTATION COMMENTS Dear Reviewer #2, I am concerned that the paper presents only a single case of GD to support the scientific work. What do you think?
__Reply: __We would like to highlight that, as we describe above, GD can be diagnosed prior to pubertal age in individuals with red flag phenotypic signs and biochemical evidence of hypogonadism.
Dear Reviewer #1: In addition to the weakness in the microarray data, what do you think about the authors using publicly available data from human GnRH neuron transcriptomics for analysis?
__Reply: __please see the above discussion on the comparison with publicly available datasets.
Reviewer #3 (Significance (Required)):
There is not high significance to this paper: This is not the first article with GnRH transcriptomes. I would argue the human data is more relevant. Developmental disability has been previously linked the GnRH deficiency (as even cited in this paper) The article presents one case of GnRH deficiency, and one pre-pubertal case -- providing some modest evidence for a candidate gene, NLGN3.
__Reply: __We would like to rebuff this assessment of the paper’s significance. To our knowledge, this is the first report of transcriptomes from primary GnRH neurons isolated at key embryonic developmental time points. Other published reports refer to iPSC-derived or adult GnRH neurons (Keen et al., 2021; Lund et al., 2020; Wang et al., 2022; Vastagh et al., 2016 and 2020).
Similarly, the association of central hypogonadism with developmental disabilities have been reported in registry-based studies, but few causative genes have been identified, nor patient variants functionally validated in order to investigate the molecular biology underpinning this association. In the Discussion, in the light of a recent paper (Manfredi-Lozano et al., 2022, doi: 10.1126/science.abq4515), we also postulate that NLGN3 might be required for neuritogenesis of extra-hypothalamic projections of GnRH neurons thus contributing to the pathogenesis of NDD (lines 294-300).
Regarding to human data, we would like to acknowledge that we had a third case that we were not able to publish due to family consent. NLGN3 deficiency is likely to be a rare disorder, but that should not obviate the impact of investigating the molecular etiology – indeed, many insights into human biology have come from private mutations in rare disease.
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Referee #3
Evidence, reproducibility and clarity
Summary:
Oleari et al used murine GnRH1, and immortalized GnRH cell lines (GT1-7, Gn11) to define genes of interest in GnRH development and used this list to filter exome sequencing data from patients with some evidence for GnRH Deficiency.
Title: I am concerned that the title of the paper overstates the results and conclusions.
Intro: use of "candidate causative genes" overstates the evidence presented.
Results:
The transcriptomic profile of the developing human GnRH neuron has been published via in vitro differentiation protocols twice (Lund et al 2020, and Keen et al 2021). Gene set data is publicly available. This should be explicitly compared in results not relegated to discussion -- two or three examples it not enough to say mouse can be used instead of human.
The authors need to comment on other GnRH1 expression in the brain of developing rodent and if they think the GnRH1 sorted neurons are just "GnRH Neurons" associated with reproduction (Parhar et al 2005) due to microdissection.
Questions about Cases/Missing Phenotypic Information:
- Case 1: the patient underwent increased testicular volume on testosterone therapy -- testosterone therapy does not increase testicular volume. Has this patient undergone or been assessed for reversal of his hypogonadism?
- Case 2: Is too young to be classified as having a pubertal defect. Microphallus is mentioned but what size, was this diagnosed at birth and treated? I think the case for GD is overstated in the results and discussion (especially with the discussion of small testes).
Genetic Information:
Since this was a candidate gene search -- what other candidate genes were uncovered in these probands? Do the probands have a clear explanation for their developmental disability other than the gene noted?
I would encourage the authors to update Table 3: they are missing IHH/KS genes such as GLI3, SEMA7A, SOX2, STUB1, TCF12. I suggest they update the Table and analyses.
Referees cross-commenting
Dear Reviewer #2, I am concerned that the paper presents only a single case of GD to support the scientific work. What do you think?
Dear Reviewer #1: In addition to the weakness in the microarray data, what do you think about the authors using publicly available data from human GnRH neuron transcriptomics for analysis?
Significance
There is not high significance to this paper: This is not the first article with GnRH transcriptomes. I would argue the human data is more relevant. Developmental disability has been previously linked the GnRH deficiency (as even cited in this paper) The article presents one case of GnRH deficiency, and one pre-pubertal case -- providing some modest evidence for a candidate gene, NLGN3.
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Referee #2
Evidence, reproducibility and clarity
Oleari et al performed comparative transcriptome analysis on the different developmental stages of GnRH neurons, as well as two immortalized GnRH neuronal cells GT1-7 and GN11 which represent mature and immature GnRH neurons. As a results, they identified a panel of differentially expressed genes (DEG). They further used top DEGs as candidate disease-related genes for GnRH-deficiency (GD), a disorder characterized with absent of delayed puberty and infertility. To this end, they found two loss-of-function mutations in NLGN3 in patients with GD combined with autism. This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. I only have some minor concerns.
- According to the pedigree, both probands (case 1 and 2) inherited their NLGN3 mutations from their unaffected mother, consistent with an X-linked recessive inheritance. However, only "parent" was used in the manuscript, therefore, it is not clear if this "parent" is the probands' mother or father.
- It is suggested to integrate Figure 2 as a panel in Figure 1.
- What is the meaning of Peak LH and Peak FSH, and how are they measured in Table 2?
- A genotyping for the elder brother of Case 2 will be a strong evidence to support NLGN3 as a GD-associated gene.
- The authors claimed neither probands carried deleterious variants in known GD genes. It is suggested to indicate the exclusion criteria (which genes? How do they define a variants is deleterious?)
- Please also include a sequence chromatogram for proband 2.
Referees cross-commenting
I agree with Reviewer 3, the genetics is not very strong, as NLGN3 mutations were only found in one GD case from their cohort and one pre-pubertal case from the literature. It will be nice to analyze the genotype and phenotype of Case 2's older brother. Further, it is important to screen NLGN3 rare sequencing variants in larger GD cohorts.
Significance
This study provide a resource for the identification of novel GD-associated genes, and suggest an intrinsic connection between GD and other neurodevelopmental diseases, such as autism. It may welcome by researchers and clinicians in the filed of neurodevelopment.
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Referee #1
Evidence, reproducibility and clarity
The current manuscript in question is well written and of general interest to the reproductive neuroendocrinology field. Overall it is a well written and substantiated.
The primary problem with the paper is the data derived from the microarray. While the experimental design included replicates (n = 3), although weak, the actual microarray data was based on a single data point. A major weakness. This experiment should be repeated using more up-to-date approaches such as RNA-seq or left out of the manuscript, because this data set is compromised due to the data collection procedure.
Referees cross-commenting
Notwithstanding the importance of neuroligin 3 during glutaminergic synaptogenesis, I agree with the reviewers on both points. Further screenings of the patient's family members should be done and the microarray data should be removed or potentially moved to a supplementary status.
Significance
The paper is of significance based on the neuroligin 3 data, which is indicative of abnormal synaptogenesis. However, these defects seem to only have a limited effect on the functionality of GnRH neuron system and do not seem to cause elimination of GnRH neurons themselves. Nevertheless these data do open end a new direction that may help explain some dysfunctions in reproductive health.
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Referee #4
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors use both cellular and single molecule assays to compare the motility properties of three human kinesin-6 proteins: MKLP1, MKLP2, and KIF20B. The presented data indicate that the three motors are primarily non-processive as single molecules, but are capable of driving plus-end directed motility in ensembles. The ability of kinesin-6 motors to move organelles in cells was also tested using an FRB-FKBP induced dimerization assay. MKLP1 and KIF20B were more capable of driving the dispersion of both peroxisomes and golgi than MKLP2. These data are interpreted to indicate that MKLP2 ensembles exhibit less force than MKLP1 or KIF20B.
Major Comments:
- The single molecule results from analyses of MKLP2 presented in this study contrast significantly with those presented in Adriaans et al. 2020. This previous study presented evidence that full-length MKLP2 moves processively (1.1 um run-length at 150 nm/s) as single molecules, and that this processivity is enhanced by binding to the chromosome passenger complex. The current study addresses these differences to some extent by indicating that a fraction of MKLP2 motors displayed slow, processive movement. However, the kymographs of MKLP2 in the two studies still look quite different (e.g. frequency of processive movement, pausing, velocity), and further explanation would be useful for understanding the apparent conflict in conclusions regarding MKLP2 motility. Does the use of a truncated MKLP2 construct in the current study change the behavior of the protein in the motility assay?
- The organelle dispersion assays shown in Figures 4 and 5 rely on co-transfection of motor-FRB and targeting-FKBP constructs. The extent of dispersion could be affected by expression levels of either construct in a particular cell. Controls indicating that similar expression levels were compared across experimental groups should be included.
- The authors speculate about the contributions of kinesin-6 extensions in the neck-linker and presence or absence of the N-latch residue to the motility properties observed. However, these predictions are not tested experimentally.
Minor Comments:
- In the legend for Figure 2B- kymographs are of fluorescent microtubules?
Significance
The presented work provides an assessment of human kinesin-6 motors in a number of different motility assays. These motors play key roles during cell division and cytokinesis, and the multifaceted investigation of kinesin-6 motility presented in this manuscript complements previous studies that examined the same motors in one type of assay or assessed the activity of kinesin-6 motors from other organisms. The work, therefore, provides a framework for future structure, function studies that will be of interest to the molecular motor, cell division, and cytoskeleton fields.
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Referee #3
Evidence, reproducibility and clarity
The manuscript, "Differences in single-motor and multi-motor motility properties across the kinesin-6 family," by Poulos, et al, is a comprehensive study of the motility properties of the kinesin-6 motor proteins.
The work has a high number of molecules, filaments, and cells used to have statistical significance and reliability of the results.
Positive aspects<br /> 1. Kinesin-6 family is an important class of motors that needed to be investigated in a systematic manner.<br /> 2. The work was performed using highly reproducible assays that revealed novel information about this family of motor proteins.<br /> 3. The data was presented in a clear and cogent manner, making the paper highly accessible to non-experts.
Negative aspects
My only suggestion is that the figures be discussed in order to make it a bit easier for the reader to follow. This is a minor suggestion.
Significance
Employing single molecule motility assays, gliding assays, and cellular transport assays, this study elucidates the physical abilities of this elusive and essential family of kinesin motors. Excitingly, it was shown that the kinesin 6 motors can move infrequently as single motors and more frequently as multi-motors. In cells, they can also transport cargos except MKLP2. These results clearly demonstrate that kinesin-6 motors have motility and can even move large objects under high load in multi-motor configurations. The work is well-articulated and should be accessible to a wide audience.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, Poulos and colleagues perform experiments aimed at understanding the in vitro behavior of kinesin-6 family motor proteins. The results, which are well supported by the experiments, show that the truncated motors show mostly diffusive behavior in single molecule assays. Surface bound motor domains drive microtubule sliding at low velocity. Assays in which motors were coupled to organelles, using rapamycin induced dimerization of the FRB-tagged motor construct and either Golgi or lysosome proteins with a FKBP domain, further revealed that the MKLP1 and Kif20B could drive transport of both peroxisomes and Golgi, although MKLP2 could not transport the high load cargo (Golgi) and showed limited transport of peroxisomes.
Comments.
These experiments are important to show the properties of the kinesin-6 family motor domain; however, the general lack of robust single molecule processive motility supports the idea that in vivo, these motors contribute to cellular processes as part of complexes with other proteins (Central spindlin (MKLP1), Chromosome passenger complex (MKLP2)) which enhance motility. In fact others have shown that motorclustering is important for plus end accumulation of MKLP1 (using C.elegans proteins). More recently Adriaans etal showed that for MKLP2 that MKLP2 is a processive plus end directed motor, using purified homodimeres of full length protein. Importantly, addition of a recombinant CPC further increased processivity. What remains unclear is why imaging of MKLP2 in cells shows predominantly diffuse behavior with only a fraction of events showing directed motility. The authors might discuss this concept in more detail - how motility is impacted by binding partners and/or regions outside of the motor domain for some kinesin families. Alternatively, they could demonstrate the changes in motility by extending the study with longer constructs and additional components.
The authors used truncated proteins for their assay, but also tested longer constructs. They state that the behavior was similar in single molecule assays, so they focus on the truncated motors. However, figure S2 looks like there are move processive motility events for MKLP1 and MKLP2, which is more in line with some other results (i.e. Adriaans et al, for MLP2). Can the authors comment on this?
The authors perform assays to study organelle motility. What is already known about kinesin-6 motor contribution to this process? For MKLP1 and 2, the best studied role is anaphase and cytokinesis.
Significance
Overall, the work is well done; however, the main result is that motor domains of kinesin-6 show mostly diffuse motility. This strongly suggests that other binding partners or other parts of the motor are needed for processive motion. The mechanism responsible for mixture of directed and diffuse motion observed in cells remains unclear. The advance from these studies is not major, for the current manuscript. The authors could submit to a journal that supports publication of work that is well-executed, and an important part of the larger picture, but that is not a major advance. Alternatively, they could continue to address the additional cellular machanisms that may contribute to regulation of processive motion in dividing cells.
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Referee #1
Evidence, reproducibility and clarity
This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.
Major points are the following:
- Quantitation and presentation of the data throughout the manuscript should be improved.
The criteria used for identifying fluorescent spots as single motors are not given. This is typically based on photobleaching experiments and fluorescence intensity measurements - the authors should show these data to validate that the motility reported is due to single motors.
A table should be included that shows the single molecule motility parameters that were analyzed and compared for the three motors, rather than just the dwell times for the assays shown in Fig. 1. Other motility characteristics should include run lengths, binding rates, detachment rates, and velocity. The percentage of time that the single motors move directionally, diffuse, or remain stationary should also be given.<br /> The authors refer to imaging rates (1 frame/50ms, p. 5), but do not state the total time of the assays, making the statements uninterpretable, as it is not clear what would be expected without knowledge of the total assay time. The authors also state that a slower imaging rate (1 frame/2 sec) was used to detect slow processive motility, but the logic underlying this statement is not clear, as a longer assay time should reveal the slow processive movement irrespective of the imaging rate. These statements should be clarified.<br /> The authors give the data for the dwell times in single motor assays and velocities in multiple motor assays as the mean + SEM, but the SD rather than SEM should be reported for these assays, given that the data are for individual single motors or individual gliding microtubules. The authors state the number of replicate experiments for the assays, but they should also state the number of data points that were obtained for each replicate. Further, they should evaluate the significance of differences in their data by giving P values obtained using appropriate statistical tests and indicate whether the differences among the motors are significant.<br /> The percentages of processive events (p. 5) are most likely dependent on the amount of inactive or denatured protein in a given preparation, rather than a motility property of the motor protein - this could be determined by analysis of whether the percentages differ from preparation to preparation of each motor and whether the mean+SD of the preparations of a given motor differs from the other motors. The statements by the authors on p. 8 that "the majority of proteins do not undergo unidirectional processive motility as single molecules but rather diffuse along the surface of the microtubule for several seconds" and "It is presently unclear why only a subset of kinesin-6 molecules are capable of directional motility (Figure 1 ..." are not meaningful, as they do not take into account the percentages of the kinesin-6 proteins that are inactivated or denatured during protein preparation.<br /> Again, given that inactive motors are produced during preparation of the proteins, it is not clear what the frequency of processive motility events means. If the authors think that the frequency of processive motility events is informative and a characteristic of each motor, they should present controls showing frequencies of processive motility events for specific well characterized motors. For example, does a control of kinesin-1 show 100% or only 95% processive motility events?<br /> For the multiple motor gliding assays, velocities are shown in Fig. 2 without controls demonstrating the dependence of the velocities on motor concentration in the assays - the gliding assays require dilution experiments to show that the velocities are within the linear range of motor concentration and do not fall within the range of higher concentrations in which motor gliding velocity is inhibited or lower motor concentrations in which the density of motors on the surface is too low to support processive movement. These control experiments of motor concentration vs velocity for the gliding assays should be shown for each of the three motors that was assayed. The authors should state whether the gliding velocities that were determined correspond to the Vmax for each of the motors that was assayed.
Again, the velocities given on p. 6 should include the SD and evaluation of the significance of the differences among the motors by obtaining P values.
Proteins for motility assays: Western blots of the purified proteins should be shown as a supplemental figure.
How are the motility characteristics of the three motors related to their spindle functions? This is the central point of the manuscript but is not clearly stated.
- Functional assays should be relevant to motor function.
Given that the kinesin-6 motors under study are mitotic spindle motors that do not normally transport vesicles, it is not clear why the authors chose to show load dependence using peroxisome and Golgi dispersion assays, rather than assays of spindle function. The authors interpret peroxisomes and Golgi to differ in dispersion load, but this appears to be based on interpretations from assays of highly processive motors, kinesin-1 and myosin V, that function in vesicle trafficking, rather than quantitative data from appropriate controls showing that peroxisomes and Golgi can be dispersed by spindle motors that bear different loads. The problems inherent in the use of these assays for spindle motors are evidenced by the authors' observations on p. 6 that MKLP1- mNG-FRB and KIF20-mNG-FRB in midbodies could not be localized to peroxisomes by rapamycin. There are no data presented showing the dependence of dispersion on protein expression/presence in the cytoplasm, making the dispersion assays difficult to interpret.
The kinesin-6 motor functional tests would be more relevant if they involved mitotic spindle assays, rather than peroxisome or Golgi dispersion assays. It is not clear how the loads involved in peroxisome or Golgi dispersion are related to kinesin motor function in the spindle. What are the implications of low- vs high-load motors in the spindle? How do the authors envision that motor loads in spindles relate to loads borne by vesicle transport motors?
Minor points needed for clarity and reproducibility of the data:
Methods
Plasmids<br /> "MKLP1(1-711) lacks the insert present in KIF23 isoform 1" - the insert present in KIF23 isoform 1 but missing in MKLP1 (1-711) should be depicted/pointed out in Fig. S1 and information provided as to its predicted or actual structure.
"KIF20B contained the protein sequence conflict E713K and natural variations N716I and H749L "- the sites of these changes should be indicated in Fig. S1 and information provided as to their effects on predicted or actual structure.<br /> Protein purification: "MKLP1(1-711)-3xFLAG-Avi was cloned by stitching four oligonucleotide primer sequences together into a digested MKLP1(1-711)-Avitag plasmid" - please explain what this means: what do the four oligonucleotide primer sequences correspond to? if they are the 3xFLAG-Avi tags, why were four sequences stitched together instead of three?<br /> The figures showing the kymographs should include labeled X and Y axes, rather than scale bars.
The significance of the statement that "All motors displayed similar behaviors when tagged with Halo and Flag tags" is not clear, as the Halo and Flag tags were also C-terminal tags, like the 3xmCit tag.
The figures (Fig. 3-5) that contain grey-scale cell depictions would be more readily interpretable by others if they were labeled with the authors' classification of the dispersion phenotype.
Significance
This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.
My expertise: motors, motor function in division, motility assays, microtubules
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Reply to the reviewers
2RE: Review Commons Refereed Preprint #RC-2022-01651
Overall author response to reviewers:
We appreciate the enthusiasm of reviewer 3, who remarks on how our study’s toolset allows us to tackle a longstanding question in the field. We also appreciate the fair criticisms of reviewers 1 and 2.
We have taken into account all the comments of the reviewers by adding new data and new figures, but also by better underlining the limitations of our study design. Here, we underline the main points (see below for points to points answer).
We have addressed the reviewer’s concerns by including sex-specific data in the supplemental for each figure. We also hope the reviewers can see the value in not making this manuscript about sex-specific lifespan effects given our arguments below. We believe that additional experiments using a co-housing design, with far more replication per sex per genotype, would be needed to make strong claims about sex-specific effects. In designing our study, we included both sexes to avoid biasing our overall results to only one sex. In our study, we did not design our experiments in a way that allows sex*genotype interactions to be distinguished from vial effects. We believe our current evidence is sufficient to make claims strictly at the genotype level. But at the sex-specific level, not only is our study not designed for a fair comparison of the sexes by virtue of keeping them in independent vials (with independent microbiomes developing over long timeframes), but such comparisons would also be less robust, effectively based on only half the data per treatment compared to comparisons at the genotype level.
Of note, we do report Cox mixed model statistics in the figures for the interested reader. We just focused on median lifespans for data presentation clarity, and to ensure we focused only on strong trends we could be confident were not statistical Type I errors (falsely rejecting a true null hypothesis). While relying on median lifespan for insights is non-standard, focusing on and showing median lifespans also allows us to better display inter-experiment variation by showing individual data points reflecting each experiment. Sum survival curves with error bars/shading would force us to make an arbitrary decision about what error range to display (SE? SD? 95%CI?), and would not permit the reader to see inter-experiment variation. Median lifespan graphs also let us show just how repeatable our experiments were. We also chose to analyse median lifespan to make it easier to consider the effect of multiple hypothesis testing, so we could better ensure that trends in our data are really striking enough to be worth comment, particularly given the genetics caveats we draw attention to (despite our isogenization efforts, e.g. DefSK3).
We hope with the revisions we provide, reviewers 1 and 2 can, if not agree, at least acknowledge our concern with claiming many sex*genotype interactions. Such effects are lost if we look at other genotypes containing those mutations (e.g. GrA vs. GrAC, or GrB vs. GrBC). It is a challenge to use the fly lines we have generated, which systematically combines 8+ mutant loci, requiring constant reflection. Taking into consideration the complexities of life span studies, we prefer to focus our attention only on the key and robust results of our study.
Nevertheless, in the revised version, we also now provide additional data, and we soften our language regarding the impact of nora virus on aging. We have included new data with additional genotypes that were nora-infected to reinforce our claim that nora virus impacts lifespan. These data greatly strengthen the correlation of nora virus and lifespan reduction. We now also make it explicit that our statement that nora virus infection shortens lifespan is strictly correlation-based, and does not reflect intentional infection experiments. Of note, our study confirms a previous observation done by Ayebed et al. (2009) in the lab of Dan Hultmark, though we find a more striking effect in the DrosDel iso w1118 background.
Overall, we believe we have reinforced the claims of our manuscript after the revisions and addressed the constructive comments of the reviewers. Changes from the original manuscript are highlighted in yellow.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary:
Whether and how age-associated immune hyperactivation affects the ageing process is a key question in the ageing and immunology fields. Using a CRISPR-based knockout approach, Hanson and Lemaitre address the effect of antimicrobial peptides (AMPs), essential immune effectors, on the ageing process. They compared the lifespan and climbing ability of flies deficient for groups of, or individual, AMPs to their isogenic control and the null mutants of the major immune pathways IMD and Toll. Although deletion of individual AMPs had limited effects, the authors detected an association between deletion of a group of AMPs on bacterial proliferation, and examined possible causality using antibiotic treatment. Interestingly, this causal link was missing in the IMD deficient strain, suggesting a potential AMP-independent mechanism of innate immune signalling in the ageing process. The topic is ambitious and exciting; however, the work has some quite serious technical limitations that would need to be addressed with further data analysis and experimentation.
We appreciate Reviewer 1 and Reviewer 2’s concerns regarding the sex-specific effects, which is an important consideration in aging studies. We de-emphasized sex specific effects for reasons outlined in the supplemental text, which we will reiterate here: Per experiment, per genotype, we used ~20 males in one vial and ~20 females in another vial. As a result, the interaction of sex*experiment within a genotype is effectively the same as vial effects. We have to be especially careful of vial effects in our study because we are studying immune-deficient flies susceptible to stochastic microbiome dysbiosis, causing vial stickiness and mass mortality events. Moreover, these mass mortality events are not randomly distributed over aging: they were most likely to occur on Mondays. This is because microbes accrue in the vial over 3 days during the weekend, but only over 2 day time periods for flies flipped between Monday-Friday. As a result, in genotypes that suffer dysbiosis (many AMP group mutants), dysbiosis vial effects can change that experiment’s male/female relative lifespan by ~7 days just by stochastic chance. This issue likely affects other aging studies less, as microbiome dysbiosis is a uniquely important consideration in our immune-deficient flies, and we emphasized this point by noting mass mortality events in Figure 3 as “{“ annotations in the survival curves. We have added these considerations in the revised version (new lines 209-214).
Microbe density in vials is also affected by remaining fly density. As an example of how this can greatly impact the final data at the sex*genotype level: let’s say 4 males died or were censored over the first 50 days randomly in a given experiment. That male vial (16 flies) is less dense than its paired female vial (20 flies), and would have persistent lower microbe load every flip as fewer flies are transferring microbes to the next vial through feces. Then on say… day 60 (a Monday) of that experiment there’s a mass mortality event. Specifically, at day 60 the female vial, which is more densely populated and has had days-to-weeks of higher microbe transfer rate during flipping, has 11/20 females die at once. On the other hand, the males pull through without mass mortality owing to their lower vial density. Suddenly, the female median lifespan in that experiment is 60. The male mortality is most likely to pass the median the next Monday, when another microbiota-induced mass mortality event occurs. Exactly when these critical weekends occurred was stochastic by experiment, but the fact that they occurred was consistent in all experiments in AMP mutant stocks. In this example, female median lifespan would be 60, while males would likely be 67 or so… Importantly, this isn’t a genuine sex-specific effect, it’s variance from how early stochastic mortality affects late-experiment vial density due to microbiome development. It is a vial effect that can look like a sex*genotype effect given our study design, which is exacerbated here because we work on immune deficient flies given that can exhibit microbiome dysbiosis (Marra et al., 2021; mBio).
Given the importance of AMPs in combatting infection, we chose to keep sexes separate to avoid random mating, which can introduce stochastic sexually-transmitted infections. We would also then have had to worry about differences in mating rate amongst genotypes impacting the likelihood of transmitting infections, and differently imposing stress on females by having constant suitors even when they were not receptive. A future study using a co-housing setup would be better equipped to tackle microbiome vial effects. We have now performed the study with sexes kept separately, that can provide a reference for that future study. A future study would also benefit from focusing on fewer genotypes, with far more replication per sex per genotype, to ensure AMP*sex*lifespan effects were robust.
We have now provided supplementary analyses of the genotype-specific ratios of male/female lifespans for each figure (new Table S1-S4). By using the ratio of male lifespan to female lifespan, we focus on how within-genotype comparisons work. We want to emphasize the stochasticity of this ratio depending on experiment batch, even within genotype. For instance, the iso w1118 wild-type male/female lifespan ratio was 0.87 for the six experiment in Figure 2, or 0.93 for the four experiments in Figure 3. It’s a reminder that any collection of experiments is just a sampling of the true population mean. Still, we do see some striking departures in the paper from these ratios. For instance, if we focused on striking departures from this sexual dimorphism, we could comment on a couple genotypes in Figure 3 (AMP groups figure): Group B had a ratio of 1.04 (Only females had reduced lifespan, while males remained comparable to iso w1118). Yet in Group AB the ratio is 0.91, with both sexes having proportionally reduced lifespan. And then Group BC, the ratio is 0.86, with both sexes living to wild-type lifespans. ∆AMP10 and ∆AMP14, which contain all Group B mutations, also have ratios of 0.87 and 0.93 respectively. We also did not see a striking effect in individual mutations from Group B, which all have lifespan and male/female ratios comparable to wild-type. What are we to make of Group B alone then? Is it a genuine effect that combining the Group B mutations uniquely reduces female lifespan? Or is this simply an outlier, perhaps caused by vial effects, that is not supported by all other individual or combinatory genotypes that include Group B mutations?
With all that said, the supplemental tables now included for each figure note these data trends, and includes our cautious view of how to best interpret them. We prefer to merge the sexes in the main article to simplify data display, which is not unique to our study (e.g. see Kounatidis et al. 2017; Cell Rep). We feel our study is not robust at the sex*genotype level to make assertions on sex-specific effects.
Major comments:
- Figure 1A shows a large increase in fly lifespan in response to deletion of 8 AMPs on Chr II. However, the labelling on the figure shows that data from different experimental runs and from the two sexes have been combined to produce the result, and this problem runs through subsequent results. This is not an acceptable approach. Males and females have different average lifespans and may respond differently to the deletion. Repeats of the same lifespan measurement at different times often give significantly different absolute lifespans between runs, even if the differences between experimental treatment are consistent. The procedure used to construct this Figure therefore lumps heterogeneous data, which in this case is both biologically and, more generally, statistically invalid. We need to see an analysis where the results for the sexes and for different runs of the experiment are disaggregated. To assess the effect of the deletion, comparison should be made only within a single replicate of the experiment, and within each sex. It needs to be made clear how many experimental runs were done and with how many flies in each, since replicability is important with these labile traits. Data should be appropriately analysed, for instance with Log Rank test, or, if the assumptions of the test are fulfilled, with Cox Proportional Hazards. The authors mention analysis of median lifespans, but it is not clear what experimental data the medians were derived from, and this is not a gold standard approach to analysis of lifespan data. This problem also applies to the comparison made between lifespans of iso w1118 flies in Figure 1A and 1B - these were evidently measured in different experiments, so direct comparison cannot be made.
We hope the above response reassures the reviewer about our statistical approach, which we agree is non-standard. We feel it is less likely to result in false-positive claims (Type I error) given the many genotypes we screen and the high stochasticity of individual vials affecting some AMP mutant genotypes more than others (i.e. statistically: unequal variance, requiring an alternate data treatment). Still, we did perform Cox mixed models, and we report those genotype-specific p-values in the figures. But we prefer to make inferences only based on trends robust enough to show in median lifespan comparisons.
- Figure 2A reports data from mutants that were not backcrossed into a standard genetic background. The authors were aware that this can be a problem because they took care of it with their own mutants, but the data reported in this Figure 2A could be entirely attributable to difference in genetic background between stocks rather than the mutants themselves.
Agreed. The reviewer may be confused regarding our intent in showing these genotypes. We used lines as used in other papers to show how control genotypes behave in our hands. This makes our study easier to compare to the broader literature encompassing immunity*aging studies. The intent is not to test if those mutations’ effects are true in another background. They are there to provide context for our lab settings compared to other studies. They are inter-study calibrator controls.
We have added some text in the study to better highlight the utility of including these control genotypes for making our work more comparable for the field at large (e.g. Line 298, Line 515).
- Sexual dimorphism is widely reported in many ageing and immunology studies (reviewed in Belmonte et al., Front. Immunol., 2020 and Garschall et al., F1000Res., 2018). This is a problem for the data discussed on line 317, where it is suggested that Group A mutations are shorter lived than the control group because Group A carries the DefSK3 mutation. However, if the results are split by sex, the lifespan difference between Group A vs control is solely contributed by the shorter-lived male flies in Group A. The female flies in Group A live as long as the female control flies. However, the lifespans of both genders of the DefSK3 mutant in Figure 2B are all shorter than control flies. There appears to be a complicated interaction, with diverse function of individual AMPs during ageing, which cannot be summarised by the statement that "deleting single AMP genes has no effect on lifespan" on line 295.
The reviewer is correct that Group A shows a male-specific lifespan reduction. But taking the example of Group B above, we could argue that Group B uniquely reduces female lifespan; except when Group B mutations are found alone, or small combinations (e.g. Dro-AttAB, DptSK1), or in the Group AB, Group BC, ∆AMP10, and ∆AMP14 backgrounds. The same contradictory results are true of the Group A result the reviewer highlights, as Group AC has the opposite effect on male/female lifespan ratios and wild-type lifespan, and ∆AMP10 and ∆AMP14 have normal male/female lifespan ratios.
- The authors claimed nora virus regulates fly lifespan but they do not produce any direct evidence that this is the case. Bleaching can remove many bacteria and viruses, including many pathogens. To establish the causal role of nora virus on lifespan, reinfection studies with a range of microbes including nora virus would be needed.
We absolutely agree. These experiments were not intended to test for the effect of nora at the outset. Nora is only highlighted because its presence is strictly correlated, in our hands, with strongly reduced lifespan and also the bloating phenotype seen upon aging. We did not previously show other nora-infected data in the manuscript, but in retrospect, we can see that additional context is needed to reassure the reader of why we suspect nora so strongly. We have modified Figure 1B to include nora-positive data from four additional genotypes that were infected with nora at various times in the lab (previously did not show).
To explain the process of how we detected nora in some of our AMP stocks: we randomly screened them for a set of common viruses as part of a research workshop in 2019 hosted by Luis Teixeira. The results of that initial screen were not formally recorded, though we screened all the individual and group AMP mutants available at the time. The viruses we screened for were: Drosophila sigma virus, DCV, DAV, and Drosophila nora virus. Out of the stocks relevant to this study (~20 genotypes), we detected nora virus in iso w1118, AttC, Group C, and Bom. The new data we provide even includes an experiment where AttC with/without nora was included at the same timeframe (re: batch effect concern). We also had instances where OR-R became newly infected with nora virus just from random lab inoculation (not intentional infection).
Importantly, we had noted AttC, Group C and Bom as lines with exceptionally poor lifespan that also showed a bloating phenotype upon aging for ~2 years before we had nora virus on our radar. We were operating on a hypothesis of cryptic microsporidia infection at the time based on a set of chitin stainings of hemolymph that didn’t fully pan out. What convinces us this is nora virus and not a co-infecting virus/microbe is that we only saw the reduced lifespan and bloating upon aging in these confirmed nora-positive lines out of the ~20 we were screening at the time: a 5/5 correlation. We were even able to go back and screen RNA collected from those stocks from earlier experiments to confirm nora infection (informing which experiments we could objectively censor from our analyses). Upon bleaching, we rescued the lifespan of iso w1118, AttC, Bom, and Group C to the levels reported in the main figures. We hope this adds weight to the correlation between nora and lifespan, even if we haven’t done proper reinfection experiments. We also thank the reviewers for commenting, as including these data improves our study by providing more context on the variability of the nora lifespan effect in different genotypes: for instance, we did not collate the nora-infected OR-R data previously, but upon analyzing those data, the negative effect is clearly present but to a lesser extent than iso w1118 (and more in line with what was seen in Habayeb et al. (2009)).
To address the reviewer’s comment, we have also softened our language to be very clear we find only an association, and do not provide a demonstration (new lines 191-193). But the results were sufficiently striking, and poor lifespan associated with bloating was perfectly predictive of nora presence in stocks in our hands. We fully believe the result and feel it is important to include this in the main text to make the field more aware of this important aging study confounding variable.
- The authors reported a potential AMPs-independent mechanism of the IMD/Relish pathway on ageing, which would be important. However, in Figure 4A and the associated raw data, the authors compared the lifespans of flies from different experiments, and this seems to be a problem. Using ΔAMP14 as an example of a more general problem: the authors assayed conventional feeding ΔAMP14 flies between 07.2021 and 11.2021; however, the antibiotic treated flies were assayed between 12.2021 and 02.2022. There is an obvious batch effect: the median lifespan of ΔAMP14 is 50d on 29.07.2021, 73d on 20.08.2021, 65d on 06.11.2021, and 61d on 10.11.2021. There is therefore a confounding of batch effects with the biological function of AMPs. Some groups seem to have extremely limited sample size, such as only two female flies and six male flies were recorded in "ATM8 23-07-2021 f" in Figure 2A-associated raw data.
We show this inter-experiment variation explicitly by presenting individual experiments as data points in median lifespan graphs. It is not hidden, it is emphasized by our median lifespan data presentation style. The experiments from 06.11.2021 and 10.11.2021 were from independently kept stocks of ∆AMP14, although naturally they come from similar timeframes and would have used food prepared at roughly the same time. If the reviewer feels we should merge the two experiments, we can do that. Merging the experiments and having only 3 entered replicates for conventionally-reared ∆AMP14 changes the one-way ANOVA result in Fig. 4B from “p = .006” to “p = .004,” so we don’t feel having those two experiments entered independently is biasing the analysis. We do feel the parents, and the flies measured, were independent at the rearing level.
ATM8 specifically had poor homozygous viability. This is the only genotype with such a significant departure in sample size per experiment. ATM8 flies are also not relevant to the core message of the study, and their reduced lifespan was extremely clear and in line with previous studies. We prefer to keep the genotype in the study to allow comparison of our lab conditions to studies using ATM8. We have added this note in the Fig. 2 caption (Line 1029).
Regarding how much can be attributed to batch effects: there are many elements of laboratory studies that can contribute to batch effects. However antibiotic food would undergo independent food preparation from standard food regardless of what we do, and parents and flies would be reared in independent vials from independent food preps regardless of what we do. This really just leaves the seasons as the batch effect, which ought to be controlled for by our incubators controlling temperature and humidity. We do appreciate the valid concern, and that incubators aren’t perfect. But we want the importance of the concern to be viewed in the light that even had we done the experiments at the same time, the conventionally-reared and antibiotic-reared flies would still have been given totally different preparations and conditions. Given this consideration, we will further note:
1) ∆AMP14 flies were already known to suffer dysbiosis with aging (Marra et al., 2021; mBio). Our present study only quantifies the effect of this dysbiosis on lifespan.
2) Dysbiosis is associated with flies gut barrier dysfunction and gut content leakage into the hemolymph (Rera et al., 2012; PNAS)(Clark et al., 2015; Cell Rep).
3) Imd-mediated barrier dysfunction leads to microbiome invasion into the hemolymph (Buchon et al., 2009; Genes Dev).
4) Systemic infection by microbiota-derived Acetobacter kills AMP mutants (Marra et al., 2021; mBio)(Hanson et al., 2022; Proc R Soc B), which suggests microbiome invasion into the hemolymph will kill AMP mutants more readily than wild-type flies.
The result that AMP mutants have improved lifespan in antibiotic conditions is therefore not especially surprising – it is important to actually test this, but it is by no means unexpected. One of the curious findings of our study is that we could not rescue Relish to the same extent. If batch effects were affecting the antibiotic rescue experiments by having different intrinsic lifespans during those months, we would expect this to also be visible in iso w1118 and Relish. Antibiotics (or time period batch effects) did not affect lifespan of our wild-type flies, which was very repeatable across all experiments across many years, agreeing with antibiotic-reared fly data from Ren et al. (2007).
Minor comments:
- The authors reported that the mutant flies lacking 14 AMPs are short-lived due to dysbiosis. Interestingly antibiotic treatment rescued the shortened lifespan of ΔAMP14 but not RelE20. Considering that some of these AMPs, such as lDef, Dro and Drs, are controlled by both the IMD and Toll pathways. It would be worth exploring if the axenic environment could improve the lifespan of Toll mutant flies, which might point to a distinct function of the Toll pathway on the microbiome and ageing process.
- Line 169: The authors stated that they screened the existence of common viruses but did not provide the results.
- Line 179: The authors need to include the quantitative results of nora virus in their 44 stocks.
- Lines 311-314 should be combined with the next paragraph as they are all about "screening".
- Line 319: Please indicate the associated panel "Fig. 2B" instead of using "Fig. 2".
- Lines 353 and 358 and in Figure 3C: The authors should provide the quantification of the sticky food in AMPs mutant and Relish mutant flies.
- Line 389: Please indicate the associated panel "Fig. 2A-B" instead of using "Fig. 2".
- Line 435: As discussed above, the authors cannot rule out an effect of individual AMPs on lifespan based on their current data and interpretation.
- Line 507: "During our study, we experienced a number of challenges to lifespan data interpretation." As discussed in the major comments, unless the authors re-perform and re-analyse the lifespan assays this problem will persist.
- Line 514: As discussed above, the authors cannot rule out the effect of other pathogens on lifespan.
We appreciate the reviewer’s request for spz axenic lifespans. The effect was more striking in Relish, and so we focused on Relish, which regulates antibacterial peptides in the gut. Repeating the experiment with spz will cause non-essential delays and would not affect our main conclusions that focus on AMPs genes.
Line 179: we have, in Fig. 1C. Negatives are not shown, but positives are included in the “Stocks” column. Is the reviewer requesting the exact stock names? We can provide most of these… although the screen was organized by having lab members submit stocks that were in use, and so the spreadsheet of those results is organized with labels provided by lab members (e.g. FM1, rather than the genotype exactly). So we cannot provide exactly which stocks were positive in our hands, but we also don’t feel this is important for the reader. We can share the spreadsheet of this screen (conducted in Dec 2020) with the reviewer if desired.
We were happy to revise the manuscript according to all other requests/comments, highlighted in yellow, and hope our explanations above are sufficient to give the full weight and meaning of the statement in Line 507.
Reviewer #1 (Significance (Required)):
Significance: The crosstalk between AMPs and ageing is a long-standing contradictory topic in the immunology and ageing field (Loch et al., PLoS One, 2017; Badinloo et al., Arch. Insect Biochem. Physiol., 2018; Garschall et al., F1000Res., 2018). This work is a potential step in determining whether and how AMPs regulate ageing. This research may open a door toward yet uncharacterized AMP-independent mechanisms of innate immune signalling in the ageing process.
I am familiar with Drosophila ageing and its relationship with innate immunity, although I am not an insect immunologist.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: In this study, Hanson and Lemaitre generated AMP single or compound mutants, and subjected them to lifespan analyses. They showed that deletion of all AMPs but not individually (except for Defensin) reduced Drosophila lifespan. The observations that ΔAMP14 suffered microbial dysbiosis and antibiotic treatment rescued the decreased lifespan in ΔAMP14 flies implied a link between AMP-controlled lifespan and host microbiome. Thus, the authors would like to conclude that AMPs contribute to fly lifespan via modulating microbiome.
Comments: This reviewer deeply appreciates that the authors generated AMP mutants (individually or with various combinations). However, these mutants have been reported in their previous publications (eLife, 2019; Genetics, 2021). It seems that the authors carefully analyzed the lifespan of indicated flies, but many pieces of data are inconsistent with previous findings (for instance the lifespan under axenic condition). The explanation about the fly medium (Line 476-477) is not convincing (if yes, who not analyze the lifespan using the same diet as described in previous studies?). The lifespan analyses are of course the most pivotal part in this study, but it is a pity that the results are not shown in an appropriate way, making them rather difficult to interpret. For example, the reviewer is surprised to note that the lifespan of males and females are not analyzed and shown separately (e.g. Figures 1A, 1B, 2A, 2B, 3A, 4A), even the authors declared that they did this before lifespan examination. It is also unclear how many males and females were utilized individually in most assays.
We do not believe the axenic lifespans are inconsistent with previous findings. There have been papers both finding an effect of antibiotics, or finding no effect of antibiotics, on wild-type flies (ex: refs 56 and 57: Ren et al., 2007 and Brummel et al., 2004). Our study would support no major effect. Given our note on nora virus, one might wonder if papers where antibiotics affected lifespan might in part be explained by pathogens (like viruses) that were confounding results in the initial stock, and cleared by the initial bleaching common to those experiments. This would imply that commensal bacteria/fungi community do not have a major role in wild-type lifespans, but rather some stocks are stochastically infected with opportunistic viral pathogens, giving the impression that antibiotic treatment was the key, when in fact it was the bleaching pre-treatment that removed an opportunistic viral pathogen. This is only speculation, but it emphasizes the importance of openly discussing the effect nora virus can have on lifespan for future studies.
Regarding the diet comment: which standard diet should we have chosen? There are many standard diets. We have provided our recipe and we have used our diet for decades. Of note, we did try a set of experiments on a molasses-based food, which showed similar lifespan for wild-type flies in our hands (Response supplemental figure below).
We hope our answers to the first reviewer regarding our care to not include sex*genotype interactions as major claims in our study will satisfy reviewer 2’s concerns.
SEE ATTACHED RESPONSE TO REVIEWERS FOR FIGURE
Response supplemental figure: rearing on molasses food (recipe per lab of Brian McCabe) does not drastically alter wild-type lifespan. In this pilot experiment (n = 1) iso w1118 and OR-R lifespan remain comparable to our standard diet, including the relative lifespans of those wild-types to each other.
Another weakness is the study regarding AMP and microbiome. The authors observed that both ΔAMP14 and RelE20 flies became sticky during aging and their foods in the vials were also sticky and discolored, implying the proliferation of microbiome in the gut and/or the external environment of these flies. To test the microbial load, the authors performed an assay of the bacterial abundance on the fly medium. They further performed antibiotic treatment in the food to remove microbiome as they declared in the study and examined the effect on the lifespan of ΔAMP14 flies. According to the knowledge of the reviewer, antibiotic treatment in food can restrict the microbiome in the gut. The authors have also mentioned in the manuscript the important role of gut microbiota in impacting Drosophila aging and lifespan. Thus, a more direct and widely-utilized way is to dissect the guts for microbial analysis (qPCR, 16S-seq, etc.), which is lacking in this study without reasonable explanations.
We published a paper in mBio on the relationship of AMPs and the microbiome with aging in much greater detail previously (Marra et al., 2021; mBio, ref25). That study did not perform lifespan experiments, but rather compared microbiome communities at 10 and 29 days. The novel aspect of this study is properly following survival. However, the reviewer is correct in their critique that our study is not especially innovative nor are our findings strikingly novel. We do have, we feel, an important set of experimental results to contribute to the field at large.
Reviewer #2 (Significance (Required)):
This study utilized various AMP mutants, but these mutants have been reported in the previous publications, making this study somehow lacking the novelty in this context. The IMD pathway has been shown to be involved in regulating fly lifespan, so the findings in this study are not that surprising. Additionally, this study doesn't show any creative improvements in terms of methodology and model system.
Admittedly, in some ways, much of our study is a “negative results” study. Given the contradictory nature of the literature claiming positive or negative effects of immunity and AMPs on lifespan, we feel this is a particularly valuable contribution to avoid publication bias focusing only on significant results in this controversial field. Still, we believe that the AMP/microbiome/lifespan interactions we uncover in our article will have an impact in the immune-aging field. Thus, while not being fully creative, our article is an important step for better characterizing of immune-aging relationships, which is of broad interest.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Gene knockouts provide definitive loss of function for individual and collective AMPs. This eliminates ambiguity caused by the partial efficiency of RNAi, and it disambiguates the pleiotropy caused by mutants of relish. Survival assays are conducted with and without the resident microbiome. This combined design leads to a clear conclusion: loss if individual (mostly so) AMPs does not affect adult survival either way, demonstrating these peptides likely function redundantly. Knock out of 14 AMPs (but still not all of those encoded in the genome) reduces survival when adults must cope with a microbiome. Depleting this microbiome sends survival back to normal. Perhaps these are not surprising results in that they show immune function is essential when challenged with pathogens, but the results are important because they unambiguously show that AMPs themselves are not a cause of intrinsic aging. This will finally put away that lingering hypothesis.
Overall, I like the scholarship of the work, of how it uses the literature, and its quality experimental execution. Cohort size, replication and survival analyses meet current, high standards. Demographic aging is supplemented with data on climbing rate as a function of age. It is a strength of the study that it is simple but comprehensive.
Reviewer #3 (Significance (Required)):
Aging across animal systems is strongly associated with changes in immunity and inflammation, both innate and adaptive. Overall, we want to understand if such changes are underlying causes of morbidity and mortality with age, or are consequences and compensation to underlying aging and cumulative pathogen exposure. These are difficult questions to address in mammalian systems but amenable using Drosophila which possesses a robust innate immune system. Researchers have used the fly for this end but still have mixed and ambiguous results. Now Hanson and Lemaitre provide a substantial design that fully controls the two essential ingredients: expression of antimicrobial peptides and the microbiome.
We thank the reviewer for their positive assessment. In particular, we appreciate the nod to the importance of the question on intrinsic contributions of AMPs to lifespan. We believe this is the key strength of our study’s mutant approach, which has been challenging to assess using previously-existing tools.
Additional Author changes (not requested by reviewers):
We realised there was a copy/paste error in data used in Figure 2. Specifically, extra OR-R experiments were included in these data for this figure that were not intended to be part of this figure. We have removed these OR-R experiments, which are experiments common to Figure S4 and remain visible in Figure S4. This does not impact any of the conclusions in the manuscript.
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Referee #3
Evidence, reproducibility and clarity
Gene knockouts provide definitive loss of function for individual and collective AMPs. This eliminates ambiguity caused by the partial efficiency of RNAi, and it disambiguates the pleiotropy caused by mutants of relish. Survival assays are conducted with and without the resident microbiome. This combined design leads to a clear conclusion: loss if individual (mostly so) AMPs does not affect adult survival either way, demonstrating these peptides likely function redundantly. Knock out of 14 AMPs (but still not all of those encoded in the genome) reduces survival when adults must cope with a microbiome. Depleting this microbiome sends survival back to normal. Perhaps these are not surprising results in that they show immune function is essential when challenged with pathogens, but the results are important because they unambiguously show that AMPs themselves are not a cause of intrinsic aging. This will finally put away that lingering hypothesis.
Overall, I like the scholarship of the work, of how it uses the literature, and its quality experimental execution. Cohort size, replication and survival analyses meet current, high standards. Demographic aging is supplemented with data on climbing rate as a function of age. It is a strength of the study that it is simple but comprehensive.
Significance
Aging across animal systems is strongly associated with changes in immunity and inflammation, both innate and adaptive. Overall, we want to understand if such changes are underlying causes of morbidity and mortality with age, or are consequences and compensation to underlying aging and cumulative pathogen exposure. These are difficult questions to address in mammalian systems but amenable using Drosophila which possesses a robust innate immune system. Researchers have used the fly for this end but still have mixed and ambiguous results. Now Hanson and Lemaitre provide a substantial design that fully controls the two essential ingredients: expression of antimicrobial peptides and the microbiome.
-
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study, Hanson and Lemaitre generated AMP single or compound mutants, and subjected them to lifespan analyses. They showed that deletion of all AMPs but not individually (except for Defensin) reduced Drosophila lifespan. The observations that ΔAMP14 suffered microbial dysbiosis and antibiotic treatment rescued the decreased lifespan in ΔAMP14 flies implied a link between AMP-controlled lifespan and host microbiome. Thus, the authors would like to conclude that AMPs contribute to fly lifespan via modulating microbiome.
Comments:
This reviewer deeply appreciates that the authors generated AMP mutants (individually or with various combinations). However, these mutants have been reported in their previous publications (eLife, 2019; Genetics, 2021). It seems that the authors carefully analyzed the lifespan of indicated flies, but many pieces of data are inconsistent with previous findings (for instance the lifespan under axenic condition). The explanation about the fly medium (Line 476-477) is not convincing (if yes, who not analyze the lifespan using the same diet as described in previous studies?). The lifespan analyses are of course the most pivotal part in this study, but it is a pity that the results are not shown in an appropriate way, making them rather difficult to interpret. For example, the reviewer is surprised to note that the lifespan of males and females are not analyzed and shown separately (e.g. Figures 1A, 1B, 2A, 2B, 3A, 4A), even the authors declared that they did this before lifespan examination. It is also unclear how many males and females were utilized individually in most assays.
Another weakness is the study regarding AMP and microbiome. The authors observed that both ΔAMP14 and RelE20 flies became sticky during aging and their foods in the vials were also sticky and discolored, implying the proliferation of microbiome in the gut and/or the external environment of these flies. To test the microbial load, the authors performed an assay of the bacterial abundance on the fly medium. They further performed antibiotic treatment in the food to remove microbiome as they declared in the study and examined the effect on the lifespan of ΔAMP14 flies. According to the knowledge of the reviewer, antibiotic treatment in food can restrict the microbiome in the gut. The authors have also mentioned in the manuscript the important role of gut microbiota in impacting Drosophila aging and lifespan. Thus, a more direct and widely-utilized way is to dissect the guts for microbial analysis (qPCR, 16S-seq, etc.), which is lacking in this study without reasonable explanations.
Significance
This study utilized various AMP mutants, but these mutants have been reported in the previous publications, making this study somehow lacking the novelty in this context. The IMD pathway has been shown to be involved in regulating fly lifespan, so the findings in this study are not that surprising. Additionally, this study doesn't show any creative improvements in terms of methodology and model system.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Whether and how age-associated immune hyperactivation affects the ageing process is a key question in the ageing and immunology fields. Using a CRISPR-based knockout approach, Hanson and Lemaitre address the effect of antimicrobial peptides (AMPs), essential immune effectors, on the ageing process. They compared the lifespan and climbing ability of flies deficient for groups of, or individual, AMPs to their isogenic control and the null mutants of the major immune pathways IMD and Toll. Although deletion of individual AMPs had limited effects, the authors detected an association between deletion of a group of AMPs on bacterial proliferation, and examined possible causality using antibiotic treatment. Interestingly, this causal link was missing in the IMD deficient strain, suggesting a potential AMP-independent mechanism of innate immune signalling in the ageing process. The topic is ambitious and exciting; however, the work has some quite serious technical limitations that would need to be addressed with further data analysis and experimentation.
Major comments:
- Figure 1A shows a large increase in fly lifespan in response to deletion of 8 AMPs on Chr II. However, the labelling on the figure shows that data from different experimental runs and from the two sexes have been combined to produce the result, and this problem runs through subsequent results. This is not an acceptable approach. Males and females have different average lifespans and may respond differently to the deletion. Repeats of the same lifespan measurement at different times often give significantly different absolute lifespans between runs, even if the differences between experimental treatment are consistent. The procedure used to construct this Figure therefore lumps heterogeneous data, which in this case is both biologically and, more generally, statistically invalid. We need to see an analysis where the results for the sexes and for different runs of the experiment are disaggregated. To assess the effect of the deletion, comparison should be made only within a single replicate of the experiment, and within each sex. It needs to be made clear how many experimental runs were done and with how many flies in each, since replicability is important with these labile traits. Data should be appropriately analysed, for instance with Log Rank test, or, if the assumptions of the test are fulfilled, with Cox Proportional Hazards. The authors mention analysis of median lifespans, but it is not clear what experimental data the medians were derived from, and this is not a gold standard approach to analysis of lifespan data. This problem also applies to the comparison made between lifespans of iso w1118 flies in Figure 1A and 1B - these were evidently measured in different experiments, so direct comparison cannot be made.
- Figure 2A reports data from mutants that were not backcrossed into a standard genetic background. The authors were aware that this can be a problem because they took care of it with their own mutants, but the data reported in this Figure 2A could be entirely attributable to difference in genetic background between stocks rather than the mutants themselves.
- Sexual dimorphism is widely reported in many ageing and immunology studies (reviewed in Belmonte et al., Front. Immunol., 2020 and Garschall et al., F1000Res., 2018). This is a problem for the data discussed on line 317, where it is suggested that Group A mutations are shorter lived than the control group because Group A carries the DefSK3 mutation. However, if the results are split by sex, the lifespan difference between Group A vs control is solely contributed by the shorter-lived male flies in Group A. The female flies in Group A live as long as the female control flies. However, the lifespans of both genders of the DefSK3 mutant in Figure 2B are all shorter than control flies. There appears to be a complicated interaction, with diverse function of individual AMPs during ageing, which cannot be summarised by the statement that "deleting single AMP genes has no effect on lifespan" on line 295.
- The authors claimed nora virus regulates fly lifespan but they do not produce any direct evidence that this is the case. Bleaching can remove many bacteria and viruses, including many pathogens. To establish the causal role of nora virus on lifespan, reinfection studies with a range of microbes including nora virus would be needed.
- The authors reported a potential AMPs-independent mechanism of the IMD/Relish pathway on ageing, which would be important. However, in Figure 4A and the associated raw data, the authors compared the lifespans of flies from different experiments, and this seems to be a problem. Using ΔAMP14 as an example of a more general problem: the authors assayed conventional feeding ΔAMP14 flies between 07.2021 and 11.2021; however, the antibiotic treated flies were assayed between 12.2021 and 02.2022. There is an obvious batch effect: the median lifespan of ΔAMP14 is 50d on 29.07.2021, 73d on 20.08.2021, 65d on 06.11.2021, and 61d on 10.11.2021. There is therefore a confounding of batch effects with the biological function of AMPs. Some groups seem to have extremely limited sample size, such as only two female flies and six male flies were recorded in "ATM8 23-07-2021 f" in Figure 2A-associated raw data.
Minor comments:
- The authors reported that the mutant flies lacking 14 AMPs are short-lived due to dysbiosis. Interestingly antibiotic treatment rescued the shortened lifespan of ΔAMP14 but not RelE20. Considering that some of these AMPs, such as lDef, Dro and Drs, are controlled by both the IMD and Toll pathways. It would be worth exploring if the axenic environment could improve the lifespan of Toll mutant flies, which might point to a distinct function of the Toll pathway on the microbiome and ageing process.
- Line 169: The authors stated that they screened the existence of common viruses but did not provide the results.
- Line 179: The authors need to include the quantitative results of nora virus in their 44 stocks.
- Lines 311-314 should be combined with the next paragraph as they are all about "screening".
- Line 319: Please indicate the associated panel "Fig. 2B" instead of using "Fig. 2".
- Lines 353 and 358 and in Figure 3C: The authors should provide the quantification of the sticky food in AMPs mutant and Relish mutant flies.
- Line 389: Please indicate the associated panel "Fig. 2A-B" instead of using "Fig. 2".
- Line 435: As discussed above, the authors cannot rule out an effect of individual AMPs on lifespan based on their current data and interpretation.
- Line 507: "During our study, we experienced a number of challenges to lifespan data interpretation." As discussed in the major comments, unless the authors re-perform and re-analyse the lifespan assays this problem will persist.
- Line 514: As discussed above, the authors cannot rule out the effect of other pathogens on lifespan.
Significance
The crosstalk between AMPs and ageing is a long-standing contradictory topic in the immunology and ageing field (Loch et al., PLoS One, 2017; Badinloo et al., Arch. Insect Biochem. Physiol., 2018; Garschall et al., F1000Res., 2018). This work is a potential step in determining whether and how AMPs regulate ageing. This research may open a door toward yet uncharacterized AMP-independent mechanisms of innate immune signalling in the ageing process.
I am familiar with Drosophila ageing and its relationship with innate immunity, although I am not an insect immunologist.
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Reply to the reviewers
RC-2022-01632
Answers to referees
First of all, we wish to thank the 3 referees for their careful evaluation of the manuscript. We see many issues that they have raised as legitimate and have tried to provide experimental or editorial answers. In contrast, some issues are presently addressed in the context of a future manuscript and we had rather not introduce these studies in the revised version.
Below, one will find the answers and the description of the revisions already introduced in the revised manuscript (questions are recalled in blue italics).
New and modified figures, plus not shown figures and tables are indicated in the text below but could not be pasted in the document and can be found in the Revision plan.
Referee # 1
Evidence, reproducibility and clarity
They then delivered 86/8 and LSBio anti-En1 antibodies, that catch En1 in the cleft and prevent it from being captured by MNs.
Perhaps we were not clear. We did not deliver the antibodies 86/8 and LSBio, we used them for western blots and immunohistochemistry (IHC) to identify EN1 and localize it. We delivered the third antibody, a single-chain anti EN1 antibody (scFvEN1), that captures extracellular EN1 and prevents it from being captured by MNs on the basis of the LSBio staining (Figure 4A-C).
Finally, heterozygotes revealed also a degeneration in dopaminergic neurons within midbrain similar to the one observed in spinal MNs, along with an upregulation of SQTSM1/p62 gene/protein, a factor in MN ageing linked to the classical genes implicated in familial forms of ALS (SOD1, TDP-43, FUS, and C9ORF72).
This is a fair comment/work description, that does not require answers.
Significance
*Major comments: *
It is unclear why levels of intensity for RNAscope were not quantified, and qPCR was preferred for quantifications in Figure 1b. RNAscope is a technique that allows for spatial distribution analysis of the markers and their level of the expression. This data can be easily quantified utilizing the QuPath software which is open access. Same concerns apply to Figure 2a.
Quantitative RT-PCR provides a quantitative measure of gene expression. Since only V1 interneurons (including, Renshaw cells) express EN1, we infer the spatial distribution, although not expression level cell by cell. Figure 2A is an actual counting at 4.5 months of En1+ cells and of Calbindin+ cells (Renshaw cells), both identified by RNAscope. Thus, it is clear that the number of En1-expressing cells (V1 interneurons) is not modified at 4.5 months when muscle weakness and death of aMNs are well advanced (around 70% of the aMNs that will eventually die, are already gone). Long-term survival of V1 interneurons is further demonstrated in Figure 2D (left panel) until 15.5 months, (see also below) whereas total En1expression is reduced by half. Quantification neuron by neuron of the amount of En1 transcribed (RNAscope) would indicate the variation, among interneurons, of En1 transcription in WT and mutant mice. This is interesting per se but would not modify the main information that these neurons do not die in the heterozygote and that En1 transcription does not decrease with time in both WT and mutant genotypes (at least until 15.5 months).
*Antibodies should be validated utilizing a reporter mouse. En1cre mice are commercially available and can be crossed with reporters (TdTomato or YFP mice). Utilizing this tissue En1 antibodies can be easily validated. The EN1 antibody shown in Figure 1c seems unspecific, staining several neuronal populations in the spinal cord. *
Indeed, antibody validation is extremely important. LSBio is commercial (CliniSciences), 86/8 was developed in the laboratory and fully characterized and used in previous studies (e.g. Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), scFv against EN1 was prepared from the 4G11 hybridoma (Developmental Hybridoma Bank, Iowa City, USA) and validated in previous studies (e.g. Wizenmann et al. Neuron 64: 355-366, 2009). In the present study, the two polyclonal were further validated inseveral ways.
In the WBs we compared ventral midbrain (VMB) and spinal cord (SC) tissues and found similar patterns. Strong evidence for antibody specificity is immunostaining extinction with the antigen and with absence of first antibody, which we carried out.
We have now used LSBio and 86/8 to perform a WB on spinal cord (SC) and ventral midbrain (VMB) extracts with or without the first antibody and we find that the absence of first antibody fully eliminates band staining. The western has been introduced in the revised manuscript in place of the cross immunoprecipitation.
Finally, we have quantified EN1 in the aMNs of the heterozygote at 3 months (before cell death), showing that EN1 content is decreased by approximately 2-fold (LSBio antibody) in both a and gMNs with no change in neuron number. This result demonstrating that EN1 is diluted by approximately twofold (concentration per neuron when all neurons are still present), in addition to further validating the antibody, is itself interesting and has been introduced in the revised manuscript as Supp. Fig. 1A.
Regarding the staining in other neuronal populations, there is always some background, in particular in the tissue treatment conditions used for RNAscope. Furthermore, given the large number and wide distribution of V1 interneurons (Fig. 1A), we cannot preclude that EN1 is present at a low concentration in the extracellular space and in several cell types (discussed in Fig. 9 of the manuscript). This does not weaken the main conclusion that it primarily accumulates in MNs which do not express En1 (RNAscope).
*Investigations of En1 expression in motor neurons from already available omics data sets would support the idea that En1 is expressed in motor neurons. *
The En1 locus is silent in MNs. Microdissection of MNs and proteomic analysis would not be definitive since the interneurons that produce EN1 are in close vicinity of the MNs and since some protein is necessarily present in the extracellular space (where it is trapped by scFvEN1), making contamination unavoidable.
Differentiation between Gamma and Alpha motor neurons should be performed using specific markers as Err3, Wnt7a or NeuN.
This is a possible way to do the distinction, but size criterion in Cresyl violet is supported in the literature (Wu et al. Journal of Biological Chemistry, 287: 27335-27344, 2012; Dutta et al. Experimental Neurology, 309: 193-204, 2018). In our study, it is further validated by the demonstration that, in 9-month-old animals, the results obtained (cell number and specific death of large neurons >300µm2, but not of intermediate size ones 200-299µm2) are replicated by counting ChAT-stained neuron (Figure 2C). It is of particular interest that the number of medium size neurons (also ChAT-positive medium size MNs) does not increase when the number of large size (Cresyl and ChAT-positive) neurons decreases, thus precluding a “shrinkage effect”. Most importantly, the size criterion (Cresyl violet) allows us not to be mistaken by a possible down-regulation of markers in the mutant, independently of cell survival. We provide for the reviewer (Revision plan) but not for publication, the evolution with time of the number of neurons based on size (above 200 µm2) showing clearly that at 15.5 months the large population (>300 µm2) is decreased in the En1-Het, with very little change for neurons between 200 and 300 µm2, and certainly not an increase which would be expected if shrinkage occurred.
We were indeed surprised by this finding and a plausible explanation is that a lower metabolic activity makes interneurons less sensitive to stress than aMNs which have to “fuel” long axons and high firing rates (not the case for gMNs). We propose this explanation in the discussion and make it clearer in our revised version. We agree that it is speculative and that the point raised by the reviewer is very interesting. We hope to address this in the future and have discussed this point.
Since the cells do not die, we did not look for signs of apoptosis.
We analyze lumbar sections from L1 to L5 as now indicated in the methods section in the manuscript
The set of experiments reported in Figure 4 is of difficult interpretation without showing the actual presence of extracellular En1, that could be assessed with protein detection or RNAscope.
This is another interesting suggestion, but we think that it will be difficult to distinguish low extracellular staining due to EN1 diffusion from some unspecific background. Since the scFvEN1 is secreted by astrocytes, it necessarily neutralizes extracellular EN1, resulting in a decrease in the MN content of the protein. This is an experiment with high specificity since the same scFv harboring a Cysteine to Serine point mutation that prevents EN1 recognition (no disulfide bound formation between the light and heavy chains) does not block EN1 capture by MNs (Fig. 4C for IHC and quantifications).
As for extracellular EN1 mRNA identified by RNAscope, we hesitate to embark on the idea as mRNAs are likely secreted in insufficient amounts to be identified, even by RNAscope. The results that we have (no En1 visible by RNAscope in MNs, loss of EN1 in MNs following extracellular scFvEN1 activity, and preferential addressing of injected EN1 to MNs) demonstrate EN1 capture by MNs. Indeed, we cannot completely preclude the transfer of tiny amounts (escaping RNAscope detection in MNs) of En1 mRNA (for example, through extracellular vesicles), but we plead for not considering this hypothesis in the present paper. However, if the reviewer wishes, the possibility can be introduced in the discussion.
Referee 2
Evidence, reproducibility and clarity
In general, most of the experiments shown in this study are well done and convincing. However, the data on p62 upregulation appear correlative and do not allow any conclusions about the mechanism and function how EN-1 modulates motoneuron survival and function. In addition, this study is not very precise on the mechanisms how motoneurons degenerate in this model so that there are only limited insights into the way how EN-1 acts on motoneurons in a physiological manner and under pathophysiological conditions.
This criticism is justified, at least in part, as we agree that p62 upregulation is correlative. However, the fact that the neutralization of extracellular EN1 by the scFv increases p62 expression, is in favor of a causative link. The increase is also seen at 3 months in the En1-Het when all aMNs are still present but not after, which is interesting because, due to aMNs death, surviving MNs receive more EN1, information provided below and now introduced and discussed in the revised manuscript (Supp. Fig. 1B).
As for p62, and as also mentioned by referee 3, Fig. 8 is very hard to follow and we propose to simplify it to make the message clearer:
We have revised Fig. 8C, D in which we focus exclusively on SQTSM1/p62 mean expression (see revision plan)
A second information is that a difference in mean p62 expression between WT and Het is seen only at 3 months in aMNs. For aMNs, we propose that this is due to the fact that they are very sensitive to EN1 dosage (in contrast with gMNs which do not die in the En1-Het). At 3 months, aMNs have only half of their normal EN1 content. Later, at 4.5 months 75% of the aMNs bound to die are already dead (Fig. 2D) and the remaining neurons receive more EN1 (even more so at 9 months), as could be measured (see above Supp. Fig. 1B). We thus can propose an accelerated aging of aMNs at 3 months due to both EN1 decrease and high metabolic activity (higher than in gMNs).
In the case of the scFv, scFvEN1, but not the mutated version induces enhanced mean p62 expression in the 80% surviving aMNs and in gMNs at 7 months (low aMN death in this model, see Fig. 4F). As can be seen also in a newly added figure (Supp. Fig. 2) that has been introduced in the revised manuscript and is shown below, 7-month-old scFv animals and 3- to 3.5-month-old En1-Het have similar phenotypes. This mild scFv phenotype (a-MN death and muscle strength loss) in 7-month-old mice in spite of a huge loss in the EN1 content of MNs (Fig. 4C) suggests that the En1-Het phenotype is not entirely due to the decrease in EN1 transport from V1 interneurons to MNs (see discussion and Fig. 9).
It remains true that we have voluntarily decided not to examine in depth the molecular mechanisms allowing EN1 to exert its protective activity, a decision that we would like to defend and maintain.
A first reason is that in previous papers on mesencephalic dopaminergic (mDA) neurons (Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), we evaluated several mechanisms involved in EN1 neurotrophic activity and we did not want this study to be a duplication of studies done on a different neuronal population, even if mechanisms might differ in part, between aMNs and mDA neurons. What has interested us more is that, in the two cases, age is an important factor in the unveiling of the degeneration phenotype (mDA neurons start dying at 1.5 months and aMNs at 3 months). It is because of this similarity that we performed the bioinformatic study that has led us to SQTSM1/p62. In this context, it is of interest that mean SQTSM1/p62 expression (variability of expression between neurons is not discussed in the revised version) increases with age in the wild type, thus can be seen as an age marker. It allows us to propose that EN1 extracellular neutralization and the loss of one En1 allele, that increases mean SQTSM1/p62 expression accelerate aging.
A second reason is that the study is oriented toward a possible use of EN1 as a therapeutic protein. This orientation also has to do with the focus on SQTSM1/p62. Indeed, there are probably many pathways downstream of EN1, but in the bioinformatic analysis of genes differentially regulated in WT and En1-Het mDA neurons and also expressed in MNs, SQTSM1/p62 is the only one that interacts with the 4 genes mutated in the major ALS familial forms. In addition, SQTSM1/p62 mutations have been observed in ALS patients (References 41 to 45 in the manuscript).
Finally, the most important point is that the main message of this paper is the discovery of a non-cell autonomous EN1 activity in the spinal cord and of its ability to travel between V1 interneurons and MNs. This specificity best explained by a targeting signal that we have identified is at the basis of the specific addressing to MNs of EN1 intrathecally injected, which also has implications for its potential therapeutic use.
Specific points of criticism
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- In Fig. 2a, the authors show that EN-1-positive interneurons are not reduced at 4.5 months in the spinal cord. No data are shown for later time points such as 9 months, the corresponding stage when motoneuron loss is observed, or at 16 months which corresponds to the data shown in Fig.1. The argument that there is no reduction of V1 interneurons between 4.5 months and 16 months because there is no decrease of EN-1 expression between 4.5 and 16 months, as shown in Fig. 1b is not convincing. EN-1 expression could change in individual cells, thus compensating for the loss. Data on numbers of EN-1-positive cells at 9 and 16 months should be included, and a potential autocrine effect of EN-1 on V1 interneurons, as observed in midbrain dopaminergic neurons, characterized in more detail. * Fig. 2A illustrates the absence of interneuron loss at 4.5 months, but this set of data is completed by those of Fig. 2D that demonstrate the maintenance of V1 interneuron number until 15.5 months, at least. It can be noted that, in contrast with interneurons, aMNs at 4.5 months have experienced massive cell death (70% approx. of total aMN death at 15.5 months). As a whole, data of Fig. 2 demonstrate that the number of small neurons (100-199 µm2) and intermediate size neurons (200-299 µm2) does not change with age, at least through 15.5 months. This is in strong contrast with large aMNs (>300 µm2). As already explained in our answers to referee 1, size is an excellent marker for the identification of neuronal subtypes and the analysis of survival (See answers to referee 1, justifying the use of neuron size).
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In Fig. 2e, the authors present data on loss of muscle strength between 4.5 and 15.5 months. They conclude that this reflects gradual neuromuscular strength loss. Since neuromuscular endplates have a very high safety factor, they can maintain full function even if transmitter release is reduced by more than 80%. Therefore, the loss of muscle strength seems to reflect the progressive loss of presynaptic terminals at neuromuscular endplates, rather than a gradual loss of neuromuscular strength. *
We apologize for the semantic confusion. What is measured is a progressive loss of muscle strength due to the progressive loss of presynaptic terminals and not a gradual loss of neuromuscular strength. This is now modified throughout the revised text.
- More detailed data on NMJ morphology should be included. How does EN-1 modulate neuromuscular endplates? Is EN-1 located at neuromuscular endplates after being taken up from motoneurons? Even if the mechanism is indirect, via upregulation of p62 under conditions when EN-1 signaling is reduced, does this situation lead to enhanced localization of p62 at neuromuscular endplates? *
We do not see expression of En1 mRNA or the presence of EN1 protein at the level of the endplate (Supp. Fig. 3 in revision plan)
- The data shown in Fig. 3 on changes in NJM morphology appear incomplete and not convincing. As SV2a is not a good marker for changes in presynaptic compartments since it does not allow conclusions on how many synaptic vesicles are released, additional markers for presynaptic active zones such as Bassoon, Piccolo, Munc-13 should be studied. The analysis of fully occupied endplates appears arbitrary, and the differences are relatively small. Additional EM pictures and quantitative analyses of active zone proteins in the presynaptic compartment would help to support the argument of the authors that presynaptic compartments degenerate before cell bodies are lost in EN-1 +/- mice. *
SV2a and NF staining (it is not only SV2a) at the level of endplates identified by a-Bungarotoxin labeling has been used in a large number of studies (Wahlin et al. J. Comp. Neurol. 506: 822-837, 2008; Hasting et al. Scientific Reports 10: 1-13, 2020; Yahata et al. J. Neurosci. 29: 6276-6284, 2009 ; Jones et al. Cell Reports 21: 2348-2356, 2017) Our goal was not to document the loss of synaptic activity through the use of the three suggested markers, Bassoon, Piccolo and Munc-13. Doing it would force us to initiate experiments taking several months to prepare the material and do a quantitative analysis in the models of EN1 loss of function (En1-Het) and neutralization (scFv), plus rescue by EN1. Nor do we wish to initiate a novel collaboration to produce a quantitative ultrastructural study. We see the latter morpho-functional studies beyond the scope of the manuscript and wish to be given the possibility to present them in a separate study (see below in “Description of the experiments that the authors prefer not to carry out”).
The distinction between fully occupied, partially occupied and denervated endplates is not arbitrary and we apologize for not having sufficiently described the methodology. As illustrated in modified Fig. 3 and explained in Material and Methods, a fully innervated endplate is defined as an endplate in which 80% or more of the green pixels (a-BGT) are covered by a red pixel (SV2a), a partially one is between 20 and 80% and a denervated one below 20% coverage. Thus at 9 months and later ages, close to 30% of the endplates are either partially innervated or denervated. In fact, it is more likely that they are partially innervated since the number of AChR clusters does not change (totally denervated clusters normally dissolve). The 80% threshold for fully innervated was selected to give a margin of security, and it is likely that the percentage of 25 to 30% of partially innervated endplates is an underestimation.
In the Revision plan is presented a table with the calculations and modified Figure 3.
We agree that we were not clear enough in our description and that it may have given the impression that the differences were relatively small. We think that retrograde degeneration is strongly supported by a loss of muscle strength that parallels the decrease in fully occupied endplates (a-BGT, NF, SV2a) and precedes aMN loss by more than 1 month. We have recently contacted an electrophysiology group to establish a collaboration that will allow us to follow functional changes at the level of the spinal cord and of the neuromuscular junction and we see the experiments proposed by the reviewer as complementary to these physiological approaches. Yet, we do not want to ignore the opinion of the reviewer and mention it in the conclusion, on the basis of his/her comment.
- The authors present evidence for a glycosaminoglycan (GAG) binding domain that appears responsible for uptake of EN-1 into motoneurons. However, it is unclear into which cellular compartment EN-1 is taken up after GAG binding on motoneurons. The authors propose this could be an alternative pathway to conventional endosomal uptake. How can the EN-1 that is taken up into cells exert transcriptional effects in motoneurons? As a minimum, more data on the subcellular distribution of endocytosed EN-1 should be included to support current hypotheses and to close the gap from cellular uptake to transcriptional regulation. *
The question is justified since we did not recall until page 12 of the Discussion that EN1 is, as most tested homeoprotein transcription factors, captured by a mechanism distinct from endocytosis. While not yet fully understood, the process involves the formation of inverted micelles that allow for direct targeting to the cytoplasm and from there to the nucleus thanks to the NLS. We now mention in the introduction that EN1 transfer and HP transfer is based on unconventional secretion and internalization processes.
- The differences in p62 expression with age in WT and EN-1 +/- mice as shown in Fig. 8c are not convincing. First, the p = 0.0499 and p = 0.0536 values for differences at 3-4 months of age appear borderline, and it is unclear what the dispersion analysis that is shown really means. Moreover, the question remains how a potential dysregulation of p62 then affects NMJ morphology and function. Is this change in p62 also detectable in presynaptic compartments? *
We agree that p values in the range of 0.05 are not extremely high and this is due to the heterogeneity in SQTSM1/p62 expression, that reflects that of MN populations, and induces a high variance. We also agree that this figure is too complicated and a simplified version has been proposed above (see answers to reviewer 1). To summarize, Fig. 8C shows that in WT animals, with no aMN death (grey) the level of SQTSM1/p62 expression in aMNs and gMNs increases between 3 and 4.5 months and between 4.5 months and 9 months, with significances varying between pThe new Fig. 8 panel D (please see above, answers to referee 1) now includes the results obtained with the scFvs. A phenotype comparison between the two models (En1-Het and scFvEN1) has been introduced in Supp. Fig. 2 (see above).
We have no evidence that EN1 modulates the SQTSM1/p62 promoter directly. The identification of this gene as a target (not necessarily a direct target) of EN1 comes from the bioinformatic analysis described in the manuscript and we were intrigued by the interaction with the 4 main familial ALS mutations and the existence of families with SQTSM1/p62 mutations. This is what led us to analyze its expression in our two models of EN1 loss of function. Although the En1-Het mouse is not an ALS model, the results support the idea that EN1 could be used as a therapeutic protein in several familial and even sporadic forms of the disease. The latter hypothesis is now being tested on MNs derived from iPSCs (sporadic patients, fALS and isogenic variants, and healthy controls). If the data lend weight to our hypothesis, as collaborative and in-house preliminary data suggest, then a complete analysis of EN1 targets in human MNs will be undertaken. Again, we really think that this is out of the scope of this study.
For Fig. 8, we fully agree that it can give headaches and we apologize. Moreover, it induces wrong interpretations (mean intensity increases with age and dispersion between 4.5 and 9 months has a calculated p__Referee #3__
Evidence, reproducibility and clarity
Nevertheless, the connection between EN-1 and p62 is not well developed by the data presented and future readers may be left with many questions regarding how EN-1 and p62 are related (e.g. direct interaction? transcriptional regulation?), whether p62 is indeed the mediator of EN-1 trophic effects, or the significance of the increased levels of p62 for motoneuron disease
The reviewer is right and we have tried to better explain and to simplify. Please see responses to referees 1 and 2.
*Figure 1C: There appears to be EN1 immunoreactivity (green) in several areas of the spinal cord, including dorsal regions. Can the authors clarify what that labeling could be representing? *
Unfortunately, there is always some background staining, in particular in the tissue treatment conditions appropriate for RNAscope. Furthermore, given the large number and wide distribution of V1 interneurons (Fig. 1A), we cannot preclude that EN1 is present at a low concentration in the extracellular space and in several cell types (now represented in Fig. 9). This does not weaken the main conclusion that it primarily accumulates in MNs which do not express En1 (RNAscope).
*Figure 1D: These immunoprecipitation results lack a negative control with irrelevant antibody to confirm that the band shown it's being recognized specifically by the antibodies reacting with the blot. *
Please see the response to reviewer 1 above with the Western blot and the absence of staining on a WB in absence of first antibody (86/8 or LSBio).
F*igure 1E: The intensity of the EN1 labeling in MNs, much stronger than in V1 interneurons, is intriguing, given that MNs do not express engrailed-1 mRNA. One would have expected the opposite. It may help here if it was possible to show that immunoreactivity in MNs is diminished in the het mutant mouse. *
We also were surprised by this intensity higher in MNs than in V1 interneurons, as if the protein was exported rapidly towards the target neurons. We have done the experiment proposed by the referee, found a twofold (approx.) immunoreactivity reduction in En1-Het MNs (see above Supp. Fig. 2A in answers to referee 2). This supplemental figure has been introduced in the revised version. The experiment was done at 3 months when no MN death has yet occurred. Later the neurons “replenish” with EN1, probably because they do not have to share the limited supply with the dead ones (see above answers to referee 2 and Supp. Fig. 2B).
*Figure 2D: There are a few possible problems with these data and their interpretation. First, this reviewer feels that 5 neurons (y-axis) is a rather small number. Are these 5 neurons per what area? From how many mice? I did not find that information in the figure legend. A larger area should be quantified so that we get numbers that are more robust. Second, such differences could also be due to hypotrophy of the MNs, namely, that MN number is the same but they are smaller. *
The differences cannot be attributed to hypotrophy. A first reason is that, at 9 months, the Cresyl violet and ChAT staining give the same results for medium size and large neurons (Fig. 2C). Furthermore, when one counts the cells throughout 15.5 months, the decrease in the number of large neurons is not compensated by an increase in the number of medium size or small ones. The reasoning and a graph, not intended for publication can be found in answers to referee 1.
*Figure 3A: It would be useful that the authors explain how these AChR clusters were defined, visualized and counted. I could not find this information in the Methods. Perhaps this could be done by showing an alpha-BTX image illustrating the clusters. *
We fully agree that the procedure was not well explained and we have introduced a correction in the Material and Methods section. For more details, please see answers to referee 2.
*Figure 3B: As each adult endplate is only innervated by one MN, one would have expected fewer clusters and/or endplates, if indeed MNs are missing in this mouse, rather than endplates that are partially occupied. This could be clarified a bit more explicitly. *
This is true and the ambiguity takes its origin in insufficient explanation of how fully innervated, partially innervated and denervated endplates were defined. Please see above and also in answers to reviewer 2. Modifications have been introduced in the text and in Fig. 3. The referee is right, the absence of change in the number of AChR clusters suggests that there are very few fully denervated endplates and that what is defined as such in the analysis corresponds to partially innervated endplates (see above). This is now discussed in the text.
Figure 6B: Would not be better to do this with a virus, like in the case of the antibody? A more robust effect on MN survival may be attainable and thus strengthen the concept.
This would be another interesting experiment and we are presently exploring this possibility (with preliminary results). The choice of the virus and of the promoters is very important. We are comparing several AAVs, including AAV2, AAV2-TT (which diffuses better) and AAV8. For the promoter, we do not want to express within MNs as the imported protein might have special properties, associated with import. V1 interneurons would be best, but we have to verify if this does not modify V1 physiology. Astrocyte is another option, but with a similar pitfall. This means that we have a long way to go before proposing a “gene therapy” approach.
In addition, in the context of future clinical studies, we were eager, on the basis of the long-lasting activity of the protein already observed in the mesencephalic dopaminergic neurons (Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Rekaik et al. Cell Reports 13: 242-250, 2015; Blaudin de Thé et al. EMBO J. 37: e97374, 2018), to try a protein therapy in the spinal cord. Interestingly, the effects are also long-lasting in the spinal cord, (12 weeks in the mouse before a second injection is needed) and, according to contacted physicians, intrathecal injections, every second month or even more frequently, could be envisaged in the human. In that case, protein injection is possibly advantageous for the following reasons:
(i) viral particles can travel far and we do not know what would be the side effects.
(ii) the protein is short-lived but specifically addressed to MNs (thanks to the presence of EN1 binding sites at their surface), thus minimizing the issues associated with permanent expression and side effects.
(iii) EN1 is a natural protein normally secreted and the immune system might not be solicited as much as with viral approaches.
*Figure 7A: The protein seems to be mainly in the cytoplasm of those cells (nuclei are dark and unlabeled), which is also unusual for a transcription factor that functions in the nucleus. Also surprising that the protein is gone in 3 days, but has effects over 24 weeks. Any explanation for that? *
The protein is imported and is thus both in the cytoplasm where it exerts an effect on protein translation (Brunet et al. Nature 438: 94-98, 2005; Alvarez-Fischer et al. Nature Neurosci. 14: 1260-1266, 2011; Yoon et al. Cell 148: 752-764, 2012) and in the nucleus where it exerts its transcriptional and “epigenetic activity (see below for the latter). In fact, different antibodies and fixation procedures can favor cytoplasmic or nuclear staining. When nuclear, the dark point at the center, probably the nucleolus is less stained.
Two images illustrating this point are shown in the revision plan.
For the second part of the question, three days are sufficient for a long-lasting activity. This was also observed in the midbrain where the protein restores the epigenetic marks jeopardized by an acute oxidative stress (Rekaik et al. Cell Reports 13: 242-250). This has led to the hypothesis that EN1 has an important action at the level of the structure of the heterochromatin, thus a long-lasting “epigenetic” activity. We are presently working on the latter effects on the chromatin structure using human MNs derived from iPSCs (patients and control).
*Figure 7B: It's not clear what the blue and red bars mean, as this is not explained in the legend. Also, the y-axis says "%Chat+" suggesting they are counting MNs, but in the text they talk about EN-1 capture. If the latter, the y-axes should indicate % EN-1 over Chat, or something like that. In general, better figure legends would improve the experience of the reader. *
In this experiment, we wanted to test the presence of a GAG-binding domain in EN1. To test its potential role in EN1 internalization and localization, we co-injected or not the RK-EN1 with hEN1 protein. Then, we counted the percentage of MNs (%ChAT+) which contain, or not, the hEN1 protein (hEN1+ in red or hEN1- in blue), allowing us to verify if the RK-EN1 alters the internalization of the hEN1 protein. So yes, we are looking at the capture of EN1 by the MNs with or without the RK-peptide (or control peptides). We have modified the text to make the point clearer.
*Statistical analyses: In principle, comparisons of data obtained in studies that involved two variable parameters (such as time and genotype/treatment) should be weighted by a 2-way ANOVA test, which is more stringent since more conditions are being tested simultaneously. Usually a t-test is reserved for a pairwise comparison in an experiment involving only two conditions of the same variable. *
The reviewer is correct. The two-way ANOVA is explained in the Statistical analyses section of the Methods. The analyses were carried out and the results listed in the legends for Figs 2, 3, 4, 6 and Supp. Fig. 1.
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Referee #3
Evidence, reproducibility and clarity
This is an interesting and provocative manuscript reporting non-cell autonomous trophic activities of a homeobox protein, a concept pioneered by Dr. Prochiantz since many years ago. The study involves a significant amount of experimental work and the authors are to be congratulated by the scope and ambition of their study. Given previous studies by this laboratory on EN-1 functions in midbrain dopaminergic neurons, the concept advanced in the present paper is not entirely novel, although it is indeed interesting to find EN-1 activities in motoneurons; these were unexpected. Given that this is a non-cell-autonomous effect (EN-1 is made and released by neurons adjacent to MNs), it would have been interesting to explore the conditions under which EN-1 synthesis, release and effects are regulated, whether by lesion, degeneration, etc. But that may be something the authors wish to leave for a future report. It is welcome that an effort was put into trying to mechanistically understand how these trophic effects are mediated. This reviewer understands that this is a major undertaking. Nevertheless, the connection between EN-1 and p62 is not well developed by the data presented and future readers may be left with many questions regarding how EN-1 and p62 are related (e.g. direct interaction? transcriptional regulation?), whether p62 is indeed the mediator of EN-1 trophic effects, or the significance of the increased levels of p62 for motoneuron disease. In its present form, this paper will be welcome, if nothing else by the provocative ideas that it advances. For this, it clearly deserves to be published in a good journal (whatever that means these days). Here below are a few questions and suggestions which the authors may want to take into consideration.
Figure 1C: There appears to be EN1 immunoreactivity (green) in several areas of the spinal cord, including dorsal regions. Can the authors clarify what that labeling could be representing?
Figure 1D: These immunoprecipitation results lack a negative control with irrelevant antibody to confirm that the band shown it's being recognized specifically by the antibodies reacting with the blot.
Figure 1E: The intensity of the EN1 labeling in MNs, much stronger than in V1 interneurons, is intriguing, given that MNs do not express engrailed-1 mRNA. One would have expected the opposite. It may help here if it was possible to show that immunoreactivity in MNs is diminished in the het mutant mouse.
Figure 2D: There are a few possible problems with these data and their interpretation. First, this reviewer feels that 5 neurons (y-axis) is a rather small number. Are these 5 neurons per what area? From how many mice? I did not find that information in the figure legend. A larger area should be quantified so that we get numbers that are more robust. Second, such differences could also be due to hypotrophy of the MNs, namely, that MN number is the same but they are smaller.
Figure 3A: It would be useful that the authors explain how these AChR clusters were defined, visualized and counted. I could not find this information in the Methods. Perhaps this could be done by showing an alpha-BTX image illustrating the clusters.
Figure 3B: As each adult endplate is only innervated by one MN, one would have expected fewer clusters and/or endplates, if indeed MNs are missing in this mouse, rather than endplates that are partially occupied. This could be clarified a bit more explicitly.
Figure 6B: Would not be better to do this with a virus, like in the case of the antibody? A more robust effect on MN survival may be attainable and thus strengthen the concept.
Figure 7A: The protein seems to be mainly in the cytoplasm of those cells (nuclei are dark and unlabeled), which is also unusual for a transcription factor that functions in the nucleus. Also surprising that the protein is gone in 3 days, but has effects over 24 weeks. Any explanation for that? Figure 7B: It's not clear what the blue and red bars mean, as this is not explained in the legend. Also, the y-axis says "%Chat+" suggesting they are counting MNs, but in the text they talk about EN-1 capture. If the latter, the y-axes should indicate % EN-1 over Chat, or something like that. In general, better figure legends would improve the experience of the reader.
Statistical analyses: In principle, comparisons of data obtained in studies that involved two variable parameters (such as time and genotype/treatment) should be weighted by a 2-way ANOVA test, which is more stringent since more conditions are being tested simultaneously. Usually a t-test is reserved for a pairwise comparison in an experiment involving only two conditions of the same variable.
Significance
see above
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Referee #2
Evidence, reproducibility and clarity
Engrailed-1 does not act only in a cell-autonomous way in neural development, but also has non-cell-autonomous functions. These functions depend on the release of this homeoprotein which has been characterized in much detail by previous work of this group. In this paper, they show that EN-1 is expressed in spinal V1 interneurons, both on the RNA and on the protein level. In spinal motoneurons, EN-1 protein but not RNA is detected. Neutralization of extracellular EN-1 with a secreted antibody apparently blocks transfer from these interneurons to motoneurons and causes motoneuron disease symptoms. A similar phenotype is also observed in EN-1 +/- mice. Most importantly, the authors also demonstrate that intrathecal injection of EN-1 into EN-1 +/- mice restores loss of muscle strength and prevents motoneuron death. The authors also show that the autophagy modulator SQTSM1/p62 is expressed at elevated levels in EN-1 +/- mice and in mice after injection of the EN-neutralizing antibody. Since p62 expression also seems to be increased in general during aging in motoneurons, the authors conclude that EN-1 from spinal V1 interneurons is a regulator of motoneuron aging. In general, most of the experiments shown in this study are well done and convincing. However, the data on p62 upregulation appear correlative and do not allow any conclusions about the mechanism and function how EN-1 modulates motoneuron survival and function. In addition, this study is not very precise on the mechanisms how motoneurons degenerate in this model so that there are only limited insights into the way how EN-1 acts on motoneurons in a physiological manner and under pathophysiological conditions.
Specific points of criticism:
- In Fig. 2a, the authors show that EN-1-positive interneurons are not reduced at 4.5 months in the spinal cord. No data are shown for later time points such as 9 months, the corresponding stage when motoneuron loss is observed, or at 16 months which corresponds to the data shown in Fig.1. The argument that there is no reduction of V1 interneurons between 4.5 months and 16 months because there is no decrease of EN-1 expression between 4.5 and 16 months, as shown in Fig. 1b is not convincing. EN-1 expression could change in individual cells, thus compensating for the loss. Data on numbers of EN-1-positive cells at 9 and 16 months should be included, and a potential autocrine effect of EN-1 on V1 interneurons, as observed in midbrain dopaminergic neurons, characterized in more detail.
- In Fig. 2e, the authors present data on loss of muscle strength between 4.5 and 15.5 months. They conclude that this reflects gradual neuromuscular strength loss. Since neuromuscular endplates have a very high safety factor, they can maintain full function even if transmitter release is reduced by more than 80%. Therefore, the loss of muscle strength seems to reflect the progressive loss of presynaptic terminals at neuromuscular endplates, rather than a gradual loss of neuromuscular strength.
- More detailed data on NMJ morphology should be included. How does EN-1 modulate neuromuscular endplates? Is EN-1 located at neuromuscular endplates after being taken up from motoneurons? Even if the mechanism is indirect, via upregulation of p62 under conditions when EN-1 signaling is reduced, does this situation lead to enhanced localization of p62 at neuromuscular endplates?
- The data shown in Fig. 3 on changes in NJM morphology appear incomplete and not convincing. As SV2a is not a good marker for changes in presynaptic compartments since it does not allow conclusions on how many synaptic vesicles are released, additional markers for presynaptic active zones such as Bassoon, Piccolo, Munc-13 should be studied. The analysis of fully occupied endplates appears arbitrary, and the differences are relatively small. Additional EM pictures and quantitative analyses of active zone proteins in the presynaptic compartment would help to support the argument of the authors that presynaptic compartments degenerate before cell bodies are lost in EN-1 +/- mice.
- The authors present evidence for a glycosaminoglycan (GAG) binding domain that appears responsible for uptake of EN-1 into motoneurons. However, it is unclear into which cellular compartment EN-1 is taken up after GAG binding on motoneurons. The authors propose this could be an alternative pathway to conventional endosomal uptake. How can the EN-1 that is taken up into cells exert transcriptional effects in motoneurons? As a minimum, more data on the subcellular distribution of endocytosed EN-1 should be included to support current hypotheses and to close the gap from cellular uptake to transcriptional regulation.
- The differences in p62 expression with age in WT and EN-1 +/- mice as shown in Fig. 8c are not convincing. First, the p = 0.0499 and p = 0.0536 values for differences at 3-4 months of age appear borderline, and it is unclear what the dispersion analysis that is shown really means. Moreover, the question remains how a potential dysregulation of p62 then affects NMJ morphology and function. Is this change in p62 also detectable in presynaptic compartments?
- Is there any molecular evidence that EN-1 modulates the p62 gene promoter directly? What is the argument to assume that increase in SQTSM1/p62 expression and dispersion is an indicator of aging? The mean intensity, if I understand Fig. 8c correctly, does not significantly increase, it is only the dispersion that changes. In general, the data shown in Fig. 8c are hard to read and interpret. For example, in the right panel, the difference between the dispersion in 4.5 and 9 month old EN +/- mice is indicated as p = 0.06, but marked with 4 stars. The presentation of these data should be changed to make them clearer.
Referees cross-commenting
I agree with all comments from the other reviewers
Significance
This study expands previous work of the authors, in particular work that has been performed and published on the effects of EN-1 on mesencephalic dopaminergic neurons. If adequately revised, it could make an interesting contribution to the general understanding how spinal V1 interneurons act on funcitonality and survival of spinal motoneurons.
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Referee #1
Evidence, reproducibility and clarity
The present work focuses on Engrailed 1 (En1), a homeoprotein that is expressed in spinal V1 interneurons that connect to α-motoneurons (MNs). The authors studied its role in neuromuscular strength and MN retention and loss with the aid of different approaches. First, they studied its expression in spinal cord with RNAscope, a novel ISH method that makes possible to detect biomarkers that would be otherwise difficult to study with traditional ISH techniques. They then delivered 86/8 and LSBio anti-En1 antibodies, that catch En1 in the cleft and prevent it from being captured by MNs; moreover, they used a heterozygotic En1 mouse model to reduce En1 levels. The behavioral assessment, studied with grip strength, inverted grip test and hindlimb extensor reflex, showed motor alterations, paralleled by α-MNs loss and with an even stronger phenotype in the heterozygotic mice. This phenotype, however, appeared weeks before the MN loss, so they used NMJ assessment to determine what it seems to be a retrograde degeneration. En1 administered intrathecally was effectively internalized by the MNs and led to a long-term amelioration of the motor impairments and renervation of the NMJ, that needed to be boosted after 12 weeks for a stable therapeutic effect. Finally, heterozygotes revealed also a degeneration in dopaminergic neurons within midbrain similar to the one observed in spinal MNs, along with an upregulation of SQTSM1/p62 gene/protein, a factor in MN ageing linked to the classical genes implicated in familial forms of ALS (SOD1, TDP-43, FUS, and C9ORF72). They authors did not observe degeneration in V1 interneurons. They conclude that En1 might have a role in regulating MN ageing in degenerative motor disorders.
Significance
Overall, the manuscript is well written however, some of the data appears too preliminary for publication. While the potential beneficial effect of En1 intrathecal administration looks promising and worth of publication, it is difficult to understand the mechanism of action. Some of the results are puzzling and require further investigations.
Major comments:
It is unclear why levels of intensity for RNAscope were not quantified, and qPCR was preferred for quantifications in Figure 1b. RNAscope is a technique that allows for spatial distribution analysis of the markers and their level of the expression. This data can be easily quantified utilizing the QuPath software which is open access. Same concerns apply to Figure 2a.
Antibodies should be validated utilizing a reporter mouse. En1cre mice are commercially available and can be crossed with reporters (TdTomato or YFP mice). Utilizing this tissue En1 antibodies can be easily validated. The EN1 antibody shown in Figure 1c seems unspecific, staining several neuronal populations in the spinal cord.
Investigations of En1 expression in motor neurons from already available omics data sets would support the idea that En1 is expressed in motor neurons.
Differentiation between Gamma and Alpha motor neurons should be performed using specific markers as Err3, Wnt7a or NeuN.
How can the authors explain the lack of loss of En1 interneurons in the En1-Het mice? Do spinal En1 interneurons show any signs of apoptosis (e.g., cleaved caspase 3 marker)? Which levels of the spinal cord were used for interneuron quantifications? Segments between L1 and L3 would be preferable.
The set of experiments reported in Figure 4 is of difficult interpretation without showing the actual presence of extracellular En1, that could be assessed with protein detection or RNAscope.
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Reply to the reviewers
1. General Statements
We would like to thank the editor for handling our manuscript entitled, “Mouse SAS‑6 is required for centriole formation in embryos and integrity in embryonic stem cells”, and the reviewers for the insightful comments and suggestions to improve our work. We aim for our manuscript to be considered for a “Short Report” format. As such, we would like to emphasize that we did not focus on the in vivo part of our study, where the Sas-6 mutant mouse embryos resemble our previously published Sas-4 mutants, as pointed out by the reviewers, because both mutants lack centrioles. In our opinion, the novelty of our work is evident in the discovery that mouse embryonic stem cells (mESCs) lacking SAS-6 are still able to form centrioles, albeit mostly abnormal, which is also shared by the reviewers. This is in contrast to Sas-4 mutant mESCs for example, which lack centrioles (Xiao*, Grzonka* et al, EMBO Reports 2021), and human cultured human cell lines without SAS-6, which have been shown to lose centrosomes. We are in the process of editing the manuscript and performing additional experiments per the reviewers’ recommendations. Below, we provide a point-by-point description of our revision plan.
2. Description of the planned revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The article by M. Grzonka and H. Bazzi entitled: Mouse Sas-6 is required for centriole formation in embryos and integrity in embryonic stem cells, describes new findings in novel mouse models of Sas-6 knockouts (KO). This is an interesting study that reports two different mouse Sas6 KO models and the depletion of Sas6 from mouse embryonic stem cells (mESCs). This type of analysis has never been done before and so it reveals and describes a role for Sas-6 in centriole biogenesis in mouse.
We thank the reviewer for highlighting the novelty of our work on the roles of SAS-6 in mice.
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The authors compare their analysis with Sas-4 KO and overall found similar results when compared to previous work from H. Bazzi, when Sas-4 was depleted in mouse embryos. Due to the mitotic stopwatch pathway, Sas6KO embryos die during development at extremely early stages and this can be rescued by depletion in p53 and other members of the pathway.
Perhaps, not so surprisingly, these embryos do not contain centrioles, showing that in vivo, Sas6 is absolutely required for centriole duplication. More surprisingly, however, in cultures of mESCs, established and propagated in vitro, Sas-6 crispr induced KO, does not result in lack of centrioles. Instead, abnormal structures that show aberrant morphologies, length, and incapacity to assemble cilia were detected. In principle, this means that centrioles can be assembled independently of Sas-6, even if not in the correct manner.
We again thank the reviewer for astutely pointing out the most surprising finding in our data, which is that mESCs lacking SAS-6 can still form centrioles.
The authors interpret these differences as possible differences in the pathways involved in centriole assembly and propose different requirements in different cell types, within the same species.
I have problems with this interpretation. To me is very difficult to understand, how the "protein" absolutely required for cartwheel assembly at the early stages of centriole biogenesis, can be essential and dispensable at the same time. Although, I may be wrong, I think the authors have not envisage other possibilities to interpret their data, which should be taken into consideration.
We agree with the reviewer that SAS-6 is currently considered in the centrosome field as one of the “core” centriole formation or duplication factors and that it is a major component of the cartwheel scaffold during the early phase of centriole biogenesis. Although, the absence of centrioles in the Sas-6 mutant mouse embryos in vivo supports the essential function of SAS-6, and perhaps the cartwheel, in centriole formation; the mere presence of centrioles in mESCs indicates that SAS-6, and again the cartwheel, is not essential for the existence of centrioles in these cells. Because this is a major finding that we would like to bring across from our study, we will better highlight and clarify it in the new version of the manuscript as described below. In fact, in points #4 and #5, we share the same possible explanation for the difference in the phenotypes between Sas-6 mutant mouse embryos and mESCs as the reviewer.
1) I do not know anything about ESC and ESC cultures. So maybe this is a stupid suggestion. But can't they be derived exactly from the same genetic background of SAS-6KO embryos? Because the way the two (or even 3 as there are 2 mouse KO lines) are generated is different. Why is that?
The reviewer is correctly suggesting that the mESC can be derived from the Sas-6 mutant blastocysts. We have initially derived mESCs from the Sas-6em4/em4 mutants and performed our analyses on the centriole phenotypes in these mutants before realizing that the allele was hypomorphic (SAS-6 staining in Fig. S2F, and the appearance of centrioles at E9 in Fig. S1B). Because the surprising finding in our study is that SAS-6 does not seem to be essential for centriole presence in mESCs, as pointed out by the reviewer, we decided to generate a more convincing Sas-6-/- null allele in mESCs by deleting the entire ORF of Sas-6 (more on this point below). We would also like to direct the attention of the reviewer that we have cultured the blastocysts (E3.5) from the Sas-6em5/em5 null mutants, which as we show lack centrioles at E3.5, and the cells indeed start to form centrioles just 24 h post-culture (Fig. 3C-D).
To build on these findings, we have already taken this a step further and generated a mESC line from the Sas-6em5/em5 mutants. These Sas-6em5/em5 –derived mESCs show CENT2-eGFP-positive centrioles, and we are currently analyzing their number and integrity, similar to our analyses of the CRISPR-generated Sas-6-/- null mESCs.
2) Still on mESCs, are the authors sure that there are no WT Sas-6 mRNAs still present in their ESC cells? Because tiny amounts are maybe sufficient to allow the initial cartwheel structure. In FigS2B, I can see a really faint band, very faint but it is there.
Due to the nature of the surprising finding that Sas-6 mutant mESCs can still form centrioles, we understand the concerns and suggestions of this reviewer and the other reviewers in this regard. We have generated several Sas-6 mutant alleles in mESCs (in exons 2, 4 or 5), in which we used Western blots to check whether they were null alleles or not. We used different commercial (Proteintech cat# 21377-1-AP, Sigma-Aldrich cat# HPA028187 and Santa Cruz cat# SC-81431) and non-commercial (kind gift from Renata Basto, Institute Curie) antibodies. The SAS-6 antibody from the Basto lab gave the most reliable and reproducible results. Using this antibody, and in our own interpretation, we were not able to detect SAS-6 by Western blots in Sas-6 mutant mESCs. We concluded that SAS-6 in mESCs (and mouse embryos, see below) is expressed at low levels. Of note, we always detected centrioles in the different Sas-6 mutant mESCs, even those derived from the Sas-6em5/em5 null mutant blastocysts, which as blastocysts had no detectable centrioles.
For a more definitive knockout in mESCs, we decided to bi-allelically delete the entire Sas-6 ORF DNA from the ATG to the TAA (over 34 Kb of DNA, Fig. S2A). According to the central dogma of molecular biology, when there is no DNA, then there should be no mRNA and no protein. In confirmation of this premise, recent RT-PCR data showed that Sas-6 mRNA is not detectable in these Sas-6-/- null mESCs. Also, immunofluorescence analyses did not detect SAS-6 in these cells. We will add the RT-PCR and immunofluorescence data to the fully revised manuscript. We will also repeat the SAS-6 Western blots to achieve better band resolution.
These Sas-6-/- mESCs started from a single cell and have been passaged up to 20 times by now without losing centrioles. SAS-6 protein was not detectable at the early passages and the mRNA is still not detectable. This is how knockouts have been and are produced. If this mutant is still not convincing, then we respectfully ask that the reviewers provide their own suggestion on what will be more convincing. In our humble opinion, this Sas-6-/- mESCs line can be used to test the specificity of the antibodies in mouse cells and not the other way around.
3) This last point goes also with the western-blot of Figure S2C- there is still a band, very tiny between the two very tick bands (marked with *). Maybe separating proteins better will help visualizing the real Sas-6 band? Have they used the Sas6 ab in other WBs from the KO embryos, for example? Can they use the Sas6 ab in immunostaining to show if the assembled abnormal centrioles completely lack Sas6. This will allow to distinguish between the hypothesis of having some (even if not much) sas6 left?
The answer to these questions is above in point #2. In addition, we have used the Basto lab antibody for SAS-6 for Western blots on mouse embryos, which detect low levels of SAS-6 in controls and no signal in the mutants. We will repeat the SAS-6 Western blots on mESCs to achieve better band resolution. Using this antibody for immunofluorescence showed that the Sas-6em4/em4 mutant is hypomorphic, whereas the Sas-6em5/em5 mutant showed very low, most likely background, staining (Fig. S1F). For mESCs, we decided to delete the entire Sas-6 ORF DNA in mESCs and generate homozygous Sas-6-/- null mutants. Immunofluorescence analyses did not detect SAS-6 in these cells.
4) Then a more theoretical point? Have the authors considered that the difference is more in the stability of the abnormal structures. Let's say, without a cartwheel and maybe enough PLK4 activity and high level of other centriolar components, the centrioles are abnormally assembled- they have no cartwheel, but they are disassembled very fast in the embryo but not in ESCs?
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We agree and share the reviewer’s interpretation for the potential requirement of SAS-6 in vivo to stabilize intermediate structures, that is compensated for by other factors in mESCs. This was not directly discussed in the first version of the manuscript and we will include it in the new version.
5) Even if there is a real difference and without Sas-6 ESCs can make centrioles that are abnormal in structure and function (at least at the cilia assemble level), the choice of words "strictly required", I am not sure it is correct. Because, since Sas-6 is described by many studies as the factor required for cartwheel assembly, which occurs very early in the pathway, this means that in mESCs centrioles can assembled without forming a cartwheel. And so that the cartwheel is actually not required for the initial building block, but more as a structure that maintains the whole centriole in an intact manner?
We agree with the reviewer on the likely requirement of SAS-6, and therefore the cartwheel as a whole, for the symmetry and integrity of the forming centrioles, which is along the same line as in point #4. In our interpretation, “centriole formation” does not necessarily mean centriole “initiation” but rather the presence of the centriole as a structure. We will use more appropriate and specific wording to match our shared interpretation with the reviewer.
6) The authors mentioned that in flies, abnormal Sas-6 structures have been described in certain cell types. Are these mutants, null mutants? In other words, do these structures assembled in a context of no Sas6 or abnormal Sas-6 protein or even low levels of Sas-6?
According to the published report (Figure S3B in Rodrigues-Martins et al, 2007, PMID: 17689959) the fly brains have no detectable DSAS-6 protein. Therefore, we assume that they are Sas-6 null fly mutants. The abnormal centrioles in Sas-6 C. Reinhardtii mutants and Sas-6-/- mESCs null mutants support the conclusion that the main role of SAS-6, and perhaps the cartwheel, is in maintaining the integrity of the forming procentriole.
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Other points:
I think the 1st sentence of the abstract appears disconnected from the rest. The same goes for the 1st sentence of the introduction. And also, what is the evidence that pluripotent stem cells rely primarily on the proper assembly of a mitotic spindle? They rely on many other things, not sure this is the first one.
The sentences were meant to highlight the importance of cell division in stem cells. We will adjust the wording in these sentences per the reviewer’s comment to not focus on pluripotency per se.
The authors mention that centrioles are lost in Sas6-/- after "differentiation" of mESCs. The term differentiation is not appropriate, and confusing here. Differentiation normally refer to cells that stopped proliferating and exited the cell cycle, which is not the case here, as NPCs are progenitor cells that keep cycling.
We believe the reviewer is referring to “terminal differentiation”, when the cells exit the cell cycle and adopt their destined cell fates. The word “differentiation” in this context refers to limiting the potency of stem cells into a subset of cell fates such as NPCs, which are proliferating progenitors.
Figure S1: Percent of cells with centrosomes was assessed by a co-staining of gtubulin and Cep164, which mark the mother centrioles. As Cep164 may be absent from centrosomes after lack of centriole maturation in sas6-/- embryos, another combination of staining should be performed to evaluate the percent of cells without centrosomes. gtubulin staining can be seen in Sas6 em5/em5 embryos, while the quantification claims total absence of centrosomes. The authors use the CENT2-eGFP transgenic line to count the number of centrioles in Figure 3, they should do the same in Figure S1.
We will follow the reviewer’s recommendation of counting Cent2-eGFP for the assessment of centrioles in Sas-6em5/em5 (Fig. S1).
The g-tubulin (TUBG) aggregates at the poles of dividing cells are assembled in the absence of centrioles, as shown in Sas-6em5/em5 embryo sections (Fig. S1H). In addition, we have previously observed these pericentriolar material aggregates in Sas-4-/- mutant embryos (Bazzi and Anderson, 2014), which do not contain centrioles in serial transmission electron microscopy. Therefore, we do not refer to them as centrosomes in the absence of centrioles at their core.
Reviewer #1 (Significance (Required)):
This study shows with a novel mouse model the requirement of centrioles during mouse development. It will be relevant to centrosome labs, the novel mouse lines will be useful to many labs working on centrioles, cilia and centrosomes.
My expertise: centrosome biology
We thank the expert reviewer for the critical comments and suggestions, and the positive evaluation of our manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
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Here, Grzonka and Bazzi present their work on characterizing the requirement of SASS6 in mouse embryo development and in embryonic stem cell (mESC) culture. In mouse, female and male gametes lack centrioles, and early divisions occur without centrioles. De novo formation typically happens at the blastocyst stage (~E3.5). The authors generated two SASS6 knock-out strains, SASS6 em4/em4 (frameshift deletion, reported as a severe hypomorphic allele), and SASS6 em5-em5 (frameshift deletion, reported as a null allele). Mutant embryos arrest development at mid-gestation unless the p53, USP28 and USP28 pathway is perturbed. As expected, centrioles do not form in SASS6 -/- mice. However, the authors report that de novo formation of centrioles is facilitated in mESC culture conditions for SASS6 CRISPR knock-out mESCs and mESCs derived from SASS6 em5/em5 blastocysts. Centrioles are lost upon differentiation of SASS6 CRISPR knock-out mESCs into neural progenitor cells (NPCs).
The presented study is relevant for scientists investigating the requirements for centriole formation during embryonic development. Further, it provides insights in possibly different requirements for centriole formation between stages of differentiation, as well as differences in in vivo and in vitro models.
We thank the reviewer for finding our work relevant and insightful into the differential requirements for centriole formation depending on the cell type.
The data represented by Grzonka and Bazzi are robust and support the manuscript and conclusions made. However, the study is predominantly descriptive, and the authors do not test the molecular pathway underlying the de novo formation of centrioles observed in SASS6 -/- mESCs. It is generally believed that de novo formation of centrioles is not possible in SASS6 knock-out cells although work from Wang and Tsou with SASS6 a oligomerization mutant suggests otherwise. A dissection of the specific factors required for the de novo formation of centrioles in the mESC context would provide more insights into de novo centriole assembly in general and would increase the impact of this work. I would support publication of the manuscript if the following points are addressed:
We again thank the reviewer for finding the data robust and support our conclusions and interpretation. We agree with the reviewer that our study opens new questions about how mESCs manage to assemble centrioles in the absence of SAS-6. Together with the phenotypes of the Sas-6 mutant D. melanogaster and C. Reinhardtii, and the SAS-6 oligomerization mutants (but not full SAS-6 mutants) in human cell lines mentioned by the reviewer and cited in our manuscript, the data open new investigations into the exact requirements of SAS-6 and the cartwheel in centriole biogenesis in the different cellular contexts.
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- One of the main figures, ideally Figure 1, should be dedicated to the characterization of the newly generated mouse strains. This should also be elaborated in the text further. I would like to see a schematic representation of the genomic modifications. The SASS6 stainings of wt and Sas-6 knock-outs (now Figure S1F) should be shown in that context as well as the Figures S2A-C. The authors should discuss why there still appears to be SASS6 protein in the SASS6-em5/em5 Sas-6 stainings visible. Also, the western blot, especially the unspecific bands so close to the SAS-6 protein, should be discussed. Adding qRTPCR results would also be good. Per the reviewer’s requests, we will move the embryo mutant characterization (Fig. S1F) and mESCs (Fig. S2A-C) to the main figures and elaborate the text accordingly. The genomic modifications in mice are described in a detailed tabular format in Table 1 in Materials and Methods. The immunofluorescence staining in Fig. S1F was performed on mouse embryonic sections, which tend to have higher backgrounds than cultured cells; Thus, we attribute the very low percentage of SAS-6 staining in Sas-6em5/em5* mutants to higher background, especially given the lack of centrioles in these mutants at all the stages examined.
For Western blots, we used different antibodies against SAS-6 that were either commercially available (Proteintech cat# 21377-1-AP, Sigma-Aldrich cat# HPA028187 and Santa Cruz cat# SC-81431) or non-commercial (kind gift from Renata Basto, Institute Curie). The SAS-6 antibody from the Basto lab gave the most reliable and reproducible results. Using this antibody, and in our own interpretation, we were not able to detect SAS-6 by Western blots in Sas-6 mutant mESCs (including hypomorphic alleles). We concluded that SAS-6 in mESCs (and mouse embryos, see below) is expressed at low levels. Thus, we decided to use the antibody provided by Renata Basto and shown in current Fig. S2C, although it shows two thick non-specific bands flanking the specific band for SAS-6.
For a more definitive knockout in mESCs, we decided to bi-allelically delete the entire Sas-6 ORF DNA from the ATG to the TAA (over 34 Kb of DNA, Fig. S2A). According to the central dogma of molecular biology, when there is no DNA, then there should be no mRNA and no protein. In confirmation of this premise, recent RT-PCR data showed that Sas-6 mRNA is not detectable in these Sas-6-/- null mESCs. Also, immunofluorescence analyses did not detect SAS-6 in these cells. We will add the RT-PCR and immunofluorescence data to the fully revised manuscript. We will also repeat the SAS-6 Western blots to achieve better band resolution.
In addition, we have used the Basto lab antibody for SAS-6 for Western blots on mouse embryos, which detect low levels of SAS-6 in controls and no signal in the mutants.
- The authors could elaborate on the topic of mESCs as a special in vitro model for centriole biology akin to the more "primitive" origins of life such as algae.*
We will elaborate on the topic of mESCs as a special system for centriole biology to stress the findings that mESCs without SAS-6 can still form centrioles, but also that these cells seem to tolerate centriolar aberrations, such as in Sas-6 mutants, or even the loss of centrioles, as in Sas-4 mutants, without undergoing apoptosis or cell cycle arrest.
- Figure 4 should show timeline of embryo development, include embryo stages (E3.5, E9 etc.), group together mESCs with corresponding embryonic developmental stage. The Figure can indicate when mESCs were derived from SASS6 em5/em5 blastocysts, when they were stained and indicate the number/state of centriole formation observed.*
We will adjust the model in Fig. 4 to accommodate the suggestions of the reviewer, but at the same time try not to overcrowd the model and dilute the main findings of the study.
- The work from Wang and Tsou using SAS-6 oligomerization mutants should be better discussed in the context of the work presented here since centriole assembly was not affected per se but structural defects were observed, like is the case in this study.*
We will elaborate on this finding from Wang et al. In this respect, we will note that the loss of the entire SAS-6 protein in human RPE-1 cells (on a p53-mutant background), leads to the loss of centrioles, but that the deletion of the oligomerization domain of SAS-6 in these cells leads to similar phenotypes to the total loss of SAS-6 in mESCs.
- The observation that the ability of forming centrioles de novo in NPCs derived from ESCs is lost is interesting but the mechanisms underpinning this differentiation remain unclear. The authors at a minimum should speculate on these further.*
We agree with the reviewer and will speculate on this finding further. This comment is along the same line as the difference in phenotype between the cells in the developing mouse embryo and mESCs, where the NPCs are more akin to the in vivo phenotype.
CROSS-CONSULTATION COMMENTS
Looks like we are all pretty much in agreement.
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Reviewer #2 (Significance (Required)):
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This is a well executed study with no major flaws that builds on similar studies on knocking out centriole components in mouse and other cell types. Although well-executed the study remains descriptive and lacks a clear mechanistic understanding of why de novo centriole assembly is ineffective in NPCs. As it stands the advances this study provides to the centrosome biogenesis field remain incremental.
We thank the reviewer for the compliments about our work and agree that it opens new questions in the field about the precise roles of SAS-6 and the cartwheel in centriole biogenesis.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this publication, Grzonka and Bazzi build upon their recent work describing the role of SAS-like protein function in centriole formation during embryonic development. More specifically, they demonstrate that loss of Sas-6 in vivo and in vitro disrupts centriole formation. To this reviewer's surprise, they found that Sas-6 is required for centriole formation in embryos, yet, stem cells form centrioles with disrupted centriole length and ability to template cilia.
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We thank the reviewer for highlighting the novel and surprising aspect of our work, which is that Sas-6 mutant mESCs are still able to form centrioles. We would like to stress that SAS-4, from our previously published work, and SAS-6, in this study, are not part of the same protein family and have different structures and roles in centriole formation. The naming has its origin in “Spindle-ASsembly abnormal/defective” mutant screens performed in C. elegans. Although the phenotypes are similar in vivo, due the lack of centrioles in both cases, only mutations in Sas-4, but not in Sas-6, lead to the lack of centrioles in mESCs.
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Likely, this occurs from the residual proteins that existed prior to CRISPR-mediated knockout.
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Due to the nature of the surprising finding that Sas-6 mutant mESCs can still form centrioles, we understand the concerns and suggestions of this reviewer and the other reviewers in this regard.
For a more definitive knockout in mESCs, we decided to bi-allelically delete the entire Sas-6 ORF DNA from the ATG to the TAA (over 34 Kb of DNA, Fig. S2A). According to the central dogma of molecular biology, when there is no DNA, then there should be no mRNA and no protein. In confirmation of this premise, recent RT-PCR data showed that Sas-6 mRNA is not detectable in these Sas-6-/- null mESCs. Also, immunofluorescence analyses did not detect SAS-6 in these cells. We will add the RT-PCR and immunofluorescence data to the fully revised manuscript. We will also repeat the SAS-6 Western blots to achieve better band resolution.
These Sas-6-/- mESCs started from a single cell and have been passaged up to 20 times by now without losing centrioles. SAS-6 protein was not detectable at the early passages and the mRNA is still not detectable. This is how knockouts have been and are produced. If this mutant is still not convincing, then we respectfully ask that the reviewers provide their own suggestion on what will be more convincing.
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Unsurprisingly, they found that Sas-6 loss in the developing mouse activates the 53BP1-USP28-p53 surveillance pathway leading to cell death and embryonic arrest at mid-gestation, similar to their findings in Cenpj knockouts. What remains to be properly elucidated is the mechanistic differences in the requirement for Sas-6 in stem cells versus the embryo, which may be beyond the scope of a short report. As it reads, the manuscript is a compliment to their Sas-4 paper but falls short of novelty and providing large strides in revealing the role of centriolar proteins in developmental processes. Moreover, the advances beyond the requirement for centriole and associated proteins in embryology is missing, therefore enthusiasm is tempered. Below are remaining concerns that must be addressed:
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Remaining concerns:
The authors should provide clear description of the embryonic region (neural plate & mesenchym) used to analyze centriole presence or loss in Figures 1 and S1. Was this in the forelimb vs hindlimb regions?
The assessment of centrosomes in Fig. 1 and S1 was performed on cell types in all three germ layers in the sections that were taken from the brachial region (forelimb and heart level). The information will be added to the Materials and Methods section.
Similar to their Cenpj-mouse data, the authors should provide data detailing the mitotic index and activation of the mitotic surveillance pathway beyond just p53 staining. As novelty is not the only criteria for publication, a thorough analysis of the Sas-6 activation of the mitotic purveyance pathway should be provided, including the crosses between Sas-6 and p53, 53bp1 and usp28 knockout crosses to demonstrate the pathway functions similarly to Cenpj loss.
We will perform the additional experiments suggested by the reviewer that are similar to our previous work in Sas-4 mutants (Xiao*, Grzonka* et al, 2021). We will perform these analyses knowing that both Sas-4 and Sas-6 mutants lose centrioles and activate the mitotic surveillance pathway, as the reviewer indicated. In particular, we will quantify the mitotic index in the Sas-6em5/em5 mutants and perform p53 and Cl. CASP3 staining in the double mutants with 53bp1 or Usp28, to show that the pathway has been suppressed in these mutants.
Centriole structure should be assessed in the embryos using EM to assess loss and confirm the structural defects. This would strengthen their argument and be a slight advance to their largely descriptive paper.
Because the Sas-6em5/em5 embryos lack centrioles, as indicated by regular immunofluorescence and Ultrastructure-Expansion Microscopy (U-ExM), using EM would be an attempt to find a structure that does not exist. In our opinion, it would again be a repetition of TEM studies that we have already performed in Sas-4-/- mutant embryos, that lack centrioles (Bazzi and Anderson, 2014). Using U-ExM has advanced the centriole biology field to a level that is approaching EM resolution and, in our opinion, can substitute for EM.
The WB for Sas-6 knockout is not convincing and should be redone. There are validated Sas-6 antibodies available from SCBT and Proteintech. It is not clear that the band is gone or if there's overlap with the non-specific band.
The answer to this comment is shown above. In addition, we have used the Basto lab antibody for SAS-6 for Western blots on mouse embryos, which detect low levels of SAS-6 in controls and no signal in the mutants. We will also repeat the SAS-6 Western blots on mESCs to achieve better band resolution as recommended by the reviewer.
The authors describe the centriolar structural defect in the mESCs in Figure 2C and D, and further characterize the phenotype in S2D-H. Given the role of the SAS6-CEP135-CPAP axis for centriole elongation, it is peculiar that they see elongation upon reduction of CEP135. The authors should find a rationale mechanism to explain their discordant findings. In addition, other centriole distal end components including CEP97 and CP110 should be examined to determine the structural end caping defect in the Sas-6 mESC.
Over 70% of the centrioles in Sas-6-/- mESCs retain CEP135, but the majority of CEP135 signals (over 80%) seem to be abnormally localized. One potential explanation for the elongated centrioles in Sas-6-/- mESCs is that the mis-localization of CEP135 impacts on the integrity of the centriole and results in parts of the centriolar walls being elongated. Per the reviewer’s suggestion, we have performed U-ExM with stainings for CP110 or CEP97, that also regulate centriole capping and elongation. The preliminary data suggest that similar to WT mESCs, they localize to the ends of the abnormal centrioles in Sas-6-/- mESCs. We will quantify the percentage of normally-localized CP110 and CEP97 in Sas-6-/- mESCs and include it along with the data interpretation in the next version of the manuscript.
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In Figure 2I, J the authors state the ciliation rate for the WT mESCs was only 11%, could the authors provide an explanation for the low ciliation rate in WT mESCs? Could cells be arrested to increase the ciliation rate? In addition, is there a rational explanation for the loss of centrioles and centrosomes upon differentiation into NPCs?
mESCs ciliation rate has been shown to be generally low (Bangs et al, 2015; Xiao et al., 2021) perhaps because the cells spend most of the cell cycle in the S-phase. mESCs require a high serum percentage and well-defined media for growth and maintenance. In our hands, attempting to arrest the cells by withdrawing serum, or reducing its percentage, resulted in cell death and a change in morphology to the differentiated phenotype (unpublished data). Our data indicate that a pluripotent state in Sas-6-/- mESCs is compatible with centriole formation but differentiation results in the loss of centrioles (for example, NPCs). Therefore, we have refrained from interfering with the cell cycle of mESCs in order to avoid these confounding effects on cellular viability and centriole formation.
Regarding the loss of centrioles upon differentiation of Sas-6-/- mESCs into NPCs, we agree with the reviewer and will speculate on this finding further. This goes along the same line as the difference in phenotype between the cells in the developing mouse embryo and mESCs, where the NPCs are more akin to the in vivo phenotype of Sas-6 mutants. The data suggest that the formation of centrioles in Sas-6-/- mESCs is associated with the in vitro pluripotent phenotype. A more comprehensive and general characterization of centriole duplication in mESCs is a future direction to elucidate their ability to form centrioles without SAS-6.
In figure 3F in the Sas-6−/− NPCs have a box around a cell without centrosomes yet in 3G here are some cells with centrosomes. While the authors are trying to demonstrate the decrease in centrosome in the Sas-6−/− NPCs, they should show the few cell that have centrosomes or centrosome-like structures.
We will add another example for the minority of cells that retain centrosomes upon differentiation of Sas-6-/- mESCs into NPCs.
CROSS-CONSULTATION COMMENTS
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As mentioned in my review; while the Sas6 model is new, it does not provide further evidence of why centriole duplication is important in developing mice aside from it causing an abortive mitosis leading to cell death. The discordant phenotype in the mESCs likely arises from residual Sas6, similar to experiments that were performed in flies with Sas-4 depletion. Moreover, the odd centriole phenotype represents a very small number of cells and is likely phenomenological.
In addition, their work from last year demonstrated a clear connection between Cenpj loss leading to the mitotic surveillance pathway activation. They performed double knockouts that partially rescued the survival phenotype. This new work falls short of that publication.
Reviewer #3 (Significance (Required)):
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The new publication adds a known component to the list of animal models for centrosome-opathies but fails to provide novel mechanistic insights. Dr. Bazzi's publication on Sas-4 was far more novel at the time of publication due to the multiple mouse crosses that could rescue the phenotypes. The recent publication fails to provide as much evidence or any novel insights into the role of Sas-6 (sufficient to be convincing).
The audience will be limited to centrosome biologists and even then it may not have enough novelty to be compelling. I would recommend with the revisions to be published in a more specialized journal.
*My expertise lies in genetic causes of microcephaly-associated with mutations in centrosome encoding proteins. *
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We thank the reviewer for taking the time to evaluate our work and provide helpful comments and suggestions. We would like to emphasize that even if a certain phenotype is expected, the experiment has to be performed to test the hypothesis, which is the case with the Sas-6 mutant embryos phenocopying the Sas-4 mutants. In our opinion, the novelty of our work goes beyond Fig. 1 to the ability of Sas-6-/- null mESCs to form centrioles. This surprising finding opens new avenues of investigation into the precise roles of SAS-6, and the cartwheel, in centriole biogenesis. We are confident that our study will provide a trigger to re-examine these roles in other cell types and organisms.
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Referee #3
Evidence, reproducibility and clarity
In this publication, Grzonka and Bazzi build upon their recent work describing the role of SAS-like protein function in centriole formation during embryonic development. More specifically, they demonstrate that loss of Sas-6 in vivo and in vitro disrupts centriole formation. To this reviewer's surprise, they found that Sas-6 is required for centriole formation in embryos, yet, stem cells form centrioles with disrupted centriole length and ability to template cilia. Likely, this occurs from the residual proteins that existed prior to CRISPR-mediated knockout. Unsurprisingly, they found that Sas-6 loss in the developing mouse activates the 53BP1-USP28-p53 surveillance pathway leading to cell death and embryonic arrest at mid-gestation, similar to their findings in Cenpj knockouts. What remains to be properly elucidated is the mechanistic differences in the requirement for Sas-6 in stem cells versus the embryo, which may be beyond the scope of a short report. As it reads, the manuscript is a compliment to their Sas-4 paper but falls short of novelty and providing large strides in revealing the role of centriolar proteins in developmental processes. Moreover, the advances beyond the requirement for centriole and associated proteins in embryology is missing, therefore enthusiasm is tempered. Below are remaining concerns that must be addressed:
Remaining concerns:
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The authors should provide clear description of the embryonic region (neural plate & mesenchym) used to analyze centriole presence or loss in Figures 1 and S1. Was this in the forelimb vs hindlimb regions?
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Similar to their Cenpj-mouse data, the authors should provide data detailing the mitotic index and activation of the mitotic surveillance pathway beyond just p53 staining. As novelty is not the only criteria for publication, a thorough analysis of the Sas-6 activation of the mitotic purveyance pathway should be provided, including the crosses between Sas-6 and p53, 53bp1 and usp28 knockout crosses to demonstrate the pathway functions similarly to Cenpj loss.
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Centriole structure should be assessed in the embryos using EM to assess loss and confirm the structural defects. This would strengthen their argument and be a slight advance to their largely descriptive paper.
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The WB for Sas-6 knockout is not convincing and should be redone. There are validated Sas-6 antibodies available from SCBT and Proteintech. It is not clear that the band is gone or if there's overlap with the non-specific band.
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The authors describe the centriolar structural defect in the mESCs in Figure 2C and D, and further characterize the phenotype in S2D-H. Given the role of the SAS6-CEP135-CPAP axis for centriole elongation, it is peculiar that they see elongation upon reduction of CEP135. The authors should find a rationale mechanism to explain their discordant findings. In addition, other centriole distal end components including CEP97 and CP110 should be examined to determine the structural end caping defect in the Sas-6 mESC.
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In Figure 2I, J the authors state the ciliation rate for the WT mESCs was only 11%, could the authors provide an explanation for the low ciliation rate in WT mESCs? Could cells be arrested to increase the ciliation rate? In addition, is there a rational explanation for the loss of centrioles and centrosomes upon differentiation into NPCs?
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In figure 3F in the Sas-6−/− NPCs have a box around a cell without centrosomes yet in 3G here are some cells with centrosomes. While the authors are trying to demonstrate the decrease in centrosome in the Sas-6−/− NPCs, they should show the few cell that have centrosomes or centrosome-like structures.
CROSS-CONSULTATION COMMENTS
As mentioned in my review; while the Sas6 model is new, it does not provide further evidence of why centriole duplication is important in developing mice aside from it causing an abortive mitosis leading to cell death. The discordant phenotype in the mESCs likely arises from residual Sas6, similar to experiments that were performed in flies with Sas-4 depletion. Moreover, the odd centriole phenotype represents a very small number of cells and is likely phenomenological.
In addition, their work from last year demonstrated a clear connection between Cenpj loss leading to the mitotic surveillance pathway activation. They performed double knockouts that partially rescued the survival phenotype. This new work falls short of that publication.
Significance
The new publication adds a known component to the list of animal models for centrosome-opathies but fails to provide novel mechanistic insights. Dr. Bazzi's publication on Sas-4 was far more novel at the time of publication due to the multiple mouse crosses that could rescue the phenotypes. The recent publication fails to provide as much evidence or any novel insights into the role of Sas-6 (sufficient to be convincing).
The audience will be limited to centrosome biologists and even then it may not have enough novelty to be compelling. I would recommend with the revisions to be published in a more specialized journal.
My expertise lies in genetic causes of microcephaly-associated with mutations in centrosome encoding proteins.
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Referee #2
Evidence, reproducibility and clarity
Here, Grzonka and Bazzi present their work on characterizing the requirement of SASS6 in mouse embryo development and in embryonic stem cell (mESC) culture. In mouse, female and male gametes lack centrioles, and early divisions occur without centrioles. De novo formation typically happens at the blastocyst stage (~E3.5). The authors generated two SASS6 knock-out strains, SASS6 em4/em4 (frameshift deletion, reported as a severe hypomorphic allele), and SASS6 em5-em5 (frameshift deletion, reported as a null allele). Mutant embryos arrest development at mid-gestation unless the p53, USP28 and USP28 pathway is perturbed. As expected, centrioles do not form in SASS6 -/- mice. However, the authors report that de novo formation of centrioles is facilitated in mESC culture conditions for SASS6 CRISPR knock-out mESCs and mESCs derived from SASS6 em5/em5 blastocysts. Centrioles are lost upon differentiation of SASS6 CRISPR knock-out mESCs into neural progenitor cells (NPCs).
The presented study is relevant for scientists investigating the requirements for centriole formation during embryonic development. Further, it provides insights in possibly different requirements for centriole formation between stages of differentiation, as well as differences in in vivo and in vitro models. The data represented by Grzonka and Bazzi are robust and support the manuscript and conclusions made. However, the study is predominantly descriptive, and the authors do not test the molecular pathway underlying the de novo formation of centrioles observed in SASS6 -/- mESCs. It is generally believed that de novo formation of centrioles is not possible in SASS6 knock-out cells although work from Wang and Tsou with SASS6 a oligomerization mutant suggests otherwise. A dissection of the specific factors required for the de novo formation of centrioles in the mESC context would provide more insights into de novo centriole assembly in general and would increase the impact of this work.
I would support publication of the manuscript if the following points are addressed:
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One of the main figures, ideally Figure 1, should be dedicated to the characterization of the newly generated mouse strains. This should also be elaborated in the text further. I would like to see a schematic representation of the genomic modifications. The SASS6 stainings of wt and Sas-6 knock-outs (now Figure S1F) should be shown in that context as well as the Figures S2A-C. The authors should discuss why there still appears to be SASS6 protein in the SASS6-em5/em5 Sas-6 stainings visible. Also, the western blot, especially the unspecific bands so close to the SAS-6 protein, should be discussed. Adding qRTPCR results would also be good.
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The authors could elaborate on the topic of mESCs as a special in vitro model for centriole biology akin to the more "primitive" origins of life such as algae.
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Figure 4 should show timeline of embryo development, include embryo stages (E3.5, E9 etc.), group together mESCs with corresponding embryonic developmental stage. The Figure can indicate when mESCs were derived from SASS6 em5/em5 blastocysts, when they were stained and indicate the number/state of centriole formation observed.
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The work from Wang and Tsou using SAS-6 oligomerization mutants should be better discussed in the context of the work presented here since centriole assembly was not affected per se but structural defects were observed, like is the case in this study.
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The observation that the ability of forming centrioles de novo in NPCs derived from ESCs is lost is interesting but the mechanisms underpinning this differentiation remain unclear. The authors at a minimum should speculate on these further.
CROSS-CONSULTATION COMMENTS
Looks like we are all pretty much in agreement.
Significance
This is a well executed study with no major flaws that builds on similar studies on knocking out centriole components in mouse and other cell types. Although well-executed the study remains descriptive and lacks a clear mechanistic understanding of why de novo centriole assembly is ineffective in NPCs.As it stands the advances this study provides to the centrosome biogenesis field remain incremental.
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Referee #1
Evidence, reproducibility and clarity
The article by M. Grzonka and H. Bazzi entitled: Mouse Sas-6 is required for centriole formation in embryos and integrity in embryonic stem cells, describes new findings in novel mouse models of Sas-6 knockouts (KO). This is an interesting study that reports two different mouse Sas6 KO models and the depletion of Sas6 from mouse embryonic stem cells (mESCs). This type of analysis has never been done before and so it reveals and describes a role for Sas-6 in centriole biogenesis in mouse.
The authors compare their analysis with Sas-4 KO and overall found similar results when compared to previous work from H. Bazzi, when Sas-4 was depleted in mouse embryos. Due to the mitotic stopwatch pathway, Sas6KO embryos die during development at extremely early stages and this can be rescued by depletion in p53 and other members of the pathway. Perhaps, not so surprisingly, these embryos do not contain centrioles, showing that in vivo, Sas6 is absolutely required for centriole duplication. More surprisingly, however, in cultures of mESCs, established and propagated in vitro, Sas-6 crispr induced KO, does not result in lack of centrioles. Instead, abnormal structures that show aberrant morphologies, length, and incapacity to assemble cilia were detected. In principle, this means that centrioles can be assembled independently of Sas-6, even if not in the correct manner.
The authors interpret these differences as possible differences in the pathways involved in centriole assembly and propose different requirements in different cell types, within the same species. I have problems with this interpretation. To me is very difficult to understand, how the "protein" absolutely required for cartwheel assembly at the early stages of centriole biogenesis, can be essential and dispensable at the same time. Although, I may be wrong, I think the authors have not envisage other possibilities to interpret their data, which should be taken into consideration.
1) I do not know anything about ESC and ESC cultures. So maybe this is a stupid suggestion. But can't they be derived exactly from the same genetic background of SAS-6KO embryos? Because the way the two (or even 3 as there are 2 mouse KO lines) are generated is different. Why is that?
2) Still on mESCs, are the authors sure that there are no WT Sas-6mRNAs still present in their ESC cells? Because tiny amounts are maybe sufficient to allow the initial cartwheel structure. In FigS2B, I can see a really faint band, very faint but it is there.
3) This last point goes also with the western-blot of Figure S2C- there is still a band, very tiny between the two very tick bands (marked with *). Maybe separating proteins better will help visualizing the real Sas-6 band? Have they used the Sas6 ab in other WBs from the KO embryos, for example? Can they use the Sas6 ab in immunostaining to show if the assembled abnormal centrioles completely lack Sas6. This will allow to distinguish between the hypothesis of having some (even if not much) sas6 left?
4) Then a more theoretical point? Have the authors considered that the difference is more in the stability of the abnormal structures. Let's say, without a cartwheel and maybe enough PLK4 activity and high level of other centriolar components, the centrioles are abnormally assembled- they have no cartwheel, but they are disassembled very fast in the embryo but not in ESCs?
5) Even if there is a real difference and without Sas-6 ESCs can make centrioles that are abnormal in structure and function (at least at the cilia assemble level), the choice of words "strictly required", I am not sure it is correct. Because, since Sas-6 is described by many studies as the factor required for cartwheel assembly, which occurs very early in the pathway, this means that in mESCs centrioles can assembled without forming a cartwheel. And so that the cartwheel is actually not required for the initial building block, but more as a structure that maintains the whole centriole in an intact manner?
6) The authors mentioned that in flies, abnormal Sas-6 structures have been described in certain cell types. Are these mutants, null mutants? In other words, do these structures assembled in a context of no Sas6 or abnormal Sas-6 protein or even low levels of Sas-6?
Other points:
- I think the 1st sentence of the abstract appears disconnected from the rest. The same goes for the 1st sentence of the introduction. And also, what is the evidence that pluripotent stem cells rely primarily on the proper assembly of a mitotic spindle? They relly on many other things, not sure this is the first one.
- The authors mention that centrioles are lost in Sas6-/- after "differentiation" of mESCs. The term differentiation is not appropriate, and confusing here. Differentiation normally refer to cells that stopped proliferating and exited the cell cycle, which is not the case here, as NPCs are progenitor cells that keep cycling.
- Figure S1: Percent of cells with centrosomes was assessed by a co-staining of tubulin and Cep164, which mark the mother centrioles. As Cep164 may be absent from centrosomes after lack of centriole maturation in sas6-/- embryos, another combination of staining should be performed to evaluate the percent of cells without centrosomes. tubulin staining can be seen in Sas6 em5/em5 embryos, while the quantification claims total absence of centrosomes. The authors use the CENT2-eGFP transgenic line to count the number of centrioles in Figure 3, they should do the same in Figure S1.
Significance
This study shows with a novel mouse model the requirement of centrioles during mouse development. It will be relevnat to centrosome labs, the novel mouse lines will be useful to many labs working on centrioles, cilia and centrosomes. My expertise: centrosome biology
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Reply to the reviewers
We thank the Reviewers for their useful feedback on our manuscript. We have addressed the Reviewers’ comments and revised our manuscript accordingly. A point-by-point response is provided below.
Reviewer comments:
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.
Major Comments:
- As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on. Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader.
We thank the reviewer for this question and suggested experiments. Following the advice, we have now show immunofluorescence data of pan-neuronal markers (i.e., b-tub III or MAP2) in mESC and iPSCs. In agreement with previously published datasets from the Noh and Meyer labs (Gehre et al., 2020; Bogetofte et al.,2019), we show that the protocols we use generate a very high percentage of neurons. We have now included these images and quantifications in our manuscript as Figs. 2B and S6A,B.
From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig. S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.
We thank the reviewer for highlighting this seemingly counterintuitive observation. We have now included a quantification of CerS mRNA from commercially available mouse tissues analysed in Sladitschek and Neveu, 2019 and compared this to the data from Gehre et al, 2020. In the mouse brain tissue, CerS1 expression is upregulated dramatically, while CerS5 and 6 are downregulated (see new panel A in Fig. S8). In contrast, during in vitro differentiation of mESCs, CerS5 is not downregulated and CerS6 is upregulated (Fig. S8B). Accordingly, we have expanded our discussion in the revised manuscript as follows:
“On the other hand, supplementing the cells with stearic acid (18:0) does not result in high levels of 18:0-sphingolipids. It is known that the Cer synthase CerS1 is specific for stearoyl-CoA (Venkataraman et al., 2002), which results in the production of 18:0-sphingolipids, while the synthases CerS5 and CerS6 are responsible for 16:0-sphingolipid production. During brain development, one observes a 35-fold increase in the expression of CerS1 and a downregulation of CerS5 and CerS6 compared to embryonic tissue (Sladitschek & Neveu, 2019) (Fig. S8A). In contrast, during in vitro neuronal differentiation, between day 8 and 12, CerS1 expression increases only by 5-fold and, contrary to expectation, CerS6 expression is upregulated and CerS5 expression is unchanged (Gehre et al., 2020) (Fig. S8B). This could underpin the observation that 16:0-sphingolipids remain elevated whereas brain-specific 18:0-sphingolipids only increase marginally, despite supplementation with stearic acid. Overall, this suggests that appropriate programming of the sphingolipid metabolic machinery is not fully established in stem cell-derived neurons.”
Minor comments:
- I find the data presentation of the LENA analysis to be difficult to follow (Fig. 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.
We thank the reviewer for this suggestion. In the revised manuscript, we now plot log2(odds ratio) on the y-axis instead of the p-value. Moreover, we have dimensioned the size and color intensity of each point as function of the p-value (Fig. 1E and 2E, shown below).
In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.
In the revised manuscript, we now include a plot of the PCA loadings of the lipids majorly influencing principal components 1 and 2 as supplemental Fig. S3.
Reviewer #1 (Significance (Required)):
This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.
Reviewer #2 (Significance (Required)):
The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.
We thank the Reviewer for the positive assessment of our work.
References
Gehre, M., Bunina, D., Sidoli, S., Lübke, M. J., Diaz, N., Trovato, M., Garcia, B. A., Zaugg, J. B., & Noh, K. M. (2020). Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy. Nature Genetics, 52(3), 273–282. https://doi.org/10.1038/s41588-020-0586-5
Levy, M., & Futerman, A. H. (2010). Mammalian ceramide synthases. IUBMB Life, 62(5), 347–356. https://doi.org/10.1002/iub.319
Sladitschek, H. L., & Neveu, P. A. (2019). A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit. Molecular Systems Biology, 15(12), 1–13. https://doi.org/10.15252/msb.20199043
Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Cameron Sullards, M., Merrill, A. H., & Futerman, A. H. (2002). Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells. Journal of Biological Chemistry, 277(38), 35642–35649. https://doi.org/10.1074/jbc.M205211200
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Referee #2
Evidence, reproducibility and clarity
The study used a quantitative lipidomics approach which I am very familiar with. The results should be highly reproducible.
Significance
The manuscript submitted by Gopalan et al. reported a quantitative and comparative lipidomics study between mouse brain samples from early embryonic to postnatal stages, and rodent and human stem cell-derived neurons. The authors found a couple of very unique characters only existing in brain samples, but not in stem cell-derived neurons, including 22:6-containing glycerophospholipids and 18:0-containing sphingolipids. The authors further found the brain-like lipotypes can only be partially established in stem cell-derived neurons after supplementing brain lipid precursors. These findings clearly suggest that stem cell-derived neurons might not be appropriately used to mechanistically study lipid biochemistry, membrane biology, and biophysics in brains. The study was well designed. and the manuscript was very informative and resourceful. I would suggest to accept the manuscript for publication.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
Gopalan et al use quantitative, comprehensive lipid mass spectrometry of mouse brain tissue isolated at various time points in embryonic and postnatal development. They then go on to use the same quantitative analysis of mouse and human stem cells differentiated in vitro into neurons to define the lipid composition of these cultures.
Major Comments:
- As mentioned above, it is difficult to assess whether the discrepancy in the lipotype acquisition between in vivo mouse brain development and stem cell differentiation is due to metabolic differences in the in vitro differentiation as the authors state or is due to a lack of the stem cells to actually acquire a neuronal phenotype. Perhaps showing more clearly that the protocols for neuronal differentiation work efficiently and/or how they compare to brains dissected would be helpful in stating that the lipotype is different. The protocol referenced here (Bogetofte et al) only gives ~30% TH+ positive DA neurons in their manuscript. What cell type the other 70% of the cells are is something that could be discussed as means of "diluting" out the lipotype seen in these cultures. Perhaps the 30% TH+ DA neurons do attain the "correct" lipotype, but the lipidomic analysis can not detect this due to the contaminating effects of the non-differentiated cells. In this work, it would be nice to see what percentage of the cells differentiate into the expected cell type rather than referencing previous manuscripts. As differentiation protocols and originating cell sources are highly variable and error-prone, it's difficult to know what the lipotype results are actually reporting on.
Furthermore, discussion about these differentiation techniques and how well they represent functional neurons is warranted. The papers referenced here don't show 100% differentiation into the phenotypes that are described in this work such that the lipotype finding is not the only suggestion of a "general failure of in vitro neuronal differentiation models". Maybe a discussion of how the lack of ability to attain the neuronal lipotype due to the metabolic deficiencies discussed here could be causative to the inability to full recapitulate the neuronal phenotype is useful for the reader. 2. From the discussion and work here is unclear why the stearate feeding of the stem cells did not result in an increase in the 18:0-containing sphingolipids. The authors state that the appropriate metabolic pathways are not fully established and go on to look at the CerS expression levels across the differentiation timeline. It appears that the results presented in Fig S7 counter the authors' interpretation of the lipotype and more discussion here would be nice to clarify this discrepancy.
Minor comments:
- I find the data presentation of the LENA analysis to be difficult to follow (Fig 1E). In my opinion, the p-value is not the most important bit of information in this graph, though having it on the y-axis with other pertinent information encoded by colors or arrows being disguised. I would rather see the data on the x-axis that is above a certain p-value (denoted in the figure legend) plotted with the direction and magnitude of change shown.
- In the PCA in Fig 1, what are the loadings that define the variable PC1 and PC2? What is predominantly changing the P21 samples that lead to such a large shift if most of the data shown in the subsequent panels are not changing much between P2 and P21.
Significance
This work provides a nice reference for the complex lipidomes in embryonic and postnatal murine brain development. The details of the lipotype changes during development are well laid out and will of no doubt be of great use across a variety of scientific fields. While I found the in vivo data to be compelling, interesting, and useful, the lack of controls for the in vitro stem cell differentiation work makes this particular data set and comparison less useful. Further work to identify the limitations of the stem cell differentiation protocols as a valid comparison to in vivo brain development need to be done and/or the discussion of the direct comparisons between the two toned down.
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Reply to the reviewers
We thank the reviewers for their constructive comments, which greatly helped us in steering the manuscript in the right direction. Below is our point to point response to their concerns:
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms. The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 (S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in Lat1 (K75R) promotes mitophagy of Mdh1. Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at higher level in the presence of Lat1-K75R. The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which is suppressed upon Pkp1/2 overexpression. Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 affected phosphorylation sites in 8 mitochondrial matrix proteins. From these data the authors propose a model in which the PDC complex controls the activity of the associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the PDC and associated factors could resemble large protein kinase complexes as TORC.
(1) The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controllingmitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physicalinteraction occurs under their experimental conditions and (b) whether the different mutants that affectmitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, although consistent, remains purely speculative.
Response: We fully agree with the reviewer that this is an important point and we are currently trying to verify these interactions under our working conditions. Naturally, we will then test whether the documented Pdh1-Aup1 and the Pdh1-Pkp1/2 interactions are affected by mutants such as the lat1K75R mutation and the lat1Δ mutation, as insightfully suggested by the reviewer.
(2) It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication (Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1- T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?*
Response: We completely agree with the reviewer, and we have tested the effect of the lat1Δ mutations on mtDHFR-GFP (Supplemental Figure 2). It is important to note, however, that while the Bockler and Westermann paper does report an increased red/green fluorescence ratio in lat1Δ cells using mtRosella as a mitophagy reporter, this is part of a general screen, and was never further investigated or validated with additional, more rigorous methods (that paper focused on the role of the HERMES complex in mitophagy). In vivo fluorescence can be affected by a multitude of physical intracellular and intra-organellar factors including local pH, ionic strength, and molecular crowding. Therefore, the result from the screen conducted by Bockler and Westermann - although intriguing- is somewhat preliminary and requires more scrutiny (e.g. validation with a GFP release assay, complementation, etc) before we can make conclusions regarding the effect of the lat1Δ mutation on mitophagy, under their experimental conditions.
Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?*
This is now addressed in Figure 3E and 3F. The results are consistent with our hypothesis, namely showing complete or near complete suppression of the phenotype when tested using the phosphomimetic reporter.
(3) The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be rescued by Pkp1/2 overexpression.
We are sorry to hear that the reviewer is not convinced by the microscopy data. We agree with the comment that it is not trivial to observe the segregation by eye. We do not intend to say that the segregation is absolute, and the differences we observe in the distributions of the two signals are relative. By that, we mean that a prominent green dot in the cell, which is brighter than other green dots in the same cell, has a red channel counterpart which is not brighter than the other red dots in the cell, or may even be dimmer than the other red dots in the same cell. However, we do provide illustrative examples of segregation with specific arrows pointing to loci of segregation between the two channels. This was not pointed out in the original figure legend and this oversight has now been corrected. More importantly, we performed a mathematical analysis of the overlap between the two signals, and demonstrate a statistically (very) significant difference between the endogenous mitochondrial protein and the artificial mitochondrially-targeted GFP chimera. We would also like to stress that under our conditions (gluconeogenic medium, stationary phase) yeast mitochondria (at least in our genetic background) are not normally found in tubules (this pattern is specifically observed in cells growing in glucose-based medium). Nonetheless, we will attempt to carry out a 3-d reconstruction using our available z-sections, as per the reviewer’s request.
(3 continued) In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from mitophagy? In other words, could the PDC form a physical unit to segregate and prevent proteins from mitophagy turnover?
Response: It was shown by others that Pda1-GFP localizes to mitochondrial puncta, and we also observe this in our system. We have also briefly looked at the mitophagic efficiency of Pda1-GFP. While it is inefficiently delivered to the vacuole (5% free GFP after 5 days, vs 20% for Mdh1), it is not excluded from mitophagic targeting. We are not sure what the reviewer is referring to as a “physical interaction between Pdh1 and Mdh1”. We do not demonstrate such an interaction, and to our knowledge such an interaction has not been reported and validated in any publication. Even if such an interaction existed, it cannot explain the rescue of the pda1Δ phenotype by the co-overexpression of Pkp1 and 2, nor the effect of the pda1Δ mutation on Mdh1 phosphorylation, or the effect of the pda1Δ mutation on other mitophagy reporters which we used, such as Aco1, Qcr2, Idp1, and Aco2. We agree with the reviewer that there is a possibility that an organizing center, be it the PDC or another complex, may regulate intra-matrix segregation and mitophagic trafficking. However, addressing this question would require significant additional time and resources, and we would beg the referee to defer this question to future investigations, as it is not central to the claims made in the current manuscript.
(4) The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome independence of Pda1 are rather limited. First, the experimental setup does not distinguish between directeffects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to include a number of additional controls/strains.
Response: We agree with the reviewer that the phosphoproteomic analysis is not comprehensive. However, it was the best that we were able to do at the time, and it is unlikely that we could distinguish direct from indirect effects using this approach. The purpose of the experiment was to test whether we could identify more global effects of Pda1 on mitochondrial protein phosphorylation. This was in no way intended to imply a direct effect. Rather, it provides an unbiased map for potentially identifying the signaling network(s) involved. This may also include downstream events outside the mitochondrial matrix and even in the cytoplasm. We previously showed a connection of the Aup1-dependent signaling pathway to the RTG retrograde signaling pathway (Journo et al, 2009), and such a global analysis may allow a future understanding of how intra-matrix events can signal to the cytoplasm. However, we do not make any specific claims regarding which effects are direct and which are not. To address the reviewer’s concern, we have now modified the text to clarify these points (Page 9 line 28-Page 10 line 1).In an effort to improve the evidence for PDC- dependent mitochondrial matrix phosphorylation, we will carry out a phosphoproteomic analysis comparing WT cells to cells expressing the lat1K75R mutation. Since this mutation is expected to increase kinase activity, we expect to obtain a clearer picture that will complement the results obtained with the pda1Δ deletion mutant.
Reviewer #1 (Significance (Required)):
The authors continue to explore the mechanisms underlying their initial observation of differential turnover rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens up new lines of research that need to be explored to critically test the proposed model. A key question to me is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this study may play a direct role in these mechanisms.
To further address the mechanism by which the PDC affects mitophagy via Aup1, Pkp1, and Pkp2, we will carry out the following experiments: 1) We will verify the Pda1-Pkp1/2 interaction and test for effects of PDC mutants on this interaction, with an emphasis on the lat1K75R mutation 3) We will analyze the effect of the lat1K75R mutation on mitochondrial phosphoproteomics 4) we will analyze the effect of the pda1Δ mutation on the phosphorylation state of additional proteins which exhibit defective mitophagy in the pda1Δ background (Figure 1B). Our basic hypothesis regarding the reviewers’ question, namely the molecular mechanism behind the observed regulation is based on mitochondrial heterogeneity. We suggest that mitochondrial heterogeneity underlies mitophagic selectivity and that factors which affect heterogeneity also affect mitophagic selectivity.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvatedehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting components of PDC and associated regulatory factors (kinases and phosphatases) interferes with phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I find this work important and overall valuable, some of the conclusions made by the authors are still preliminary and could require additional experimental testing.
We thank the reviewer for these supportive comments
*In this regard, some of my major suggestions include: *
1. Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase assay.
We agree with the reviewer that, theoretically, the PDC could be necessary for the stability of Pkp1, Pkp2 and Aup1. However, have now tested this, and found that deletion of PDA1 has no effect on the expression levels of Aup1, Pkp1 and Pkp2 under our conditions (see Supplementary Figure 2).
2. Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1- compromised cells and see whether they are able to phosphorylate their model substrate in vitro. Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect the Mdh1 phosphorylation levels?
We present the hypothesis that the PDC allosterically regulates Aup1 and Pkp1/2 , as an explanation for the data. We agree with the reviewer that further experimental work will be necessary to verify this hypothesis. While the Pkp1/2 kinase assays could be useful in this regard, several issues temper our enthusiasm for this experiment:
i) This assay is far from “classical” with respect to this study. Neither of the two yeast mitochondrial kinases (Pkp1 and Pkp2) was previously characterized using in vitro kinase assays. While the mammalian assay conditions may work here, this is not guaranteed.
ii) A negative result will have no value
iii) A positive result (e.g. phosphorylation of recombinant Mdh1 by recombinant Pkp1/2) could arise due to low stringency conditions in the assay
iv) The nature of the kinase activity is unclear. We do not know if it is Pkp1, Pkp2 or a heterodimer of Pkp1 and Pkp2, or whether currently unknown ancillary factors are necessary for activity. This greatly complicates the analysis.
In lieu of the direct in vitro kinase assay, we have carried out an experiment demonstrating that an Mdh1-GFP variant with a phosphomimetic threonine to aspartate mutation at position 199, which we previously demonstrated to suppress both the aup1Δ and Pkp2Δ phenotypes (Kolitsida et al, 2019), can also suppress the pda1Δ phenotype (New Figure 3E, F). We suggest that this result provides sufficient evidence of a phosphorylation cascade linking the PDC, Pkp1/2 and Mdh1 mitophagic trafficking.
In addition, we will also attempt to further support the allosteric regulation hypothesis, by testing whether mutations in LAT1 such as lat1K75R modulate the known Aup1-Pda1 and Pkp1/2-Pda1 interactions (also see response to referee #1).
We greatly appreciate the suggestion to test the effect of DCA on our assays, and this experiment will be carried out in the near future. However DCA has not been shown to affect Pda1 phosphorylation in yeast or to modulate kinase activity of Pkp1/2, and it is not clear whether this approach will work. Many reagents and inhibitors which act in mammalian cells, are unable to cross the yeast cell wall.
3. Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein quality control is maintained.
We agree with the referee that this is an excellent question. Our current hypothesis posits that the established physical interaction between Pkp1/2 and Pda1 is crucial for activity of the kinases towards "third party" clients. To test this we will assay whether the catalytically inactive lat1K75R mutation affects the Pkp1/2 - Pda1 interaction. If we cannot find evidence for such a direct mechanism using these straightforward hypothesis-driven experiments, then we will also analyze effects the pda1Δ mutation on the general interactomes of Pkp1 and Pkp2.
Minor points:
1. The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in this study.
Thank you for the suggestion. We have now expanded our explanation of the GFP release assay (see Methods section, Page 13 lines 8-16).
2. MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly,the authors did not discuss these data extensively. Although changes in the phosphorylation status of some proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model substrate - Mdh1; Figure 1).
In our experiment, the coverage of the mitochondrial phosphoproteome was not complete. Some proteins, such as Mdh1, were observed only in a subset of the replicates, and therefore are not included in the figure. In the revision, we plan to add an analysis of the mitochondrial phosphoproteome in the lat1K75R mutation, and hopefully this will provide further insights.
Furthermore, many significant changes occur in the proteins that do not belong to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of Pkp1/Pkp2/Aup1. How do authors interpret these data?
We previously showed that the Aup1 signaling pathway converges with the RTG retrograde signaling pathway, which signals from the mitochondrial matrix to the nucleus (Journo et al, 2009). We would like to suggest that these effects on non-matrix proteins may indicate a possible role in transducing this signal. We have now added this comment to the discussion on Page 9 lines 28- Page 10 line 1.
3. Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).
Thank you. Fixed.
*Reviewer #2 (Significance (Required)): *
*This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism. *
We thank the reviewer for these encouraging comments.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The topic is very timely as the subject of mitochondrial quality control is of great interest. The main strength of this study is the creative ideas and the very out-of-the-box concept of selective elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will be well received after these issues are addressed.
We thank the reviewer for this positive assessment of the manuscript.
Specific comments:
Major comments:
1. It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is downstream of mitophagy, by how? Is there any data on that?
We presume the referee is referring to Figure 4A and B, where we demonstrate suppression of the pda1Δ phosphorylation phenotype by co-overexpression of Pkp1 and Pkp2. As indicated in the manuscript, we interpret these results as indicating that Pkp1 and Pkp2 function downstream of the PDC in the regulation of mitophagic trafficking. To further bolster this conclusion we now also show that a phosphomimetic mutation in Mdh1 that suppresses the pkp2Δ mutation as well as the aup1Δ mutation (Kolitsida et al, 2019), is also able to suppress the pda1Δ mutation. In addition, we will use co-IPs to test whether mutations in Lat1, and specifically the lat1K75R mutation which increases mitophagy and Mdh1 phosphorylation, can affect the known Pkp1/2-Pda1 interaction.
2. Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low glucose and if this was required for the effect.
As pointed out in the legend to Figure 4, the cells were grown in SL medium. As per the “Methods” section, SL contains, in addition to 2% lactate, 0.1% glucose. This small amount of glucose is always added to gluconeogenic media because initial growth in the total absence of glucose is very sluggish. 0.1% glucose does not induce the crabtree effect (does not inhibit respiration) and is consumed during the initial growth phase.
3. It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?
The purpose of this experiment was to untangle the apparent paradox wherein Aup1/Ptc6 is the opposing phosphatase countering Pkp1/2 on Pda1, but this is clearly not the case for the signaling pathway uncovered in this study. This finding further differentiates our signaling pathway from the conventional PDC regulation cascade, and provides a previously unidentified function for Ptc5. We do not claim that these effects are direct. There are 3 documented phosphatases in yeast mitochondria: Ptc5, Ptc7 and Ptc6/Aup1. Since we have previously shown that the aup1Δ phenotype is very similar to that of pkp2Δ, it disqualifies Aup1 from being a candidate for the opposing phosphatase acting on Mdh1-GFP. The experiment shown in Figure 4D and E indicates that Ptc5 is the prime candidate for a phosphatase that targets Mdh1-GFP. We now clarify this point in the discussion (Page 10, lines 14-24), and we suggest that a more definitive identification will be made in future studies.
4. Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather crude approach since the point the authors are trying to make is that it is the regulation rather than the expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation rather than delete the gene.
We fully agree with this suggestion. We will now test the effect of the lat1K75R mutation on the mitochondrial phosphoproteome, in order to address this deficiency.
Minor comments:
1. The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract too.
We have now added this information to the abstract. We do not wish to do the same for the title, as most journals have strict limits on the number of characters in the title.
2. In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in cells that shrink or rebuild their mitochondrial proteome.
Thank you. We have now made these changes (Page 3, lines 8-11)
3.Are pkp1 and pkp2 found only in the mitochondria?
As per the literature, Pkp1 and 2 are exclusively mitochondrial
4. In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other
proteins (currently in the supplementary)
We have added these blots to Figure 1
5. It is not clear how the reporter in fig 1C is different from the reporter in 1A.
This has now been clarified in the text (Page 4 line 28- Page 5 line 2)
Figure 4C. Please add an image of the colonies.
Thank you. This will be added to Figure 4.
6. It is not clear what the author defines as "increased segregation of Mdh1-GFP relative to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.
Thank you. We have now dropped the qualification “generic” and explain the difference. We now also explain the protocol for quantifying the overlap between the two signals (Page 8 lines 9-12, Page 16 lines 19-21 and 27-31).
Reviewer #3 (Significance (Required)):
This is a very hot topic and this lab is at the front of it It is for broad cell biology and biochemistry audience
We thank the referee for his/her generous and supportive comments
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Referee #3
Evidence, reproducibility and clarity
In this elegant and creative study, Dr. Abeliovich and his collaborators describe a novel pathway for the selective elimination of specific proteins. The flow of the experiments is logical and the writing is clear. The topic is very timely as the subject of mitochondrial quality control is of great interest. The main strength of this study is the creative ideas and the very out-of-the-box concept of selective elimination of specific proteins. The main shortcoming of this study is the incomplete direct evidence for a mechanism and conclusions that exceed the results. I believe these are fixable and I am sure this study will be well received after these issues are addressed.
Specific comments:
Major comments:
- It is not clear what is the mechanism for the recovery of pkp1 and pkp2 shown in figure 4. Supposedly, it is downstream of mitophagy, by how? Is there any data on that?
- Pertaining to the experiment shown in figure 4, it is not clear if this was conducted in the presence of low glucose and if this was required for the effect.
- It is not clear if the effect is directly mediated by pct 5&7 acting on MDH1 or if this is an indirect effect. Can this be stated and then addressed experimentally? Are pct5 and pct7 the only phosphatases in the matrix?
- Figure 6 uses deletion of PDA1 as a loss of function experiment. However in this case, this is a rather crude approach since the point the authors are trying to make is that it is the regulation rather than the expression levels that is critical. If possible, the authors should try and affect the regulatory site by mutation rather than delete the gene.
Minor comments:
- The title should include that this was done in yeast. Similarly, yeast should be mentioned in the abstract too.
- In the introduction, it is stated that mitophagy is a process degrading dysfunctional mitochondria. It will be more accurate to say that it is degrading dysfunctional mitochondria as well as removing mitochondria in cells that shrink or rebuild their mitochondrial proteome. 3.Are pkp1 and pkp2 found only in the mitochondria?
- In figure 1A/B only the blots of MDH1 are shown. It will be informative to show the blots of the other proteins (currently in the supplementary)
- It is not clear how the reporter in fig 1C is different from the reporter in 1A. Figure 4C. Please add an image of the colonies.
- It is not clear what the author defines as "increased segregation of Mdh1-GFP relative to generic mtRFP". Please clarify "generic mtRFP" and explain how the relativeness was deducted.
Significance
This is a very hot topic and this lab is at the front of it It is for broad cell biology and biochemistry audience
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Referee #2
Evidence, reproducibility and clarity
The manuscript by Kolitsida et al. unravels an interesting link between mitochondrial pyruvate dehydrogenase complex (PDC) and protein sorting for mitophagy-mediated clearance. Using yeast genetics combined with immunoblotting-based mitophagy protein sorting reporter assay, authors show that targeting components of PDC and associated regulatory factors (kinases and phosphatases) interferes with phosphorylation status of at least some mitochondrial matrix proteins and prevents their degradation by autophagic machinery. Experimental design is sound, and statistic evaluation seems appropriate. Although I find this work important and overall valuable, some of the conclusions made by the authors are still preliminary and could require additional experimental testing.
In this regard, some of my major suggestions include:
- Authors propose that the interaction of dedicated kinases and phosphatases with pyruvate dehydrogenase complex changes their affinity/activity toward the non-PDC substrates (possibly by allosteric regulation). Yet, alternatively, it can also be considered that the PDC complex acts as a docking station that stabilizes the associated regulatory factors inside the mitochondria and allows their additional functions. Indeed, simultaneous overexpression of Pkp1/2 increases the free GFP signal and Mdh1 phosphorylation even in the absence of core E1 PDC component (pda1, figure 3 C, D and figure 4 A, B), suggesting that the interaction of phosphatases and kinases with pda1 is not essential to mediate the phosphorylation of other mitoproteins. Are the protein levels of regulatory PDC factors (Pkp1, Pkp2, Aup1) somewhat altered by depletion of their target E1a subunit (pda1)? Could depletion of pda1 trigger destabilization of these factors and decrease their half-life? This can be tested by western-blotting and combined with cycloheximide chase assay.
- Allosteric regulation of specificity of PDC kinases and phosphatases is an intriguing yet still slightly unbacked hypothesis. To provide further experimental evidence for this explanation, authors could develop the classical in vitro kinase assay for Pkp1/2 on their model substrate (Mdh1) in the absence or presence of the Pda1 subunit. Even more excitingly, authors could isolate the Pkp1/2 from wild-type and Pda1-compromised cells and see whether they are able to phosphorylate their model substrate in vitro. Additionally, does specific inhibition of pyruvate dehydrogenase kinases (e.g., with dichloroacetate) affect the Mdh1 phosphorylation levels?
- Are the Pkp1/Pkp2 changing their interactome upon PDC alternation (e.g., Pda1 depletion)? Do their interactome alters upon stimulation of mitophagy? Co-immunoprecipitation or bioID assay could shed light on how the partitioning of the Pkp1/Pkp2/Aup1 functions between the metabolic regulation and protein quality control is maintained.
Minor points:
- The paper would benefit from a brief explanation of the principles of the mitophagy reporter assay used in this study.
- MS phosphoproteomics is indeed a great approach to tackle many questions in this study. Surprisingly, the authors did not discuss these data extensively. Although changes in the phosphorylation status of some proposed targets are apparent (as QCR2), many others were not detected (including Aco1/2, Idp1, and model substrate - Mdh1; Figure 1). Furthermore, many significant changes occur in the proteins that do not belong to the mitochondrial matrix compartment (as TOMM20 or YAT1), questioning the direct involvement of Pkp1/Pkp2/Aup1. How do authors interpret these data?
- Minor spelling mistakes should be reviewed (e.g., "mutophagic" pg. 7).
Significance
This work further expands on the previous observation by the authors (Kolitsida et al., 2019) and provides a novel and interesting hypothesis that may potentially impact our understanding of mitophagy selectivity toward particular mito proteome content. Furthermore, it unravels the additional function of PDC kinases and phosphatases behind the well-established regulation of energy metabolism. Therefore, it could interest the broad field of researchers interested in mitochondrial quality control and its interplay with cellular metabolism.
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Referee #1
Evidence, reproducibility and clarity
The Abeliovich lab has discovered differential degradation rates for mitochondrial proteins by mitophagy, a selective form of autophagy of mitochondria. In previous publications, they identified two protein kinases Pkp1/2 and the protein phosphatase Aup1(Pct6), which regulate phosphorylation patterns of mitochondrial proteins and affect their degradation rates by mitophagy. In the current manuscript, the authors now analyze the role of the pyruvate dehydrogenase complex (PDC) in mitochondria in the regulation of these regulatory link between Pkp1/2 and Aup1 and their substrate proteins. They find that the deletion of pda1, a core subunit of PDC, inhibits the degradation of previously identified mitophagy substrates Mdh1, Aco2, Qcr2, Aco1, and Idp1, while the turnover of mtDHFR-GFP, a model substrate diffusely localized in the mitochondrial matrix, is not affected. These data indicate that tested mitochondrial proteins are excluded from mitochondrial turnover by mitophagy in the absence of Pda1 by as yet unknown sorting mechanisms.
The authors next show that the mutation of a known Pkp1/2 and Aup1 phosphorylation site within Pda1 (S313A) drastically reduces mitophagic turnover of Mdh1, which is completely blocked by the additional deletion of AUP1. A mutation blocking Pda1 activity (R322C) also almost completely blocked Mdh1 degradation. However, PDC activity does not generally affect mitophagy, since an inactivating mutation in Lat1 (K75R) promotes mitophagy of Mdh1. Published work has shown that Pkp1/2 and Aup1, which affect the phosphorylation and degradation of Mdh1, physically interact with the PDC. Thus, the authors tested whether the absence of Pda1 affected the phosphorylation state of Mdh1 and thus its turnover. Indeed, Mdh1 showed globally reduced phosphorylation in the absence of Pda1, which was rescued by the overexpression of Pkp1/2 promoting degradation of Mdh1. Consistent with the effects on mitophagic turnover, Mdh1 was phosphorylated at higher level in the presence of Lat1-K75R. The authors analyzed the mitochondrial distribution of Mdh1 in dependence of Pda1. They provide cytological data suggesting that Mdh1 might segregate from a diffuse matrix localized mtRFP reporter, which is suppressed upon Pkp1/2 overexpression.
Finally, the authors perform a mitochondrial phospho-proteome analysis and found that the absence of Pda1 affected phosphorylation sites in 8 mitochondrial matrix proteins.
From these data the authors propose a model in which the PDC complex controls the activity of the associated protein kinase Pkp1/2 and the phosphates Aup1 by allosteric changes, which in turn regulates the phosphorylation and differential turnover of mitochondrial proteins by mitophagy. They speculate that the PDC and associated factors could resemble large protein kinase complexes as TORC.
Critical points:
- The authors postulate a structural role for the PDC in regulating Pkp1/2 and Aup1 for controlling mitophagy turnover of certain substrates. While the physical association of Pkp1/2 and Aup1 with PDC has been shown previously, the authors need to critically test their model and assess (a) whether this physical interaction occurs under their experimental conditions and (b) whether the different mutants that affect mitophagy of Mdh1 also affect the physical interaction of Pkp1/2 and Aup1 with PDC. Otherwise, their model, although consistent, remains purely speculative.
- It is important to test every mutant that affects Mdh1 turnover for effects on mitophagy in general using the mtDHFR-GFP reporter to be able to conclude specific effects on Mdh1 mitophagy. For example, the deletion of Lat1 has been shown to induce mitophagy of a mitochondrial matrix reporter during nitrogen starvation (Bockler and Westermann, 2013). While the authors observe a complete block of Mdh1 turnover in lat1 deletion cells, they do observe increased Mdh1 degradation in Lat1-K75R cells. In line with this reasoning, the authors have identified a number of Mdh1 variants in a previous publication (Kolitsida et al. 2019) that can be used to further explore the observed phenomenon. For example, the Mdh1-T199A could be used to test whether the expression of Pkp1/2 in pda1 deficient cells has specific or general effects on mitophagy. Furthermore, can Mdh1-T199D, which is turned over independent of Pkp1/2, be degraded in the absence of Pda1?
- The cytological data are not convincing. The imaging quality is low and it is very difficult for the reader to appreciate the suggested segregation of Mdh1-GFP and mtRFP. Thus, it is important to provide cortical sections in order to visualize mitochondrial tubules/networks and Mdh1 distribution. This is particularly important, because the authors mention effects of Pda1 on mitochondrial morphology, which appears to be rescued by Pkp1/2 overexpression. In this context it would be very informative to follow the localization of PDC in mitochondria. Previous work has shown that Pda1 forms punctate structures in mitochondria in proximity to ER-mitochondria contact sites marked by ERMES (Cohen et al. 2014). Is Pda1 itself degraded by mitophagy or is it excluded? Could the physical interaction of Mdh1 with Pda1 explain mitophagy phenotypes if Pda1 is excluded from mitophagy? In other words, could the PDC form a physical unit to segregate and prevent proteins from mitophagy turnover?
- The conclusions that can be drawn from the analysis of the mitochondrial phospho-proteome in dependence of Pda1 are rather limited. First, the experimental setup does not distinguish between direct effects of PDC on Pkp1/2 or Aup1 activity and the effects of simply PDC dysfunction on mitochondrial proteins. Along those lines, it is unclear whether the few identified proteins with altered phosphorylation states are indeed targets of Pkp1/2 or Aup1. Thus, to be able to support their conclusion, the authors need to include a number of additional controls/strains.
Significance
The authors continue to explore the mechanisms underlying their initial observation of differential turnover rates for mitochondrial proteins by mitophagy. This current work specifically builds on the previous publication identifying Pkp1/2 and Aup1 as regulators of the phosphorylation state of specific substrate proteins (Kolitsida et al. 2019). Here the authors now explore how these regulators might be organized and controlled by PDC. Thus, the study adds another layer of regulation. However, the molecular mechanisms of how PDC might regulate Pkp1/2 and Aup1 is not directly addressed. In conclusion, the current study opens up new lines of research that need to be explored to critically test the proposed model. A key question to me is of course how mitochondrial proteins can be excluded from mitophagy and whether the proteins in this study may play a direct role in these mechanisms.
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Reply to the reviewers
Reviewer #1
SUMMARY
The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.
MAJOR POINTS
Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly. To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).
We appreciate the reviewer’s criticisms on our strategy for identifying Hil proteins. In response, we have significantly revised our pipeline. In particular, we now combine the search results from three queries: in addition to C. auris Hil1’s Hyphal_reg_CWP domain (XP_028889033), we added the Hyphal_reg_CWP sequences from C. albicans Hyr1 and C. glabrata Hyr1. They were chosen as representatives in the two phylogenetic groups distinct from the one containing C. auris in order to avoid the bias due to the query’s phylogenetic position. Using the same criteria as we did for the original search, we identified three additional hits compared with the original 104 homologs list. In response to the criticism of the arbitrary exclusion of some species, we now include any species from the BLASTP search results as long as it is part of the 332 yeast species studied by Shen et al. 2018 (PMID: 30415838). The reason for this criterion is so that we can use the high-quality species phylogeny generated by Shen et al. 2018 to properly study the gene family evolution by reconciling the gene tree with the species tree. We additionally include the species in the MDR clade closely related to C. auris and used Muñoz et al. 2018 (PMID: 30559369) as the basis for the species phylogeny in the clade. Lastly, we no longer require the particular domain organization in classifying Hil family members. All BLASTP hits satisfying the E-value cutoff of 1x10-5 and query coverage > 50% are included.
A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families. For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.
We appreciate the reviewer’s comment. In the original manuscript, we performed two different types of analyses, one on the conserved and well-aligned Hyphal_reg_CWP domain and another on the rapidly evolving repeat region. For the former, we performed phylogenetic dN/dS analyses using maximum-likelihood, for which a reliable alignment is crucial and is the case here. The Hyphal_reg_CWP domain alignment for C. auris Hil1-Hil8 is shown below and also included as Fig. S7 in the revised manuscript: (figure in the response file)
In the text, we added this sentence to emphasize this point: “We chose to focus on the Hyphal_reg_CWP domain because of its potential importance in mediating adhesion and also because the high-quality alignment in this domain allowed us to confidently infer the evolutionary rates (Fig. S7).”
For the repeat domain, what we did in the original version was to calculate the pairwise dN/dS between individual repeat units found in Hil1 and Hil2. This didn’t require aligning the entire repeat regions in the two proteins, but instead relied on the alignment of the individual ~44 aa repeat units, which were highly conserved (see below). In the revised manuscript, however, we decided to focus our analyses on the Hyphal_reg_CWP domain because of a different concern, namely gene conversions between paralogs could distort the evolutionary history of the repeats (the same concern was addressed for the effector domain using an additional step of detecting recombination breakpoints, but the same analysis would be challenging for the repeat region due to alignment issues).
(figure in the response file)
It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!
We appreciate the reviewer’s suggestion. Based on the reviewer’s suggestion, we removed Fig. S12 and describe the model set up in the Materials and Methods section. We were not sure if the last point was a comment or a suggestion. We didn’t perform a population level selective sweep scan in C. auris. Such an analysis has in fact been attempted by Muñoz et al. 2021, who identified several members of the Hil family as the top candidates for positive selection (PMID: 33769478). We cited this in our Discussion:
“Lastly, scans for selective sweep in C. auris identified Hil and Als family members as being among the top 5% of all genes, suggesting that adhesins are targets of natural selection in the recent evolutionary history of this newly emerged pathogen (Muñoz et al. 2021).”
A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees. Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection.
We appreciate reviewer’s comment and agree with the concern about alignment quality affecting phylogenetic reconstruction. To clarify, all phylogenetic analyses in this work are based on the alignment of the Hyphal_reg_CWP domain, which is well aligned (shown above for the subset of eight homologs in C. auris). Alignment of all 215 homologs is provided for readers to review (shorturl.at/kDEJ3). To clarify this choice, we now include the following in Results:
“To further characterize the evolutionary history of the Hil family, including among closely related Candida lineages, we reconstructed a species tree-aware maximum likelihood phylogeny for the Hil family based on the Hyphal_reg_CWP domain alignment (Fig. 1C, Fig. S2).”
We also included detailed steps for reconstructing the gene tree in Materials and Methods.
To test the effect of gaps in the alignment on phylogenetic tree inference, we used two trimming programs, ClipKit (PMID: 33264284) and BMGE (PMID: 20626897), with author-recommended modes. They resulted in consistent gene tree results. We present the tree based on the ClipKit trimmed alignment in the main results. The root of the gene tree was inferred by jointly maximizing the likelihood scores for the gene tree based on the alignment and the evolution of the gene family within the species tree, using GeneRax (Morel et al. 2020, PMID: 32502238).
Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here.
We removed Figure 1 entirely based on this and another reviewer’s comment. We note that there is broad consensus that opportunistic yeast pathogens have independently arisen multiple times, such as C. auris, C. albicans and C. glabrata. Whether Candida pathogens that are more closely related evolved separately or not are subjects of ongoing research (PMID: 24034898).
It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.
We agree with the reviewer’s concern. In the revised manuscript and with more species included, we now only analyze genomes assembled to a chromosomal level, with the exception of C. auris B8441, which is supported by Muñoz et al. 2021 as having chromosome-length sequences. The revised Figure 7 now only includes these results. We also removed the accompanying supplementary figure that showed results based on scaffold-level assemblies.
MINOR POINTS
Line 18: "spp." Should be "spps."
Addressed throughout the revised manuscript.
Line 41: I might rephrase this as "how pathogenesis arose in yeast..."
Accepted (line 43 in revised manuscript).
I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.
Accepted (lines 47-48)
The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species
Changed (line 48)
The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032
We appreciate the reviewer pointing this important reference to us. We now cite it in the introduction (line 64) and discussion (line 340)
The first paragraph of the Results could be condensed
Addressed.
How was the species tree in Figure 1A obtained?
The previous figure 1 is now removed. The species tree used throughout the manuscript is based on Shen et al. 2018 with MDR clade species added, based on Muñoz et al. 2018.
Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader.
We removed the DH and SS labels. Instead, we highlighted three clades, which were defined in previous studies. These are the Candida/Lodderomyces clade (based on NCBI taxonomy database), the MDR clade (e.g., Muñoz et al. 2018, PMID: 30559369) and the glabrata clade (e.g., Gabaldón et al. 2013, PMID: 24034898).
How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?
We now provide the following details in the Materials and Methods section:
“To infer the evolutionary history of the Hil family, we reconstructed a maximum-likelihood tree based on the alignment of the conserved Hyphal_reg_CWP domain. First, we used hmmscan (HmmerWeb version 2.41.2) to identify the location of the Hyphal_reg_CWP domain in each Hil homolog. We used the “envelope boundaries” to define the domain in each sequence, and then aligned their amino acid sequences using Clustal Omega with the parameter {--iter=5}. We then trimmed the alignment using ClipKit with its default smart-gap trimming mode (Steenwyk et al. 2020). RAxML-NG v1.1.0 was compiled and run on the University of Iowa ARGON server with the following parameters on the alignment:
raxml-ng-mpi --all --msa $align --model LG+G --seed 123 --bs-trees autoMRE
.”The parameters for the ML tree reconstruction is listed on the last line above. The main parameter was the evolutionary model (LG+G), which accounts for rate variations using a gamma distribution. Other protein evolution models, e.g., VT+I+G, were tested and resulted in nearly identical tree topologies.
Figure 3C/D could be just one panel.
The structure predictions are now reorganized and presented on their own in the new Figure 3.
Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?
We appreciate the reviewer’s comment, which referred to CgAwp1 and CgAwp3, whose effector domain structures were reported in a recent study (Reithofer et al. 2021, PMID: 34962966). We now discuss them in relation to the predicted Hyphal_reg_CWP structure, by showing them in Figure 3 and describing them in the Results (lines 181) “crystal structures for the effector domains of two Adhesin-like Wall Proteins (Awp1 and Awp3b) in C. glabrata, which are distantly related to those in the Hil family were recently reported, and the predicted structure of one of C. glabrata’s Hil family members (Awp2) was found to be highly similar to the two solved structures (Reithofer et al. 2021)”
Line 168: Should read "Hence, ..."
The original sentence was removed, but this grammatical error was checked for and corrected.
Label proteins along the top of Figure 4 too.
Accepted (in new Figure 4).
Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?
In the revised manuscript, we provide the multiple sequence alignment for the Hyphal_reg_CWP domain used for the selection analysis as Fig. S7 to illustrate the level of conservation. The black numbers at the internal nodes are numeric indices used to refer to those nodes. In the revised manuscript, we use some of them to refer to the internal branches, e.g., 12…14 in the legend. In the new Figure 5, we do not list the numeric values of Dn and Ds (aka Ka, Ks). Instead, we use a color gradient to represent the estimated dN/dS ratios. The raw estimates are available in the project github repository. Panel B in the original Figure 5 and other panels related to the evolution of the repeats are now removed.
It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?
These results pertaining to the evolution of the repeats are now removed.
Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).
Table 2 is now Table S5. We also simplified the result section in the revised version. While the functional implications of the intraspecific variable number of tandem repeats (VNTR) is speculative, it is founded on two bases: 1) the identification of the VNTR is credible, as the copy number variation is consistent within clades but differ between clades, which is not expected if they are caused by assembly errors; 2) experimental studies in S. cerevisiae for the Flo family strongly supported a direct impact of adhesin length on the adhesive phenotype of the cells (PMID: 16086015).
Table 3 is not needed.
Table 3 is now removed.
Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.
In the revised Figure 6A, the coloring scheme is consistent with what we used in Figure 1 based on the three clades, and a legend is provided.
Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.
We have removed the original Figure 1.
SIGNIFICANCE
The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing.
We really appreciate the reviewer's positive comments. We agree and plan to explore the relationship between the adhesin family evolution and virulence phenotypes.
Expertise: gene families, evolution dynamics, human fungal pathogens
Reviewer #2
SUMMARY
Gene duplication and divergence of adhesin proteins are hypothesized to be linked with the emergence of pathogenic yeasts during evolution; however, evidence supporting this hypothesis is limited. Smoak et al. study the evolutionary history of Hil genes and show that expansion of this gene family is restricted to C. auris and other pathogenic yeasts. They identified eight paralogous Hil proteins in C. auris. All these proteins share characteristic domains of adhesin, and the structural prediction supports that their tertiary structures are adhesin-like. Evolutionary analysis of protein domains finds weak evidence of positive selection in the ligand-binding domain, and the central domain showed rapid changes in repeat copy number. However, performed tests cannot unambiguously distinguish between positive selection and relaxed selection of paralogs after gene duplication. Some alternative tests are suggested that may be able to provide more unambiguous evidence. Together with these additional tests, the detailed phylogenetic analyses of Hil genes in C. auris might be able to better support the hypothesis that the expansion and diversification of adhesin proteins could contribute to the evolution of pathogenicity in yeasts.
We appreciate the reviewer’s comments and will address specific points below.
MAJOR COMMENTS
The authors present extensive analyses on the evolution of Hil genes in C. auris. There is significant merit in these analyses. However, the analyses conducted so far are incomplete, lacking proper consideration of other confounding factors. Detailed explanations of our major comments are listed below.
- First, the authors restricted genes in the Hil family to those only containing the Hyphal_reg_CWP domain. Yet, previous work included genes containing the ligand-binding domain or the repeat domain as Hil genes. More justification is needed whether the author's choice represents the natural evolutionary history of Hil genes appropriately. For instance, are the genes only containing the ligand-binding domain monophyletic or polyphyletic? We recommend including the phylogeny of all the Hil candidate genes, to discern whether evolutionary histories of the repeat domain and ligand-binding domain are congruent. Authors can use this phylogeny as justification to focus only on the ligand-binding domain containing genes.
Butler et al. 2009 (PMID: 19465905) defined the Als family and the Hyr/Iff family as having either the N-terminal effector domain or the intragenic tandem repeats (ITR). Their rationale for the latter was that the ITS sequences were often conserved across species. Upon close inspection (Fig. S19,20 in that paper), however, we found that the ITS tend to be conserved in closely related species, but diverged among more distantly related species. Moreover, proteins in those figures that only contain the ITS and not the ligand-binding domains are all missing either the signal peptide, the GPI-anchor or both. This raises questions as to whether these proteins sharing the ITS sequence alone act as adhesins.
More generally, defining the evolutionary history of proteins with multiple domains is complicated by recombination, which causes different parts (e.g., domains) of the protein to have distinct evolutionary histories. In fact, our study and others show that there exist “chimeras” that combine the effector domain from one adhesin family and the repeat sequence found in another (Zhao et al. 2011, PMID: 21208290, Oh et al. 2019, PMID: 31105652). In these cases, one phylogenetic tree is insufficient to describe the evolutionary history of the whole protein. We chose to define the Hil family by the Hyphal_reg_CWP domain and thus focus on the evolutionary history of this region because 1) while tandem repeat regions also contribute to adhesion in yeasts (Rauceo et al. 2006, PMID: 16936142), the effector domain likely plays a more important role in ligand binding and specificity. Therefore, we believe using the effector domain to define a protein family is more likely to group proteins with similar functional properties than if the repeat sequences were used. Also, while putative fungal adhesins lacking a recognizable ligand-binding domain exist, they are rare (Lipke 2018, PMID: 29772751); 2) The repeat region evolved much more rapidly than the effector domain, as we illustrate in Figures 2, 4 and 6 in our revised manuscript. While some repeat units are highly conserved, e.g., the ~44 aa unit found in Hil1-4 in C. auris and close relatives in the MDR clade, many others are short and degenerate, making it difficult to reliably identify homologs sharing the repeat. Besides, since each protein could contain many distinct repeats, it is not clear how one defines two sequences as belonging to the same family if they share one out of six types of repeats. We acknowledge that this definition leaves out the evolutionary history defined by the tandem repeats, which may reveal intriguing evolutionary dynamics, with functional implications. A recent review for the Als family discussed similar definition challenges and partly supported our choice (Hoyer and Cota, 2016, PMID: 27014205).
In the analysis of positive selection, the authors do not adequately control for the effect of recombination on the evolutionary histories of protein sequences, especially given that Hil genes are rich in repetitive sequences. To account for recombination, GARD, an algorithm detecting recombination, should be performed to detect any recombination breakpoints within a protein domain. If recombination did occur within a protein domain, the authors should treat the unrecombined part as a single unit and use the phylogenetic information of this part to proceed with PAML analysis, instead of using the phylogeny of the entire protein domain. The authors should consider doing GARD analysis for the ligand-binding and repeat domains. For the repeat domain, low BS values in Fig. 5C indicate recombination between repeat units. Thus, the authors should analyze each repeat unit with GARD and re-analyze dN/dS.
We deeply appreciate the reviewers’ criticism here. In the revised manuscript, we removed the analysis of the repeat units and followed the reviewers’ suggestion to carry out GARD analysis on the effector domain, which we now show reveals evidence of intra-domain recombination. Using the inferred breakpoints (Fig. S8), we identified two putatively non-recombining partitions and performed all downstream phylogenetic analyses on them separately. The results are presented in Fig. 5 and Table S6. Compared with the previous result based on the entire Hyphal_reg_CWP domain alignment, the new results reveal clearer patterns, including significantly elevated dN/dS on a subset of the branches. Newly added branch-site test results support a role of positive selection on the effector domain during the expansion of the Hil family in C. auris, suggesting functional diversification following gene duplications.
The authors concluded positive selection in the ligand-binding domain based on the branch-wise model of PAML. Yet, w values were not higher than one, and it's unclear whether the difference in selective pressures the authors claimed here is biologically significant. Overall, what the authors present so far seems to be weak evidence of positive selection but is much more consistent with variation in the degree of purifying selection or evolutionary constraint. Using the site-wise model (m7 vs. m8) in PAML would allow the authors to detect which residues of the ligand-binding domain underwent recurrent positive selection. Combining the evolutionary information of protein residues and the predicted 3D structure will provide molecular insights into the biological impact of rapidly evolving residues. This would be a significant addition and raise the significance of the study, besides providing potentially stronger evidence of positive selection.
We appreciate the reviewers’ criticism and suggestions. In the revised manuscript, we performed site tests comparing models M2a vs M1a, M8 vs M7 and M8a vs M8. For partition 1 (P1-414), all three tests were insignificant. For partition 2 (P697-987), the M2a vs M1a test was insignificant (P > 0.05) but M8 vs M7 and M8a vs M7a were both significant at a 0.01 level, and the omega estimate for the positively selected category was estimated to be ~15. The site tests require all branches to evolve under the same selection regime. To relax this constraint, we performed additional branch-site tests by designating the branches with an estimated dN/dS > 10 as the foreground (based on the free-ratio model estimates). This test was significant for both branches at a 0.01 level and the Bayes Empirical Bayes (BEB) procedure identified a total of 5 residues as having been under positive selection. Although three of the five residues, located in the C-terminus of the Hyphal_reg_CWP domain, are part of the α-crystallin domain, we refrain from drawing any functional conclusions because 1) the BEB procedure is known to be lacking power in identifying positively selected residues and 2) we still lack structure-function relationship for the α-crystallin domain. But we agree and believe that this line of analysis is promising in yielding functional insight into the evolution of the effector domain in the family.
MINOR COMMENTS
-
In Fig 1c, the figure legend should include more specific details: which adhesin proteins are shown here? Please specify species names on the species tree
Figure 1 is removed in the revised manuscript
In Fig 3E, secondary structures are labeled with the wrong colors. Sheet: purple, helix: yellow
In the revised manuscript, the structures of SRRP-BR (original 3E) is now colored in a single color.
What's the ligand-binding activity of the b-solenoid fold? How structurally similar are C. auris PF 11765 domains compared to C. glabrata Awp domains? This information will support the role of adhesin for the ligand-binding domain of Hil genes.
We discuss the ligand-binding activity of the β-solenoid as follows in Discussion:
“The elongated shape and rigid structure of the β-helix are consistent with the functional requirements of adhesins, including the need to protrude from the cell surface and the capacity for multiple binding sites along its length that facilitate adhesion. In some bacterial adhesins, such as the serine rich repeat protein (SRRP) from the Gram-positive bacterium, L. reuterii, a protruding, flexible loop in the β-helix was proposed to serve as a binding pocket for its ligand (Sequeira et al. 2018). Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”
We also compare the structures of the C. auris Hil1/Hil7 Hyphal_reg_CWP domain and the CgAwp1/3 in Figure 3, with this in the legend “(C) Crystal structure of the C. glabrata Awp1 effector domain, which is highly similar to C. auris Hil1 and Hil7, but with the disulfide bond in a different location.”
We added a section in the Discussion to comment on the structure-function relationship based on known β-helix (aka β-solenoid) structures. The main insight comes from similar structures identified through DALI searches, many of which are bacterial and viral surface proteins mediating adhesion. The ligand binding pocket and specificity would require additional structural studies to elucidate.
In lines 248-249, the authors should also consider the influence of evolutionary history. For instance, repeats within the same Hil protein appeared later in evolution, compared to Hil gene duplication, and therefore these repeats experience less time for sequence divergence.
In the revised manuscript, we removed the analyses pertaining to the evolution of the repeat regions due to multiple challenges including alignment, potential of gene conversion and recombination. This is an important and intriguing aspect of adhesin family evolution that we plan to follow up in future work.
Although the bioinformatic evidence of C. auris Hil genes acting as adhesins is strong, it is still worthwhile to discuss the experiments of confirming the function of adhesins.
We agree with the reviewer and acknowledge in the revised manuscript the limitation of our work:
“Future experimental tests of these hypotheses will be important biologically for improving our understanding of the fungal adhesin repertoire, important biotechnologically for inspiring additional nanomaterials, and important biomedically for advancing the development of C. auris-directed therapeutics.”
SIGNIFICANCE
Overall, this study is interesting to investigate the evolutionary history of a crucial virulent gene in C. auris. Such evolutionary understanding will help us identify critical molecular changes associated with the pathogenicity of an organism during evolution, providing insights into the emergence of pathogens and novel strategies to cure fungal infections. The research question is important; however, the current analyses on the positive selection are incomplete, so the conclusion is modest so far. We recommend that the authors re-do the PAML analysis with the above considerations. This work will bring more significance to the mycology field if the functional impact of rapid evolution in protein domains can be supported or inferred.
This manuscript is well-written, and the authors also did a great job specifying all the necessary details in the M&M.
We appreciate the reviewers’ positive comments.
Reviewer #3
Summary:
The manuscript by Smoak et al. provides considerable information gleaned from analysis of HYR/IFF genes in 19 fungal species. A specific focus is on Candida auris. The main conclusion is that this gene family repeatedly expanded in divergent pathogenic Candida lineages including C. auris. Analyses focus on the sequences encoding the protein's N-terminal domain and tracts of repeated sequences that follow. The authors conclude with the hypothesis that expansion and diversification of adhesin gene families underpin fungal pathogen evolution and that the variation among adhesin-encoding genes affects adhesion and virulence within and between species. The paper is easy to read, includes clear and attractive graphics, as well as a considerable number of supplementary data files that provide thorough documentation of the sources of information and their analysis.
We appreciate the positive comment.
MAJOR COMMENTS:
- Are the key conclusions convincing?
Overall, the authors' conclusions are supported by the information they present. However, the overall conclusion is stated as a hypothesis and that hypothesis is not particularly novel. The idea that expansion of gene families associated with pathogenesis occurs in the pathogenic species dates back at least to Butler et al. 2009, who first presented the genome sequences for many of the species considered here.
We appreciate the reviewer’s comment. Our main conclusions are 1) the Hil family is strongly enriched in distinct clades of pathogenic yeasts after accounting for phylogenetic relatedness. This enrichment results from independent duplications, which is ongoing between closely related species; 2) the protein sequence of the Hil family homologs diverged rapidly following gene duplication, driven largely by the evolution of the tandem repeat content, generating large variation in protein length and β-aggregation potentials; 3) there is strong evidence for varying levels of selective constraint and moderate evidence for positive selection acting on the N-terminal effector domain during the expansion of the family in C. auris as our focal species. Based on these observations, we propose that expansion of adhesin gene families is a key preliminary step towards the emergence of fungal pathogenesis.
Indeed, some version of this hypothesis has been proposed by several groups before us. We fully acknowledged this in our previous as well as the revised manuscript, by citing Butler et al. 2009 (PMID: 19465905), Gabaldón et al. 2013, 2016 (PMID: 24034898, 27493146). Our study built on these earlier efforts and extended them by addressing several limitations. First, we performed phylogenetic regression to test for the association between gene family size and the life history trait (pathogen or not) in order to properly account for the phylogenetic relatedness. This was not done in previous studies. Second, most earlier studies didn’t construct a family-wide gene tree to fully investigate the evolutionary history of the family. Gabaldón et al. 2013 did a phylogenetic analysis for the Epa family and a few others within the Nakaseomycetes, revealing highly dynamic expansions. In the present study, we expanded this effort by comprehensively identifying homologs within the Hil family in all yeasts and beyond. Third and perhaps the most important novelty in our study is our detailed analysis of sequence divergence and role of natural selection during the evolution of the family post duplication. This allowed us to present a complete picture of the family’s evolution, not just in its increase in copy number but also its diversification after the duplications, which is a key part of how gene duplications contribute to the evolution of novel traits. As such, we believe our study provides strong support for the above hypothesis.
One key issue with a manuscript of this type is whether genome sequence data are accurate. The authors are not the first research group to take draft genome sequence data at face value and attempt to draw major conclusions from it. The accuracy of public genome data continues to improve, especially with the emergence of PacBio sequencing. Because the IFF/HYR genes contain long tracts of repeated sequences, genome assemblies from short-read data are frequently inaccurate. For example, is it reasonable to have confidence that the number of copies of a tandemly repeated sequence in a specific ORF is exactly 21 (an example taken from Table 2) when each repeat is 40+ amino acids long and highly conserved? Table S6 would benefit from inclusion of the type of sequence data used to construct each draft genome sequence. It is also reasonable to question whether the genome of the type strain is used as a template to construct the draft genomes of the other strains. If that was standard practice, conservation of the repeat copy number among strains might be an artefact. Conservation of repeat sequences to the degree shown is not a feature of the ALS family, a point of contrast between gene families that could be explored in the Discussion.
We appreciate the reviewer’s comment and agree strongly that a key limitation in gene family evolution studies like ours is the quality of the genome assembly. In the original manuscript, we took several steps to ensure the completeness and accuracy of the Hil family homologs, primarily by basing our results on the high quality RefSeq collection of assemblies, and supplementing it with two fungi-specific databases. In the revised manuscript, we performed further quality analyses to assess and correct for inaccuracy in the BLASTP hits. Because RefSeq aims to provide a stable reference, it is often slow in replacing older assemblies with newer ones based on improved technologies. We thus compared the RefSeq hits for species in which a newer, long-read based assembly had become available. The results are documented in Text S1 and in summary, while we did find examples of missing homologs and inconsistent sequences, the problems were isolated to specific species and the inconsistency pertains only to the tandem repeat regions. Regarding the specific example of within-species variable number of tandem repeats (VNTR) in C. auris Hil1-Hil4, we are confident of both the copy number and the sequence variation for two reasons. First, all C. auris strain genomes analyzed in this study were assembled de novo rather than based on a reference genome, and all were long-read based (PacBio) (Table S4). Second, empirically, we found the VNTR identified in Hil1-Hil4 agree among strains within one of the four clades of C. auris while differing between clades (Table S5). Since assembly errors are not expected to produce clade-specific patterns, we believe this is strong evidence for the VNTR identified being real.
We also appreciate the reviewer’s suggestion on discussing the conservation of the repeats as an interesting trait for a group of Hil proteins in comparison to the Als family. We now added a section in Discussion focusing on the special properties of this group of Hil proteins.
- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
Due to the nature of my comments, this review will not be anonymous. I will include some of the data from my laboratory to further illustrate the point about the quality of draft genome sequences, especially for gene families that contain repeated sequences. My laboratory group has spent the past several years looking at the families of cell wall genes in these species and know that the C. tropicalis genome sequence used in the current analysis is highly flawed. There is even a manuscript from several years ago that documents problems in the assembly (doi: 10.1534/g3.115.017566). There is a new PacBio sequence available that has considerably improved data for this group of genes, but still is not perfect. We designed primers and amplified the various coding regions to verify whether the IFF/HYR were correct in the draft genome sequences. For C. tropicalis, we know that 7 of the genes listed in this paper are broken (i.e. prematurely terminated) giving a false impression of their construction. The current study did not verify any gene sequences, so broken/incomplete genes are a stumbling block for developing conclusions.
We deeply appreciate the reviewer pointing out the flaws in the C. tropicalis genome. Using the PacBio sequence-based new assembly, we were able to confirm the reviewer’s comment on the sequence and annotation error in the RefSeq assembly for C. tropicalis. We listed the comparisons between the two assemblies in Table S8. Because the differences reside outside of the Hyphal_reg_CWP domain, they don’t impact our phylogenetic analyses, which are based on the effector domain alignment. To determine if this is a widespread issue affecting all genome assemblies based on older technologies, and in response to the reviewer’s criticism, we systematically checked the sequences of BLASTP hits based on the RefSeq assemblies against newer, long-read based ones when available. As detailed in Text S1 in the revised manuscript, the problems seen in C. tropicalis were not observed in four other species. While the sample size is small, we believe the issues with C. tropicalis are likely due to a combination of specific issues with the original assembly and special properties of the genome.
Similarly, the recent work from Cormack's lab features a PacBio C. glabrata sequence (doi: 10.1111/mmi.14707). The paper details how the authors focused on accurate assembly of the types of genes studied here. Sequences from the current project should be compared to the PacBio assembly to determine if they provide the same results.
We compared the sequences of the three C. glabrata Hil homologs identified in the RefSeq assembly (GCF_000002545.3) to the best BLAST hits in one of the new Cormack lab assemblies for (BG2 strain, GCA_014217725.1). Two of the three proteins showed identical sequences between the assemblies. One of them is longer in the new assembly than in the RefSeq (1861 vs 1771 aa, XP_447567.2, QHS67215.1). The main difference, however, was the number of hits recovered. Performing BLASTP searches in the new assembly recovered 13 hits vs 3 from the RefSeq assembly, of which 12 were in the subtelomeric region. For this reason, we used the new assembly as the basis for the Hil homologs in our subsequent analyses. To determine if we missed homologs in other genomes due to incomplete subtelomeric regions in the RefSeq assemblies, we repeated the BLASTP search in four other genomes (Text S1). In one of the four species, C. nivariensis, we recovered one more homolog than in the RefSeq. In all other three, we identified the same number (S. cerevisiae: 0, K. lactis: 1, C. albicans: 12), suggesting that the issues seen in C. glabrata is likely specific to this species and its RefSeq assembly.
Another part of the study that deserves additional attention or perhaps altered presentation is the idea that the Iff/Hyr N-terminal domain binds ligands. The literature on the Iff/Hyr proteins is limited. In my opinion, though, the authors of this paper could more completely present the information that is known. The paper by Uppuluri et al. is cited (doi: 10.1371/journal.ppat.1007056), but I did not see any information about their data regarding interaction of C. albicans Hyr1 with bacterial proteins mentioned in the manuscript under review. It is formally possible that the N-terminal domain of Iff/Hyr proteins does not bind a ligand. The current manuscript includes a great deal of speculation on that point, suiting it better to a Hypothesis and Theory format rather than other types of publications.
We appreciate the reviewer’s criticism and suggestion. We made two revisions based on the comments. First, we no longer refer to the Hyphal_reg_CWP domain as ligand-binding. Instead, we refer to it as the effector domain, following existing practices in the field (Lipke 2018, PMID: 29772751, de Groot et al. 2013, PMID: 23397570). Second, during the description of the predicted structure for the domain, we mentioned that it lacks an apparent binding pocket as suggested/identified in other β-solenoid proteins with carbohydrate binding abilities. Therefore, we suggest that the potential substrate and mechanism of binding by this domain remain to be determined with further experiments. We do, however, believe that there is strong evidence for the domain being involved in adhesion. A recent study (Reithofer et al. 2021) presented structural and phenotypic characterization of three Adhesin wall-like proteins (Awp1,2,3) in C. glabrata. In particular, experimental studies of CgAwp2, a Hil family protein, showed that its deletion resulted in the reversion of the hyperadhesive phenotype in one of the C. glabrata strains. Plastic was one of the substrates being evaluated, although, as the reviewer’s work pointed out, adhesion to plastics doesn’t indicate ligand binding, as it can be mediated by non-specific hydrophobic interactions (Hoyer and Cota 2016, PMID: 27014205). Nonetheless, the results presented in Reithofer et al. 2021 and other lines of evidence presented in the current work strongly supported adhesin functions of the Hil family.
Table 1 attempts to offer evidence that the Iff/Hyr N-terminal domain has adhesive function but falls short of convincing the reader. One of the example structural templates is a sugar pyrophosphorylase that seems irrelevant to the current discussion. In the column called "Function", the word adhesin is found several times, but no detail is presented. The only entry that offers an example ligand indicates that the domain binds cellulose which is not likely relevant for mammalian pathogenesis, the main focus of the work. Other functions listed include self-association and cell aggregation--using the N-terminal domain. It is formally possible that Iff/Hyr proteins drive aggregation using the N-terminal domain and beta-aggregation sequences in the repeated region. The authors should develop these ideas further. Discussion of adhesive/aggregative function related to the ALS family can be found in Hoyer and Cota, 2016 (doi: 10.3389/fmicb.2016.00280).
We appreciate the reviewer’s comments. In the revised manuscript, we removed Table 1, which was based on I-TASSER identified templates. Instead, we identified similar structures in the PDB50 database to the AlphaFold2 prediction for the Hyphal_reg_CWP domain in C. auris Hil1 using DALI (Table S3). We described the functional implications based on this list as follows:
“We identified a number of bacterial adhesins with a highly similar β-helix fold but no α-crystallin domain (Table S3), e.g., Hmw1 from H. influenzae (PDB: 2ODL), Tāpirins from C. hydrothermalis (PDB: 6N2C), TibA from enterotoxigenic E. coli (PDB: 4Q1Q) and SRRP from L. reuteri (PDB: 5NY0). For comparison, the binding region of the Serine Rich Repeat Protein 100-23 (SRRP100-23) from L. reuteri was shown in Fig. 3F (Sequeira et al. 2018). Together, these results strongly suggest that the Hyphal_reg_CWP domain in the C. auris Hil family genes mediate adhesion.”
One line of evidence that suggest the Hyphal_reg_CWP domain may have ligand-binding activity is from the L. reuteri SRRP-BR, which is one of the bacterial adhesins identified as having a highly similar β-helical structure (but missing the α-crystallin domain). In Sequeira et al. 2018 (PMID: 29507249), the authors showed via both in-vitro and in-vivo experiments that this domain “bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH”. However, the predicted structure for the Hyphal_reg_CWP domain in C. auris Hil1 and Hil7 lack a protruding, flexible loop in the β-helix, which was proposed to serve as a binding pocket for the ligand in SRRP-BR. We therefore commented in the text “Such a feature is not apparent in the predicted structure of the Hyphal_reg_CWP domain. Further studies are needed to elucidate the potential substrate for this domain and its mechanism of adhesion.”
We also appreciate the reviewer’s suggestion to discuss the potential role of the Hil proteins in mediating adhesion vs cell aggregation. We now have a section in Discussion that focuses on the potential role of the β-aggregation sequences especially in the subset of Hil proteins led by C. auris Hil1-Hil4, which have an unusually large number of such sequences. We discuss the recent literature suggesting the potential of such features mediating cell-cell aggregation.
The incredibly large number of figures that focus on the repeated sequences in the genes does not appear to include mention of the idea that these regions are frequently highly glycosylated. Knowing how much carbohydrate is added to these sequences in the mature protein would also have bearing on whether the beta-aggregation potential is realized. The Iff/Hyr proteins could stick to other things based on ligand binding (adhesion), hydrophobicity, aggregative activity, etc. Not much is really known about protein function so the conclusions are only speculative. The authors are largely accurate in presenting their conclusions as speculative, but the conclusions are not developed fully and always land on the idea that the N-terminal domain has adhesive function when that aspect clearly is not known.
We appreciate the reviewer’s comment. We have performed N- and O-glycosylataion predictions for the Hil family proteins in C. auris as a focal example and presented the results in Figure 2 of the revised manuscript. Briefly, we found that all eight proteins are predicted to be heavily O-glycosylated (Fig. 2C). N-glycosylation is rare except in Hil5 and Hil6, in regions with a low Ser/Thr content (Fig. 2C). We also deemphasized the ligand-binding ability of the effector domain and its importance in assessing the adhesin function of the Hil family proteins. At the same time, we highlighted other mechanisms as the reviewer pointed out, such as aggregative activities, in our discussion on the potential importance of the large number of β-aggregation motifs.
Another aspect of the analysis that is not mentioned is that several of the species discussed are diploid. What effect does ploidy have on the conclusions? Most draft genomes for diploid species are presented in a haploid display, so are not completely representative of the species. Additionally, some species such as C. parapsilosis are known to vary between strains in their composition of gene families, with varying numbers of loci in different isolates.
We appreciate the reviewer raising this issue. The potential impact of having diploid genomes represented as haploids is twofold. First, if the genome sequencing was performed on a diploid cell sample with some highly polymorphic regions, that would present difficulties to the assembly and could result in poorly assembled sections. Second, either because of the first issue, or because the researchers used the haploid phase of the organism for sequencing, the representative haploid genome will not be “completely representative of the species” as the reviewer suggested. The second problem is not specific to diploids – even for haploids, any single or collection of genomes would represent just a slice of the genetic diversity in the species. We did two things to look into this. First, we analyzed multiple strains in C. auris to reveal both Hil family size variation and also sequence polymorphism, particularly in the tandem repeat region. We also, as part of the quality control, compared and searched assemblies for different strains of some species when available. We agree that characterizing multiple genomes in a species is important for fully revealing the gene pool diversity and could have important consequences on our understanding of the emergence of novel yeast pathogens.
Regarding the first issue, we checked the original publications for two large-scale yeast genome sequencing projects that included 10 of the 32 species in the present study (Dujon et al. 2004, PMID: 15229592 and Butler et al. 2009, PMID: 19465905). In Dujon et al. 2004, the authors stated that haploid cells were used in cases where the species has both haploid and diploid phases. In Butler et al. 2009, the authors said in the Methods that “for highly polymorphic regions of diploid genomes, initial sequence assemblies were iteratively re-assembled in regions of high polymorphism to minimize read disagreement from the two haplotypes while maximizing coverage.”. Therefore, the potential issue of heterozygosity is likely minimal. In addition, many diploid yeasts have large regions in their genomes being homozygous, both as a result of clonal expansion and also due to loss of heterozygosity (LOH), as documented in C. albicans and other Candida species (e.g., PMID: 28080987). Nonetheless, we acknowledge that this issue is yet another challenge to having high-quality, complete genome assemblies. In the discussion, we fully acknowledge the limitation of our study by genome assemblies, and believe that ongoing improvement thanks to the development of long-read technologies will allow more in-depth studies, particularly in the subtelomeric regions and for repeat-rich sequences.
The manuscript concludes that having more genes is better, that the gene family represents diversification that must be driven by its importance to pathogenesis, without recognizing that some species evolve toward lower pathogenesis. This concept could be explored in the Discussion. …My own experience makes me wonder if the authors found any examples of species that provide an exception to the idea that having more genes is better and positively associated with pathogenesis. The parallel between IFF/HYR and ALS genes is made many times in the manuscript. Spathaspora passalidarum, a species that is not pathogenic in humans, but clearly within the phylogenetic group examined here, has 29 loci with sequence similarity to ALS genes. How many IFF/HYR genes are in S. passalidarum?
We appreciate the reviewer’s comment. We will address the two comments above together as they are related. First of all, S. passalidarum is now included in our extended BLAST search list and we identified a total of 3 homologs in this species. When compared with the related Candida/Lodderomyces clade, which includes C. albicans, the Hil family in this species is relatively small (3 vs. >10). More generally, we observe a significant correlation between the Hil family size and the species’ pathogenic potential (Figure 1B and the phylogenetic regression result in the text).
Regarding the first comment, we did identify two species that had a large Hil family (>8 based on C. auris) and yet were not known to infect humans. One of them, M. bicuspidata, has 29 Hil homologs and is interestingly a parasite for freshwater animals, such as Daphnia. The other species, K. africana, has 10 homologs and its ecology is not well described in the literature. With respect to the relationship between adhesin family and pathogenicity, we would like to make two points. First, as mentioned above, we observed a strong correlation between the Hil family size and the pathogen status, after correcting for phylogenetic relatedness, suggesting that expansion of the Hil family is a shared trait among pathogenic species. This doesn’t rule out the possibility that some species may have an expanded adhesin family, such as the example the reviewer mentioned, for reasons other than infecting a human host. Second, a key point in our work is that expansion of the adhesin family is only the first step – the crucial contribution of gene duplications to adaptation is not just in the increase in copy number, but also in providing the raw materials for selection to generate novel phenotypes. On that front, we documented the rapid divergence of the central domains both between and within species, as well as signatures of relaxed selective constraint and positive selection acting on the effector domain following gene duplications in C. auris, both of which support the above theme.
There are several current taxonomies for the species in this region of the tree. The source of the names used in this paper could be specific more completely.
We appreciate the reviewer’s comment. We now gave the complete Latin names for all species in Figure 1 and only use abbreviated names, e.g., C. auris, after the first occurrence. For species with multiple names in the literature, we followed the species name and phylogenetic placement in Shen et al. 2018 (PMID: 30415838).
The Results and Discussion sections are largely redundant. The tone of the paper is conversational, making it easy to read, but there seems little left to say in the Discussion that has not already been mentioned as the background for the various types of analyses. The authors should revise the paper to eliminate discussions of published literature from the Results and expand the Discussion to include some of the themes that have not been mentioned yet.
We appreciate the reviewer’s comment. In the revised manuscript, we have moved discussion points from the Result to the Discussion section. We also overhauled the Discussion to focus on the implications based on, but not already covered, in the Result part, including the points the reviewer suggested, e.g., the implications of the structure on adhesion mechanism.
Another point that the authors do not mention is documented recombination between IFF and ALS genes (doi: 10.3389/fmicb.2019.00781) and the effect of that process on evolution among these gene families.
We appreciate the reviewer’s comment. We now mention this and related observations in Discussion as part of the discussion on the mutational mechanisms for the evolution of the family:
“Diversification of adhesin repertoire within a strain can arise from a variety of molecular mechanisms. For example, chimeric proteins generated through recombination between Als family members or between an Als protein’s N terminal effector domain and an Hyr/Iff protein’s repeat region have been shown (Butler et al. 2009; Zhao et al. 2011; Oh et al. 2019). Some of the adhesins with highly diverged central domains may have arisen in this manner (Fig. S10).”
My reading of the work by Xu et al. 2021 (doi: 10.1111/mmi.14707) does not match the direction of its presentation in the current paper. Oh et al., 2021 (doi: 10.3389/fcimb.2021.794529) discussed that point recently, providing another point for the Discussion in the current paper.
We appreciate the reviewer’s comment and agree that our original reading of Xu et al. 2021 was incorrect. Instead of suggesting a higher mutation rates in the subtelomeric region, the authors instead suggested the evolution of the Epa family in the subtelomere was driven by Break-Induced Replication. We now update our discussion in the following way, also citing Oh et al. 2021
“Finally, as reported by (Muñoz et al. 2021), we found that the Hil family genes are preferentially located near chromosomal ends in C. auris and also in other species examined (Fig 7), similar to previous findings for the Flo and Epa families (Teunissen and Steensma 1995; De Las Peñas et al. 2003; Xu et al. 2020; Xu et al. 2021) as well as the Als genes in certain species (Oh et al. 2021). This location bias of the Hil and other adhesin families is likely a key mechanism for their dynamic expansion and sequence evolution, either via ectopic recombination (Anderson et al. 2015) or by Break-Induced Replication (Bosco and Haber 1998; Sakofsky and Malkova 2017; Xu et al. 2021). Another potential consequence of the subtelomeric location of Hil family members is that the genes may be subject to epigenetic silencing, which can be derepressed in response to stress (Ai et al. 2002). Such epigenetic regulation of the adhesin genes was found to generate cell surface heterogeneity in S. cerevisiae (Halme et al. 2004) and leads to hyperadherent phenotypes in C. glabrata (Castaño et al. 2005).”
I might have missed it, but I could not find what constitutes a BLAST-excluded sequence (Table S7). Additional explanation (or making the explanation easier to find) would help the reader.
We apologize for the inadvertent mistake of leaving out Table S7. In the revised manuscript, we include all hits from species that are part of the 322 species phylogeny in Shen et al. 2018. Thus, we removed the original Table S7.
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Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
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Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
Ideally, validation of all sequences would provide a stronger foundation for the work. However, that request is not realistic in terms of time or resources.
We agree with the reviewer and appreciate the understanding. In the revised manuscript, we performed additional analyses to evaluate the accuracy and correct the sequences of the BLASTP hits from RefSeq database by comparing them to long-read based assemblies when possible. Please see previous replies to reviewers’ comments and Text S1 for details.
- Are the data and the methods presented in such a way that they can be reproduced?
Yes, the data and methods are documented clearly and perhaps too thoroughly in many places. A considerable amount of confidence is placed in sequences that might not be accurate and tracking details down to the amino acid residue may not be reasonable in this context. A disclaimer might help--everyone probably already knows that genome sequences are not perfect but stating that the analysis is only as good as the genome sequence acknowledges that fact.
We appreciate the reviewer’s comment. In the revised manuscript, we tried to strike a balance between providing enough methodological details for the readers to assess the conclusions and yet also keeping the flow of the paper. We also accepted the reviewer’s suggestion by adding a disclaimer in the Discussion:
“we acknowledge the possibility of missing homologs in some species and having inaccurate sequences in the tandem-repeat region. We believe the expected improvements in genome assemblies due to advances in long-read sequencing technologies will be crucial for future studies of the adhesin gene family in yeasts.”
- Are the experiments adequately replicated and statistical analysis adequate?
The idea of replicates does not really apply to this analysis. I think that the species sampled are reasonable to represent the region of the phylogenetic tree on which the analysis is focused. The authors clearly documented their computational methods in an admirable way.
We appreciate the reviewer’s comment.
MINOR COMMENTS:
Figure 1 has elements that would make a nice graphical summary, but most of it should not be part of the final manuscript. For example, Panel A is repeated in Figure 2. It is not clear what Panel C means until the reader gets to Figure 2. Panel D is unnecessary. The image in Panel B is a good graphic. Endothelial adhesion is not mentioned, though. It is also debatable whether the proteins bind directly to plastic or to the body fluids that coat the plastic.
Based on this and another reviewer’s comments, we removed Figure 1 from the revised manuscript.
Compared to Figure 1, the information in Figure 3 is inconsistent. The "central domain" in Panel A is not central to anything as drawn, located at the end of the protein. The figure should be revised to be consistent with the majority of the authors' results.
We appreciate the reviewer’s suggestion. The terminologies used to describe the different parts of a typical yeast adhesin vary in the literature. In the Als family literature, central domain refers to the region after the N-terminal effector domain and before the C-terminal Ser/Thr-rich stalk domain. In the Hil family proteins, there is not a clear distinction between a “central” and a “stalk” region. In Boisramé et al. 2011 (PMID: 21841123), the authors referred to the region between the Hyphal_reg_CWP domain and the GPI-anchor as the central domain. We adopted that use. We realize that this can lead to confusion especially for Als researchers. In some other literature, e.g., Reithofer et al. 2021, this part of the protein is referred to as the B-region. But we couldn’t find wide use of that term. We decided to stay with “central domain” in this work and hope that by defining the term in Figure 2A, we would avoid any confusion within the scope of this work.
Are the low-complexity repeats mentioned in the Figure 4 legend present anywhere else in the C. auris genome or elsewhere among the species used in this study? The answer to that question may also provide evolutionary clues.
We did find one other putative GPI-anchored cell wall protein containing this ~44aa repeat unit, but with a different effector domain (GLEYA, PF10528). This protein (PIS58185.1 in C. auris B8441), appears to be a hybrid between the repeat region of C. auris Hil1 and an N-terminal effector domain of a different family. This result fits the theme of the reviewer’s work in C. albicans and C. tropicalis on the chimeric adhesins formed between the Als and Hyr/Iff families. Due to the scope of the current work, we omitted this finding from the main result.
Figure S1 legend. How was the distance to C. glabrata measured to call it equal?
The original Figure S1 was removed in the revised manuscript. A consistent set of criteria was employed in deciding which BLASTP hits to include as Hil family members.
Figure S4 could be presented better. Both diagonals have the same information. One could be emptied or could alternatively present nucleotide identity.
The original Figure S4 was removed in the revised manuscript.
Italicize the species names in Panel C of Figure S8.
The original Figure S8C is now Figure S9 and we systematically checked to make sure that species Latin names are italicized. Thanks for pointing this out.
Lines 256-257: The paper selectively samples the Iff/Hyr family and does not examine the "entire" family. Please revise.
We appreciate the reviewer’s comment. In the revised manuscript, we no longer selectively sample species. Instead, we only exclude three species that are not part of the 322-yeast species phylogeny in Shen et al. 2018 and Muñoz et al. 2018, namely Diutina rugosa, Kazachstania barnettii and Artibeus jamaicensis. Our extensive BLASTP searches also indicated that the family as defined in this work is specific to the budding yeast subphylum. We therefore believe it is accurate to describe the work as examining the entire Hil family.
- Are prior studies referenced appropriately?
I was disappointed to see that the paper does not reference my laboratory's work at all. When ALS genes are featured so strongly in a report, it seems reasonable to include something we have done over 30+ years. Our most-recent ALS paper (Oh et al., 2021 doi: 10.3389/fcimb.2021.794529) would be a reasonable source for defending the gene numbers used in Figure 2A. Other examples of our work that directly relate to concepts in this paper were mentioned above.
We sincerely apologize for our negligence. We are new to the fungal adhesin field through an accidental finding, and despite our effort to digest the relevant literature, we did unfortunately overlook the extensive work done on the Als family, much of which came from the reviewer’s lab. We have carefully read the papers suggested by the reviewer as well as others, and now have better incorporated prior foundational and insightful work into our result and discussion sections (see previous replies to the reviewer’s comments).
- Are the text and figures clear and accurate?
Suggestions for improvement are incorporated into the comments above.
- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Please present Methods and Results in the past tense. I still make the same mistake when I try to get my ideas on the page but proofread one more time and ensure the verb tenses are accurate.
We appreciate the reviewer’s comments and have edited the Methods and Results sections accordingly.
SIGNIFICANCE
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
The paper reads as if it is presenting preliminary data for a grant proposal. Perhaps Prof. He's lab wants to seek functional evidence for the role of the Iff/Hyr proteins. The current paper provides an exhaustive background for such a pursuit. As presented, there is little functional data for these proteins, genome sequences are not 100% accurate, but the trends noted are defendable.
We appreciate the reviewer’s comments. We acknowledge that experimental studies will be needed to prove and further establish the functional importance of our findings. However, we believe our gene family evolutionary studies provided important novel insights and serve as an example for adhesin family evolution.
- Place the work in the context of the existing literature (provide references, where appropriate).
The ideas presented here are similar to those pioneered in the Butler et al. Nature paper in 2009 (doi: 10.1038/nature08064). We now have the benefit of more genome sequences so the analysis can encompass more species. C. auris adds a newer focus on part of the phylogenetic tree that was not previously emphasized. The idea of "more is better" is very simplistic, though. Parallel work for the ALS family shows complexity in gene expression levels, suggesting that some adhesins are poised to make a large contribution while others are likely to have a scant presence on the cell surface. Those concepts are not really explored in the current paper, either. See Hoyer and Cota 2016 (doi: 10.3389/fmicb.2016.00280); Oh et al. (doi: 10.3389/fmicb.2020.594531).
We appreciate the reviewer’s comments and have included a discussion about the potential diversity of the duplicated Hil family proteins, in terms of function and their regulation in the Discussion. Also see our response to the first comment of the reviewer regarding the novelty of our hypothesis and the significance of our findings.
- State what audience might be interested in and influenced by the reported findings.
Potential readers would come from the fields of fungal adhesion and pathogenesis, as well as evolutionary biology.
We appreciate the reviewer’s comments.
- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
I discovered and named the ALS gene family in C. albicans and have spent 30+ years characterizing it. Most recently, my lab has focused on providing an accurate gene census and validated gene sequences for the cell wall "adhesinome" in the pathogenic Candida species. Some families are expanded and some are not. Some proteins appear only in a few species and demonstrate key roles in host-fungus interactions. There are many nuances to interpretation of what these fungi are doing from the standpoint of cell-surface adhesins and we look forward to exploring these ideas across many genomes, using validated gene sequences. We have a tremendous dataset that might make good fuel for a collaboration with Prof. He, given his enthusiasm for this area of study, as well as his outstanding expertise and perspectives on evolutionary analyses.
We sincerely thank the reviewer for the critical analysis of our manuscript and appreciate the many suggestions for improving the manuscript.
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Referee #3
Evidence, reproducibility and clarity
Summary:
The manuscript by Smoak et al. provides considerable information gleaned from analysis of HYR/IFF genes in 19 fungal species. A specific focus is on Candida auris. The main conclusion is that this gene family repeatedly expanded in divergent pathogenic Candida lineages including C. auris. Analyses focus on the sequences encoding the protein's N-terminal domain and tracts of repeated sequences that follow. The authors conclude with the hypothesis that expansion and diversification of adhesin gene families underpin fungal pathogen evolution and that the variation among adhesin-encoding genes affects adhesion and virulence within and between species. The paper is easy to read, includes clear and attractive graphics, as well as a considerable number of supplementary data files that provide thorough documentation of the sources of information and their analysis.
Major comments:
- Are the key conclusions convincing?
Overall, the authors' conclusions are supported by the information they present. However, the overall conclusion is stated as a hypothesis and that hypothesis is not particularly novel. The idea that expansion of gene families associated with pathogenesis occurs in the pathogenic species dates back at least to Butler et al. (2009; doi: 10.1038/nature08064) who first presented the genome sequences for many of the species considered here.
One key issue with a manuscript of this type is whether genome sequence data are accurate. The authors are not the first research group to take draft genome sequence data at face value and attempt to draw major conclusions from it. The accuracy of public genome data continues to improve, especially with the emergence of PacBio sequencing. Because the IFF/HYR genes contain long tracts of repeated sequences, genome assemblies from short-read data are frequently inaccurate. For example, is it reasonable to have confidence that the number of copies of a tandemly repeated sequence in a specific ORF is exactly 21 (an example taken from Table 2) when each repeat is 40+ amino acids long and highly conserved? Table S6 would benefit from inclusion of the type of sequence data used to construct each draft genome sequence. It is also reasonable to question whether the genome of the type strain is used as a template to construct the draft genomes of the other strains. If that was standard practice, conservation of the repeat copy number among strains might be an artefact. Conservation of repeat sequences to the degree shown is not a feature of the ALS family, a point of contrast between gene families that could be explored in the Discussion. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
Due to the nature of my comments, this review will not be anonymous. I will include some of the data from my laboratory to further illustrate the point about the quality of draft genome sequences, especially for gene families that contain repeated sequences. My laboratory group has spent the past several years looking at the families of cell wall genes in these species and know that the C. tropicalis genome sequence used in the current analysis is highly flawed. There is even a manuscript from several years ago that documents problems in the assembly (doi: 10.1534/g3.115.017566). There is a new PacBio sequence available that has considerably improved data for this group of genes, but still is not perfect. We designed primers and amplified the various coding regions to verify whether the IFF/HYR were correct in the draft genome sequences. For C. tropicalis, we know that 7 of the genes listed in this paper are broken (i.e. prematurely terminated) giving a false impression of their construction. The current study did not verify any gene sequences, so broken/incomplete genes are a stumbling block for developing conclusions.
Similarly, the recent work from Cormack's lab features a PacBio C. glabrata sequence (doi: 10.1111/mmi.14707). The paper details how the authors focused on accurate assembly of the types of genes studied here. Sequences from the current project should be compared to the PacBio assembly to determine if they provide the same results.
Another part of the study that deserves additional attention or perhaps altered presentation is the idea that the Iff/Hyr N-terminal domain binds ligands. The literature on the Iff/Hyr proteins is limited. In my opinion, though, the authors of this paper could more completely present the information that is known. The paper by Uppuluri et al. is cited (doi: 10.1371/journal.ppat.1007056), but I did not see any information about their data regarding interaction of C. albicans Hyr1 with bacterial proteins mentioned in the manuscript under review. It is formally possible that the N-terminal domain of Iff/Hyr proteins does not bind a ligand. The current manuscript includes a great deal of speculation on that point, suiting it better to a Hypothesis and Theory format rather than other types of publications.
Table 1 attempts to offer evidence that the Iff/Hyr N-terminal domain has adhesive function but falls short of convincing the reader. One of the example structural templates is a sugar pyrophosphorylase that seems irrelevant to the current discussion. In the column called "Function", the word adhesin is found several times, but no detail is presented. The only entry that offers an example ligand indicates that the domain binds cellulose which is not likely relevant for mammalian pathogenesis, the main focus of the work. Other functions listed include self-association and cell aggregation--using the N-terminal domain. It is formally possible that Iff/Hyr proteins drive aggregation using the N-terminal domain and beta-aggregation sequences in the repeated region. The authors should develop these ideas further. Discussion of adhesive/aggregative function related to the ALS family can be found in Hoyer and Cota, 2016 (doi: 10.3389/fmicb.2016.00280).
The incredibly large number of figures that focus on the repeated sequences in the genes does not appear to include mention of the idea that these regions are frequently highly glycosylated. Knowing how much carbohydrate is added to these sequences in the mature protein would also have bearing on whether the beta-aggregation potential is realized. The Iff/Hyr proteins could stick to other things based on ligand binding (adhesion), hydrophobicity, aggregative activity, etc. Not much is really known about protein function so the conclusions are only speculative. The authors are largely accurate in presenting their conclusions as speculative, but the conclusions are not developed fully and always land on the idea that the N-terminal domain has adhesive function when that aspect clearly is not known.
Another aspect of the analysis that is not mentioned is that several of the species discussed are diploid. What effect does ploidy have on the conclusions? Most draft genomes for diploid species are presented in a haploid display, so are not completely representative of the species. Additionally, some species such as C. parapsilosis are known to vary between strains in their composition of gene families, with varying numbers of loci in different isolates.
The manuscript concludes that having more genes is better, that the gene family represents diversification that must be driven by its importance to pathogenesis, without recognizing that some species evolve toward lower pathogenesis. This concept could be explored in the Discussion.
The Results and Discussion sections are largely redundant. The tone of the paper is conversational, making it easy to read, but there seems little left to say in the Discussion that has not already been mentioned as the background for the various types of analyses. The authors should revise the paper to eliminate discussions of published literature from the Results and expand the Discussion to include some of the themes that have not been mentioned yet.
My own experience makes me wonder if the authors found any examples of species that provide and exception to the idea that having more genes is better and positively associated with pathogenesis. The parallel between IFF/HYR and ALS genes is made many times in the manuscript. Spathaspora passalidarum, a species that is not pathogenic in humans, but clearly within the phylogenetic group examined here, has 29 loci with sequence similarity to ALS genes. How many IFF/HYR genes are in S. passalidarum?
There are several current taxonomies for the species in this region of the tree. The source of the names used in this paper could be specific more completely.
Another point that the authors do not mention is documented recombination between IFF and ALS genes (doi: 10.3389/fmicb.2019.00781) and the effect of that process on evolution among these gene families.
My reading of the work by Xu et al. 2021 (doi: 10.1111/mmi.14707) does not match the direction of its presentation in the current paper. Oh et al., 2021 (doi: 10.3389/fcimb.2021.794529) discussed that point recently, providing another point for the Discussion in the current paper.
I might have missed it, but I could not find what constitutes a BLAST-excluded sequence (Table S7). Additional explanation (or making the explanation easier to find) would help the reader. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
Ideally, validation of all sequences would provide a stronger foundation for the work. However, that request is not realistic in terms of time or resources. - Are the data and the methods presented in such a way that they can be reproduced?
Yes, the data and methods are documented clearly and perhaps too thoroughly in many places. A considerable amount of confidence is placed in sequences that might not be accurate and tracking details down to the amino acid residue may not be reasonable in this context. A disclaimer might help--everyone probably already knows that genome sequences are not perfect but stating that the analysis is only as good as the genome sequence acknowledges that fact. - Are the experiments adequately replicated and statistical analysis adequate?
The idea of replicates does not really apply to this analysis. I think that the species sampled are reasonable to represent the region of the phylogenetic tree on which the analysis is focused. The authors clearly documented their computational methods in an admirable way.
Minor comments:
- Specific experimental issues that are easily addressable.
Figure 1 has elements that would make a nice graphical summary, but most of it should not be part of the final manuscript. For example, Panel A is repeated in Figure 2. It is not clear what Panel C means until the reader gets to Figure 2. Panel D is unnecessary. The image in Panel B is a good graphic. Endothelial adhesion is not mentioned, though. It is also debatable whether the proteins bind directly to plastic or to the body fluids that coat the plastic.
Compared to Figure 1, the information in Figure 3 is inconsistent. The "central domain" in Panel A is not central to anything as drawn, located at the end of the protein. The figure should be revised to be consistent with the majority of the authors' results. Structures in Panels C to E would benefit from the "through the spiral" view that is featured in Figure S9. What experimental technique was used to solve the structure in Panel E? Adding that information to the legend would be helpful to the reader. Also, the secondary structure colors seem to be reversed between the legend and domain structure. Adding the coordinates of the domains shown would help the reader to understand their location in the mature protein.
Are the low-complexity repeats mentioned in the Figure 4 legend present anywhere else in the C. auris genome or elsewhere among the species used in this study? The answer to that question may also provide evolutionary clues.
Figure S1 legend. How was the distance to C. glabrata measured to call it equal?
Figure S4 could be presented better. Both diagonals have the same information. One could be emptied or could alternatively present nucleotide identity.
Italicize the species names in Panel C of Figure S8.
Lines 256-257: The paper selectively samples the Iff/Hyr family and does not examine the "entire" family. Please revise. - Are prior studies referenced appropriately?
I was disappointed to see that the paper does not reference my laboratory's work at all. When ALS genes are featured so strongly in a report, it seems reasonable to include something we have done over 30+ years. Our most-recent ALS paper (Oh et al., 2021 doi: 10.3389/fcimb.2021.794529) would be a reasonable source for defending the gene numbers used in Figure 2A. Other examples of our work that directly relate to concepts in this paper were mentioned above. - Are the text and figures clear and accurate?
Suggestions for improvement are incorporated into the comments above. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Please present Methods and Results in the past tense. I still make the same mistake when I try to get my ideas on the page but proofread one more time and ensure the verb tenses are accurate.
Significance
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
The paper reads as if it is presenting preliminary data for a grant proposal. Perhaps Prof. He's lab wants to seek functional evidence for the role of the Iff/Hyr proteins. The current paper provides an exhaustive background for such a pursuit. As presented, there is little functional data for these proteins, genome sequences are not 100% accurate, but the trends noted are defendable. - Place the work in the context of the existing literature (provide references, where appropriate).
The ideas presented here are similar to those pioneered in the Butler et al. Nature paper in 2009 (doi: 10.1038/nature08064). We now have the benefit of more genome sequences so the analysis can encompass more species. C. auris adds a newer focus on part of the phylogenetic tree that was not previously emphasized. The idea of "more is better" is very simplistic, though. Parallel work for the ALS family shows complexity in gene expression levels, suggesting that some adhesins are poised to make a large contribution while others are likely to have a scant presence on the cell surface. Those concepts are not really explored in the current paper, either. See Hoyer and Cota 2016 (doi: 10.3389/fmicb.2016.00280); Oh et al. (doi: 10.3389/fmicb.2020.594531). - State what audience might be interested in and influenced by the reported findings.
Potential readers would come from the fields of fungal adhesion and pathogenesis, as well as evolutionary biology. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
I discovered and named the ALS gene family in C. albicans and have spent 30+ years characterizing it. Most recently, my lab has focused on providing an accurate gene census and validated gene sequences for the cell wall "adhesinome" in the pathogenic Candida species. Some families are expanded and some are not. Some proteins appear only in a few species and demonstrate key roles in host-fungus interactions. There are many nuances to interpretation of what these fungi are doing from the standpoint of cell-surface adhesins and we look forward to exploring these ideas across many genomes, using validated gene sequences. We have a tremendous dataset that might make good fuel for a collaboration with Prof. He, given his enthusiasm for this area of study, as well as his outstanding expertise and perspectives on evolutionary analyses.
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Referee #2
Evidence, reproducibility and clarity
Summary
Gene duplication and divergence of adhesin proteins are hypothesized to be linked with the emergence of pathogenic yeasts during evolution; however, evidence supporting this hypothesis is limited. Smoak et al. study the evolutionary history of Hil genes and show that expansion of this gene family is restricted to C. auris and other pathogenic yeasts. They identified eight paralogous Hil proteins in C. auris. All these proteins share characteristic domains of adhesin, and the structural prediction supports that their tertiary structures are adhesin-like. Evolutionary analysis of protein domains finds weak evidence of positive selection in the ligand-binding domain, and the central domain showed rapid changes in repeat copy number. However, performed tests cannot unambiguously distinguish between positive selection and relaxed selection of paralogs after gene duplication. Some alternative tests are suggested that may be able to provide more unambiguous evidence. Together with these additional tests, the detailed phylogenetic analyses of Hil genes in C. auris might be able to better support the hypothesis that the expansion and diversification of adhesin proteins could contribute to the evolution of pathogenicity in yeasts.
Major Comments
The authors present extensive analyses on the evolution of Hil genes in C. auris. There is significant merit in these analyses. However, the analyses conducted so far are incomplete, lacking proper consideration of other confounding factors. Detailed explanations of our major comments are listed below.
- First, the authors restricted genes in the Hil family to those only containing the Hyphal_reg_CWP domain. Yet, previous work included genes containing the ligand-binding domain or the repeat domain as Hil genes. More justification is needed whether the author's choice represents the natural evolutionary history of Hil genes appropriately. For instance, are the genes only containing the ligand-binding domain monophyletic or polyphyletic? We recommend including the phylogeny of all the Hil candidate genes, to discern whether evolutionary histories of the repeat domain and ligand-binding domain are congruent. Authors can use this phylogeny as justification to focus only on the ligand-binding domain containing genes.
- In the analysis of positive selection, the authors do not adequately control for the effect of recombination on the evolutionary histories of protein sequences, especially given that Hil genes are rich in repetitive sequences. To account for recombination, GARD, an algorithm detecting recombination, should be performed to detect any recombination breakpoints within a protein domain. If recombination did occur within a protein domain, the authors should treat the unrecombined part as a single unit and use the phylogenetic information of this part to proceed with PAML analysis, instead of using the phylogeny of the entire protein domain. The authors should consider doing GARD analysis for the ligand-binding and repeat domains. For the repeat domain, low BS values in Fig. 5C indicate recombination between repeat units. Thus, the authors should analyze each repeat unit with GARD and re-analyze dN/dS.
- The authors concluded positive selection in the ligand-binding domain based on the branch-wise model of PAML. Yet, w values were not higher than one, and it's unclear whether the difference in selective pressures the authors claimed here is biologically significant. Overall, what the authors present so far seems to be weak evidence of positive selection but is much more consistent with variation in the degree of purifying selection or evolutionary constraint. Using the site-wise model (m7 vs. m8) in PAML would allow the authors to detect which residues of the ligand-binding domain underwent recurrent positive selection. Combining the evolutionary information of protein residues and the predicted 3D structure will provide molecular insights into the biological impact of rapidly evolving residues. This would be a significant addition and raise the significance of the study, besides providing potentially stronger evidence of positive selection.
Minor Comments
- In Fig 1c, the figure legend should include more specific details: which adhesin proteins are shown here? Please specify species names on the species tree
- In Fig 3E, secondary structures are labeled with the wrong colors. Sheet: purple, helix: yellow
- What's the ligand-binding activity of the b-solenoid fold? How structurally similar are C. auris PF 11765 domains compared to C. glabrata Awp domains? This information will support the role of adhesin for the ligand-binding domain of Hil genes.
- In lines 248-249, the authors should also consider the influence of evolutionary history. For instance, repeats within the same Hil protein appeared later in evolution, compared to Hil gene duplication, and therefore these repeats experience less time for sequence divergence.
- Although the bioinformatic evidence of C. auris Hil genes acting as adhesins is strong, it is still worthwhile to discuss the experiments of confirming the function of adhesins.
Significance
Overall, this study is interesting to investigate the evolutionary history of a crucial virulent gene in C. auris. Such evolutionary understanding will help us identify critical molecular changes associated with the pathogenicity of an organism during evolution, providing insights into the emergence of pathogens and novel strategies to cure fungal infections. The research question is important; however, the current analyses on the positive selection are incomplete, so the conclusion is modest so far. We recommend that the authors re-do the PAML analysis with the above considerations. This work will bring more significance to the mycology field if the functional impact of rapid evolution in protein domains can be supported or inferred.
This manuscript is well-written, and the authors also did a great job specifying all the necessary details in the M&M.
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Smoak et al., provides an analysis of the Hyr/Iff-like (Hil) genes in Candida species with a strong focus on C. auris. The authors demonstrate a repeated expansion of these genes in unique lineages of fungal species, many of which are associated with stronger clinical disease. There is evidence of selection operating on the gene family in the primary domain used for identification. These genes include a repeat just downstream of that core domain that changes frequently in copy number and composition. The location of these genes tends to cluster at chromosome ends, which may explain some aspects of their expansion. The study is entirely in silico in nature and does not include experimental data.
Major points
Altogether, many of the general findings could be convincing but there are some aspects of the analysis that need further explanation to ensure they were performed correctly.
To start, a single Hil protein from C. auris was used as bait in the query to find all Hil proteins in yeast pathogens. Would you get the same outcome if you started with a different Hil protein? What is the basis for using Hil1 as the starting point? It also doesn't make sense to me to remove species just because there are already related species in the list. This may exclude certain evolutionary trends. Furthermore, it would be helpful to know how using domain presence and the conservation of position changes the abundance of the gene family across species? (beginning of results).
A major challenge in the analysis like this one is in dealing with repetitive sequences present in amplified gene families.
- For example, testing modes of selection on non-conserved sites is fraught. It's not clear if all sites used for these tests are positionally conserved and this should be clarified. Alignments at repeat edges will need to maintain this conservation and relatively good alignments as stated in lines 241-242 are concerning that this includes sequence that does not retain this structure necessary for making predictions of selection.
- It's also unclear to me why Figure S12 is here. The parameters for this analysis should be tested ahead of building models so only one set of parameters should be necessary to run the test. The evolutionary tests within single genes and across strains is really nice!
- A major challenge for expanded gene families is rooting based on the inability to identify a strong similarity match for the full length sequence. The full alignment mentioned would certainly include significant gaps. If those gaps are removed and conserved sites only are used, does it produce the same tree? Inclusion of unalignable sequences would be expected to significantly alter the outcomes of those analysis and may produce some spurious relationships in reconciling with the species trees.
- Whether or not there are similar issues in the alignment of PF11765 need to be addressed as well. There's nothing in the methods that clarifies site selection. Figure 1A: the placement of evolved pathogenesis is a little arbitrary. It's just as feasible that a single event increased pathogenesis in the LCA of C. albicans and C. parapsilosis that was subsequently lost in L. elongisporus. These should be justified or I'd suggest removing. The assignment of Candida species here also seems incomplete. The Butler paper notes both D. hansineii and C. lusitaniae as Candida species whereas they are excluded here. It is tricky to include scaffolds in analysis of chromosomal location of the HIL genes. The break in the scaffold may be due to the assc repeats of these proteins alone or other, nearby repeats. Any statistics would be best done to include only known chromosomes or those that are strongly inferred by Munoz, 2021. This will change the display of Figure 7, but is unlikely to change the take home message.
Minor points
Line 18: "spp." Should be "spps."
Line 41: I might rephrase this as "how pathogenesis arose in yeast..."
I might use a yeast-centric example around line 40 for duplication and divergence. This could include genes for metabolism of different carbon sources in S. cerevisiae.
The Butler paper referenced on line 51 compared seven Candida species and 9 Saccharomyces species The autors state no other evolutionary analysis of adhesins has been performed but do not acknowledge this study: https://academic.oup.com/mbe/article/28/11/3127/1047032
The first paragraph of the Results could be condensed
How was the species tree in Figure 1A obtained?
Figure 2: In panel A, "DH" and "SS" are not defined. I'd be careful with use of "non-albicans Candida" in Figure 2B. This usually includes C. tropicalis and C. dubliniensis and may confuse the reader. How was the binding domain defined to extract those sequences are produce a phylogeny? In building a ML model, how were parameters chosen?
Figure 3C/D could be just one panel.
Can you relate more the fungal hit to the Hil proteins conveyed in lines 152-154?
Line 168: Should read "Hence, ..." Label proteins along the top of Figure 4 too.
Figure 5: for tests of selection, were sites conserved across the group? What does the black number at each node indicate? Dn and Ds are given as decimals. This is based on what attribute? For panel B, it is unclear what each tip denotes i.e., Hil1_tr6. Hil1 is the gene but what is "tr6"?
It's unclear why comparison of the PF11765 domain includes the MRD proteins when those aren't included in the comparison to the repeats alone. Could that skew the comparison due to unequal sample numbers or changed variation frequencies in MDR relative to the other two groups?
Table 2 doesn't add much. This section could probably be reduced to a few sentences since it's highly speculative (intraspecies variation).
Table 3 is not needed.
Figure 6 - color coding in 6A needs to be explained. I'm assuming this is a taxonomical coding.
Figure 1B is unnecessary. A Model of the protein indicating domains is sufficient here. Figure 1C needs labels for all termini, not just the pathogenic red branches. The figure doesn't provide clear association between adhesin families and the associated species. This could be omitted, especially since Flo is often associated with Saccharomyces species. Figure 1D is unnecessary.
Significance
The work here is sorely needed in expanded gene families and in fungi specifically. No analysis at this level has been performed, to the best of my knowledge, in any fungal associated gene family and certainly not in relationship to pathogenic potential. The authors do a good job in citing the foundational literature upon which their study builds in most cases (one exception is noted above). It would be of general interest to those interested in the evolution of virulence, but the analysis is tricky. This is the biggest drawback I currently have as some of the information to assess the results is missing. Expertise: gene families, evolution dynamics, human fungal pathogens
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Reply to the reviewers
We would like to thank the Reviewers for their valuable comments and constructive suggestions concerning our manuscript entitled " Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes" (RC-2022-01629).
Please find below our responses to the Reviewers' questions and comments. We have revised the Manuscript following the Reviewers' suggestions. The changes in the Manuscript are indicated in blue.
Reviewer #1 (Evidence, reproducibility and clarity (Required)): ____ This manuscript by Uhlirova and colleagues identified an unwanted off-target effect in the pVALIUM10 TRiP RNAi lines that are commonly used in the fly community. The pVALIUM10 lines use long double-stranded hairpins and are useful vectors for somatic gene knock-down, hence they are widely used.
Here the authors find that any pVALIUM10 TRiP RNAi line can create the silencing of any transgenes that were cloned with the commonly used Gateway system. this is caused by targeting attB1 and attB2 sequences, which are also present in other Drosophila stocks including the transgenic flyORF collection. Hence, this is an important and useful information for the fly community that should be published quickly. All experiments are well documented and well controlled. I only have a few minor comments.
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I recommend to mention the number of 1800 pVALIUM10 lines in Bloomington in the abstract rather than 11% to make clear that this is an important number of lines. (1800 of 13,698 lines in Bloomiongton are 13 and not 11 per cent?)
We now include the absolute number of pVALIUM10 lines in the manuscript abstract. The percentages have been corrected. Furthermore, we updated/corrected the total number of RNAi lines available from various stock centers in the Discussion, L153-L156.
The status on 23.10.2022
VDRC - 23,411 in total (12,934 GD lines; 9,674 KK lines; 803 shRNA lines)
Bloomington - 13,410 TRiP lines based on pVALIUM vectors (13,674 in total, including 264 non-pVALIUM, and 48 non-fly genes targeting lines)
NIG - 12,365 in total (5,676 TRiP lines; 7,923 NIG RNAi lines)
The authors may consider to call the 'unspecific' silencing effect an 'off-target' effect compared to intended 'on-target'. Such a nomenclature would be more consensus.
We changed the wording in the manuscript as suggested by the reviewer.
Ideally, all the imaging results in Figure 2 and 3 would be quantified. The simple 'V10' label in the Figure 3L and 3M is not the most intuitive, at least it took me a while to figure out what the authors compare.
The labeling in the charts has been changed. We now provide quantifications for the data shown in Figure 2 and 3.
Does the silencing also affect attR sequences? These are present after cassette exchange in many transgenes, most of the time not in the mRNA though, so it might not be so relevant.
A 22 nucleotide stretch of the attB2 site indeed shows a 100% match to the attL2 site. See the example alignment below (availbale in word/PDF version of the Letter). While we did not assess this possibility experimentally, attL sites would likely be susceptible to the same undesirable off-target silencing effects if present in the nascent or mature transcript.
Reviewer #1 (Significance (Required)): This is an important and useful information for the fly community that should be published quickly.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): ____ Stankovic, Csordas, and Uhlirova show that a specific subset of the TRiP RNAi lines available, namely the pVALIUM10 subset, can cause a knockdown of certain co-expressed transgenes that contain attB1 and attB2 sites. The authors demonstrate that while pVALIUM20 or Vienna KK lines for BuGZ or myc RNAi do not affect RNase H1:GFP expression, pVALIUM10 RNAi lines against BuGZ or myc significantly decrease expression of the RNAseH1:GFP transgene. The authors propose that, due to how these RNAi lines were constructed, the siRNA products could be targeting to attB1 and attB2 sites in transgenes that were made using similar methodology. To support this idea, they ubiquitously express mCherry transgenes encoding mRNAs either containing or lacking attB sites. They find that the knockdown of mCherry seen with several different pVALIUM10 RNAi lines is observed with the reporter mRNA containing attB sites, but is suppressed when the attB sites are removed from mCherry mRNA. They also find that the pVALIUM10 RNAi lines reduce the expression of the FlyORF transgene SmD3:HA.
The paper is very clearly written and the data presented is convincing.
Minor suggestions:
1. Figure 3 L+M The labels for the ubi-mcherry and ubiΔattb-mcherry are switched in these graphs (i.e. ubiΔattb-mcherry should be the one with a higher intensity in the pouch compared to the notum).
Figure 3M the labels don't match the RNAi lines used in H-K.
We corrected the labelling in the charts.
Figure 2 and 3. For the images of the transgenes, it seems as if the BuGZ RNAi line has a more drastic effect on RNaseH1 than mCherry, and vice versa for the myc RNAi lines. Did the authors notice a pattern with the decreased expression. Do some of the RNAi lines have a more consistent/severe impact, or might different transgenes be impacted to different extents?
Throughout the study and multiple experimental trials, we did not observe that the BuGZRNAi and mycRNAi silencing efficiency would depend on whether the monitored reporter was RNase H1::GFP or mCherry. What has been reproducible is the differential impact of the three tested mycRNAi lines on ubi-RNaseH1::GFP transgene. While pVALIUM10-based mycRNAi[TRiP.JF01761] reduces RNaseH1::GFP signal Valium20 mycRNAi[TRiP.HMS01538] enhances it and GD mycRNAi[GD2948] has no effect, although the number of replicates for the latter is lower compared to the other tested lines. Why Valium20 mycRNAi[TRiP.HMS01538] increases RNaseH1::GFP signal remains unclear for now.
We would like to refrain from directly quantitatively comparing the effects of phenotypically different RNAi lines on differently tagged mRNAs/proteins. As the RNAseH1::GFP fusion protein is nuclear while the mCherry is cytoplasmic, their distinct subcellular localization and/or turnover rate may give a different overall impression on the change in fluorescence intensity (Boisvert et al, 2012; Mathieson et al, 2018). Another confounding factor is the described roles of Drosophila Myc in regulating transcription, translation, and cell growth (Gallant, 2007).
Line 150 unnecessary comma after Both Line 131 knockdown should be knocked down Line 133 should be "using an additional" Figure legend 1 wing disc should be at least written out when the abbreviation (WD) is first used.
We thank the reviewer for pointing these out, the relevant corrections were performed.
Reviewer #2 (Significance (Required)):
Overall, this manuscript is an informative reminder that RNAi lines can have weaknesses that have not yet been considered, and we appreciate the authors work to inform the fly community about this specific issue. These insights are crucial for fly labs to consider when planning experiments that will use the pVALIUM10 RNAi lines in combination with other transgenesis modalities. The manuscript also provides a cautionary note for the usage of similar resources in other model organisms.
Reviewer #3 (Evidence, reproducibility and clarity (Required)): Summary: In their manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes", Stankovic et al. describe off-target silencing of transgenes expressed from Gateway systems when expressed in transgenic RNAi drosophila lines from the VALIUM10 collection. Using fluorescence microscopy and immunostaining, the authors show that this unintended silencing is specific to VALIUM20 lines and is not observed with VALIUM20, KK or GD lines that also allow gene-specific RNAi silencing. This pleiotropic silencing effect was observed in 10 different VALIUM20 lines and affected Gateway-based transgene expressed from an ubiquitous promoter (poly-ubiquitin, ubi) or from Gal4/UAS systems. Finally, the authors identify the molecular basis of VALIUM20 pleiotropic silencing on Gateway transgenes as being due to the presence of short sequences used for PhiC31-based recombination in the Gateway and the VALIUM systems, and could lead to the production of siRNAs against PhiC31 recombination sites in VALIUM10 lines. Using Gateway transgenes lacking the recombination sites (attB1 and attB2), the authors could abrogate silencing of the transgene in VALIUM10 lines, confirming the recombination as shared targets between the Gateway and the VALIUM systems.
Major comments: - The study is well designed and the key conclusions are convincing. - However, the authors provide only fluorescence microscopy data to show decreased transgene expression. To confirm pleiotropic RNAi effect on Gateway transgenes in VALIUM10, the authors should assess silencing with another technique. For instance, expression levels of proteins from Gateway transgenes could be measured by Western blot (e.g.: by assessing protein levels of GFP or other tags present in the Gateway transgenes).
In the manuscript, we present microscopy data as this is the typical use case for fluorescent reporters. The strength of the microscopy, in contrast to Western Blot or RT-qPCR approach, is that it allows us to directly compare the impact of RNAi silencing on cells that express the dsRNA transgene (cell-autonomous) to surrounding neighbor cells. The fluorescent imaging of WDs where all cells express the reporter construct, but only a subset of cells trigger RNAi-mediated silencing, provides spatial resolution and means for normalization while minimizing artifacts that can arise during tissue processing for WB and RT-qPCR. We provide data on GFP and HA-tagged transgenes, respectively, and untagged mCherry expressed from Gateway vectors under ubiquitin or UAS regulatory sequences with the explicit reason to show that the silencing effect is independent of the type of the protein tag or the expression regulator sequence.
In addition, the claim on line 141,"These results strongly indicate that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs" , should be modified. The authors only present fluorescence microscopy data to show decreased transgene expression and do not actually provide data on siRNA expression in the pVALUM20 lines. Therefore, with the current data, the authors should only say that their results suggest that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs.
In order to substantiate their claim about pleiotropic RNAi effects from VALIUM lines on Gateway transgenes due to the production of attB1- and attB2 -siRNAs, the authors should perform an experiment to show attB1- and attB2 -siRNAs production in VALIUM10 lines and not in VALIUM20, KK or GD lines. Deep-sequencing analysis of siRNA (i.e.: miRNA-seq) from tissue expressing the corresponding RNAi transgenes would be an excellent approach to assess siRNA production in multiple samples at once. Alternatively, the authors could search published miRNA-seq datasets from VALIUM10 and other RNAi lines to assess the presence of attB1- and attB2 -siRNAs only in VALIUM10 lines. This would be free and require only a few days of data mining and analysis, if such datasets exist already. Another cheaper and faster approach (if lacking easy access to sequencing platform or bioinformatics capability) would be to perform small RNA northern blots analysis from fly tissues expressing VALIUM10 vs VALIUM20 (or KK or GD lines) and should take only a few days to do as described in doi: 10.1038/nprot.2008.67.
If such experiments or analyses cannot be performed, then the authors can only conclude that their data suggest that the unintended silencing of Gateway transgenes in VALIUM10 is likely due to the production attB1- and attB2 -siRNAs production.
We thank the reviewer for the valuable suggestions on experimental approcahes to identify the exact interfering RNAs produced by the VALIUM10-based RNAi constructs, which can be useful for controlling the specificity of knockdown of transgenes in studies using the resources mentioned in this report.
We believe the fluorescence micrographs and quantifications demonstrate the off-target silencing effects of pVALIUM10-based RNAi lines on transgenic reporters generated using the Gateway LR cloning approach. Furthermore, we provide genetic evidence that removing the attB1 and attB2 sites from the reporter construct, which is otherwise identical to the original transgene (same promoter, same position of insertion, same genetic background), is sufficient to abolish the off-target effect. We would argue that the functional genetic experiments we performed with the original and mutated reporters represent the strongest possible evidence to confirm that silencing is taking effect via the attB sites.
As we do not attempt to detect siRNA complementary to attB1/attB2 sites directly, we have changed the statements in question as per the recommendation of the reviewer.
- The current data and methods are adequately detailed and presented, and the statistical analysis adequate.
Minor comments:
- The current manuscript does not have specific experimental issues.
- Prior studies are referenced appropriately
- Overall the text and figures are clear and accurate except for the following issues with Figure 3 and its legends On lines 396, 397, 399 and 403, the authors refer to "wild-type" ubi-mCherry. This transgene directs the ubiquitous expression of an heterologous reporter gene and thus can not as "wild type". It could instead be referred to as the "original" or "unmodified" transgene.
We removed "wild-type" from the text.
Fig.3 L: the x-axis labels are wrong. Decrease in the mCherry intensity ratio is observed with the ubi-mCherry construct and not in the ubi∆attB-mCherry, where the attB sequences thought to be targeted by the pVALIUM10 have been deleted.
More space should be added between the first row of images (B-G), the second (H-L) and also the third (M-P) to avoid confusion between the labeling of the figures. Finally, to help contextualize their findings and gauging the extent of the risk of using VALIUM10 lines in RNAi screen where a Gateway transgene is involved, the authors could provide information on the overlap between the VALIUM10 collection and VALIUM20, GD and KK collections. Knowing how many genes are uniquely targeted by VALIUM10, could be helpful.
We corrected the Figure panels according Reviewer 1 and 3’s observation.
Of the TRiP pVALIUM-based RNAi stocks currently available in BDSC, 686 genes are targeted exclusively by pVALIUM10 RNAi lines. Considering KK, GD and shRNA transgenic lines from VDRC and NIG RNAi collection, 17 genes remain unique targets for pVALIUM10 lines. The graphical overview of the availbale lines is availbale in the word/PDF file of the Response to Reviewers Letter.
Reviewer #3 (Significance (Required)):
- The manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes" by Stankovic et al. is a technical study that sheds light on potential limitations of using common RNAi drosophila lines, namely the VALIUM10 collection.
- The study provides information about very specific genetic screens conditions in Drosophila, that are likely to be rare. A rapid Pubmed search with the following terms: "drosophila TRiP screen" returns only 11 citations, while a similar search with "drosophila CRISPR screen" returns 99 citations. This suggests that in vivo RNAi screen in Drosophila using TRiP RNAi collections might not be as common or powerful as CRISPR-based screens.
- The reported findings might be of interest mostly to a small group of scientists working with Drosophila melanogaster that specifically rely on VALIUM10 lines to perform in vivo RNAi screen in combination with Gateway transgene expression. This very specific combination of parameters is rare, since other RNAi fly stock collections exist (e.g.: VALIUM20, 21, KK, GD...). Furthermore, the advent of CRISPR tools that allows tissue-specific gene knock-out has led to the rapid expansion of CRISPR fly stock collections (https://doi.org/10.7554/eLife.53865). Regardless of the limited scope of the study, this kind information is still valuable, albeit to a very limited audience.
- My relevant fields of expertise for this study are : insect RNAi, RNAi of RNAi screens and drosophila genetics.
We would like to raise some points concerning the above comments.
While TRiP-screen may not be an often-used keyword combination, the use of the TRiP lines is, in fact, ubiquitous in the Drosophila community. The tissue-specific RNA interference is still commonly utilized as a rapid, first-generation screening method that can be performed in a tissue-specific manner, representing one of the key advantages of the Drosophila model. To illustrate, since the submission of our manuscript a new study published by Rylee and co-workers investigated Drosophila pseudopupil formation by screening 3971 TRiP RNAi lines (Rylee et al, 2022). In contrast, genetic screens relying on mutant alleles usually require at least one additional cross, effectively doubling the time of the experiment. In addition, tissue-specific or temporarily restricted knockdown is sometimes required in screens, as full-body loss of function is often lethal or has developmental phenotypes incompatible with assessing gene function later in life.
The use of tissue-specifically driven Cas9 with integrated gRNA-expressing vectors is indeed becoming more common. However, this technique, much like RNA interference, is not without flaws. First, this produces knockout instead of knockdown, which means it has to be induced early in order for the resulting mutation to take effect. Otherwise, the remaining mRNA/protein may prevent the development of a phenotype. Second, the Cas9 must be titrated as high Cas9 levels have adverse phenotypes (Huynh et al, 2018; Meltzer et al, 2019; Poe et al, 2019; Port et al, 2014). Third, in our personal experience, as well as literature reports (Mehravar et al, 2019; Port & Boutros, 2022), indicate that the resulting phenotype can produce mosaics in the tissue.
Although the combination of Gateway-based reporters with TRiP-RNAi lines may seem like a fringe case, there are popular reporters that could be screening targets. Potentially the most well-known is the live cell cycle indicator fly-FUCCI system (Zielke et al, 2014), which allows the analysis of the cell cycle in real-time thanks to the expression of two fluorescently tagged degrons. As FUCCI transgenes were constructed with Gateway recombination, they represent targets of the pVALIUM10 TRiP lines. We now include the fly-FUCCI system as an example in addition to 3xHA-tagged FlyORF collection in the Discussion.
REFERENCES
Boisvert FM, Ahmad Y, Gierlinski M, Charriere F, Lamont D, Scott M, Barton G, Lamond AI (2012) A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol Cell Proteomics 11: M111 011429
Gallant P (2007) Control of transcription by Pontin and Reptin. Trends Cell Biol 17: 187-192
Huynh N, Zeng J, Liu W, King-Jones K (2018) A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues. G3 (Bethesda) 8: 3593-3605
Mathieson T, Franken H, Kosinski J, Kurzawa N, Zinn N, Sweetman G, Poeckel D, Ratnu VS, Schramm M, Becher I et al (2018) Systematic analysis of protein turnover in primary cells. Nature Communications 9: 689
Mehravar M, Shirazi A, Nazari M, Banan M (2019) Mosaicism in CRISPR/Cas9-mediated genome editing. Developmental Biology 445: 156-162
Meltzer H, Marom E, Alyagor I, Mayseless O, Berkun V, Segal-Gilboa N, Unger T, Luginbuhl D, Schuldiner O (2019) Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila. Nature Communications 10: 2113
Poe AR, Wang B, Sapar ML, Ji H, Li K, Onabajo T, Fazliyeva R, Gibbs M, Qiu Y, Hu Y et al (2019) Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila. Genetics 211: 459-472
Port F, Boutros M (2022) Tissue-Specific CRISPR-Cas9 Screening in Drosophila. In: Drosophila: Methods and Protocols, Dahmann C. (ed.) pp. 157-176. Springer US: New York, NY
Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111: E2967-2976
Rylee J, Mahato S, Aldrich J, Bergh E, Sizemore B, Feder LE, Grega S, Helms K, Maar M, Britt SG et al (2022) A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation. G3 Genes|Genomes|Genetics: jkac257
Zielke N, Korzelius J, van Straaten M, Bender K, Schuhknecht GFP, Dutta D, Xiang J, Edgar BA (2014) Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues. Cell Rep 7: 588-598
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Referee #3
Evidence, reproducibility and clarity
Summary:
In their manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes", Stankovic et al. describe off-target silencing of transgenes expressed from Gateway systems when expressed in transgenic RNAi drosophila lines from the VALIUM10 collection. Using fluorescence microscopy and immunostaining, the authors show that this unintended silencing is specific to VALIUM20 lines and is not observed with VALIUM20, KK or GD lines that also allow gene-specific RNAi silencing. This pleiotropic silencing effect was observed in 10 different VALIUM20 lines and affected Gateway-based transgene expressed from an ubiquitous promoter (poly-ubiquitin, ubi) or from Gal4/UAS systems. Finally, the authors identify the molecular basis of VALIUM20 pleiotropic silencing on Gateway transgenes as being due to the presence of short sequences used for PhiC31-based recombination in the Gateway and the VALIUM systems, and could lead to the production of siRNAs against PhiC31 recombination sites in VALIUM10 lines. Using Gateway transgenes lacking the recombination sites (attB1 and attB2), the authors could abrogate silencing of the transgene in VALIUM10 lines, confirming the recombination as shared targets between the Gateway and the VALIUM systems.
Major comments:
- The study is well designed and the key conclusions are convincing.
- However, the authors provide only fluorescence microscopy data to show decreased transgene expression. To confirm pleiotropic RNAi effect on Gateway transgenes in VALIUM10, the authors should assess silencing with another technique. For instance, expression levels of proteins from Gateway transgenes could be measured by Western blot (e.g.: by assessing protein levels of GFP or other tags present in the Gateway transgenes). In addition, the claim on line 141,"These results strongly indicate that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs" , should be modified. The authors only present fluorescence microscopy data to show decreased transgene expression and do not actually provide data on siRNA expression in the pVALUM20 lines. Therefore, with the current data, the authors should only say that their results suggest that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs. In order to substantiate their claim about pleiotropic RNAi effects from VALIUM lines on Gateway transgenes due to the production of attB1- and attB2 -siRNAs, the authors should perform an experiment to show attB1- and attB2 -siRNAs production in VALIUM10 lines and not in VALIUM20, KK or GD lines. Deep-sequencing analysis of siRNA (i.e.: miRNA-seq) from tissue expressing the corresponding RNAi transgenes would be an excellent approach to assess siRNA production in multiple samples at once. Alternatively, the authors could search published miRNA-seq datasets from VALIUM10 and other RNAi lines to assess the presence of attB1- and attB2 -siRNAs only in VALIUM10 lines. This would be free and require only a few days of data mining and analysis, if such datasets exist already. Another cheaper and faster approach (if lacking easy access to sequencing platform or bioinformatics capability) would be to perform small RNA northern blots analysis from fly tissues expressing VALIUM10 vs VALIUM20 (or KK or GD lines) and should take only a few days to do as described in doi: 10.1038/nprot.2008.67.<br /> If such experiments or analyses cannot be performed, then the authors can only conclude that their data suggest that the unintended silencing of Gateway transgenes in VALIUM10 is likely due to the production attB1- and attB2 -siRNAs production.
- The current data and methods are adequately detailed and presented, and the statistical analysis adequate.
Minor comments:
- The current manuscript does not have specific experimental issues.
- Prior studies are referenced appropriately
- Overall the text and figures are clear and accurate except for the following issues with Figure 3 and its legends On lines 396, 397, 399 and 403, the authors refer to "wild-type" ubi-mCherry. This transgene directs the ubiquitous expression of an heterologous reporter gene and thus can not as "wild type". It could instead be referred to as the "original" or "unmodified" transgene. Fig.3 L: the x-axis labels are wrong. Decrease in the mCherry intensity ratio is observed with the ubi-mCherry construct and not in the ubi∆attB-mCherry, where the attB sequences thought to be targeted by the pVALIUM10 have been deleted.
- More space should be added between the first row of images (B-G), the second (H-L) and also the third (M-P) to avoid confusion between the labeling of the figures. Finally, to help contextualize their findings and gauging the extent of the risk of using VALIUM10 lines in RNAi screen where a Gateway transgene is involved, the authors could provide information on the overlap between the VALIUM10 collection and VALIUM20, GD and KK collections. Knowing how many genes are uniquely targeted by VALIUM10, could be helpful.
Significance
- The manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes" by Stankovic et al. is a technical study that sheds light on potential limitations of using common RNAi drosophila lines, namely the VALIUM10 collection.
- The study provides information about very specific genetic screens conditions in Drosophila, that are likely to be rare. A rapid Pubmed search with the following terms: "drosophila TRiP screen" returns only 11 citations, while a similar search with "drosophila CRISPR screen" returns 99 citations. This suggests that in vivo RNAi screen in Drosophila using TRiP RNAi collections might not be as common or powerful as CRISPR-based screens.
- The reported findings might be of interest mostly to a small group of scientists working with Drosophila melanogaster that specifically rely on VALIUM10 lines to perform in vivo RNAi screen in combination with Gateway transgene expression. This very specific combination of parameters is rare, since other RNAi fly stock collections exist (e.g.: VALIUM20, 21, KK, GD...). Furthermore, the advent of CRISPR tools that allows tissue-specific gene knock-out has led to the rapid expansion of CRISPR fly stock collections (https://doi.org/10.7554/eLife.53865). Regardless of the limited scope of the study, this kind information is still valuable, albeit to a very limited audience.
- My relevant fields of expertise for this study are : insect RNAi, RNAi of RNAi screens and drosophila genetics.
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Referee #2
Evidence, reproducibility and clarity
Stankovic, Csordas, and Uhlirova show that a specific subset of the TRiP RNAi lines available, namely the pVALIUM10 subset, can cause a knockdown of certain co-expressed transgenes that contain attB1 and attB2 sites. The authors demonstrate that while pVALIUM20 or Vienna KK lines for BuGZ or myc RNAi do not affect RNase H1:GFP expression, pVALIUM10 RNAi lines against BuGZ or myc significantly decrease expression of the RNAseH1:GFP transgene. The authors propose that, due to how these RNAi lines were constructed, the siRNA products could be targeting to attB1 and attB2 sites in transgenes that were made using similar methodology. To support this idea, they ubiquitously express mCherry transgenes encoding mRNAs either containing or lacking attB sites. They find that the knockdown of mCherry seen with several different pVALIUM10 RNAi lines is observed with the reporter mRNA containing attB sites, but is suppressed when the attB sites are removed from mCherry mRNA. They also find that the pVALIUM10 RNAi lines reduce the expression of the FlyORF transgene SmD3:HA.
The paper is very clearly written and the data presented is convincing.
Minor suggestions:
- Figure 3 L+M The labels for the ubi-mcherry and ubiΔattb-mcherry are switched in these graphs (i.e. ubiΔattb-mcherry should be the one with a higher intensity in the pouch compared to the notum).
- Figure 3M the labels don't match the RNAi lines used in H-K.
- Figure 2 and 3. For the images of the transgenes, it seems as if the BuGZ RNAi line has a more drastic effect on RNaseH1 than mCherry, and vice versa for the myc RNAi lines. Did the authors notice a pattern with the decreased expression. Do some of the RNAi lines have a more consistent/severe impact, or might different transgenes be impacted to different extents?
- Line 150 unnecessary comma after Both Line 131 knockdown should be knocked down Line 133 should be "using an additional" Figure legend 1 wing disc should be at least written out when the abbreviation (WD) is first used.
Significance
Overall, this manuscript is an informative reminder that RNAi lines can have weaknesses that have not yet been considered, and we appreciate the authors work to inform the fly community about this specific issue. These insights are crucial for fly labs to consider when planning experiments that will use the pVALIUM10 RNAi lines in combination with other transgenesis modalities. The manuscript also provides a cautionary note for the usage of similar resources in other model organisms.
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Referee #1
Evidence, reproducibility and clarity
This manuscript by Uhlirova and colleagues identified an unwanted off-target effect in the pVALIUM10 TRiP RNAi lines that are commonly used in the fly community. The pVALIUM10 lines use long double-stranded hairpins and are useful vectors for somatic gene knock-down, hence they are widely used.
Here the authors find that any pVALIUM10 TRiP RNAi line can create the silencing of any transgenes that were cloned with the commonly used Gateway system. this is caused by targeting attB1 and attB2 sequences, which are also present in other Drosophila stocks including the transgenic flyORF collection. Hence, this is an important and useful information for the fly community that should be published quickly. All experiments are well documented and well controlled. I only have a few minor comments.
- I recommend to mention the number of 1800 pVALIUM10 lines in Bloomington in the abstract rather than 11% to make clear that this is an important number of lines. (1800 of 13,698 lines in Bloomiongton are 13 and not 11 per cent?)
- The authors may consider to call the 'unspecific' silencing effect an 'off-target' effect compared to intended 'on-target'. Such a nomenclature would be more consensus.
- Ideally, all the imaging results in Figure 2 and 3 would be quantified. The simple 'V10' label in the Figure 3L and 3M is not the most intuitive, at least it took me a while to figure out what the authors compare.
- Does the silencing also affect attR sequences? These are present after cassette exchange in many transgenes, most of the time not in the mRNA though, so it might not be so relevant.
Significance
This is an important and useful information for the fly community that should be published quickly.
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Reply to the reviewers
Response to reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)): The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.
My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?
We thank the reviewer for this comment. However, we would like to reiterate (as stated in the manuscript) that knockout of Pfs16 in P. falciparum is transmission lethal, i.e. you do not get progression of male gametogenesis. Thus, whilst re-generation of a Pfs16 KO would be interesting in terms of comparing phenotypically with the drug treated parasites, we are not convinced it would add any further evidence of support for or against our conclusion in terms of the ability of the N-4HCS scaffold to target this protein. E.g. we could drug treat a Pfs16 KO but this would not be expected to show gametogenesis irrespective of treatment. Therefore, whilst of academic interest, we believe it is satisfactory to judge our phenotypic work based on published accounts of the Pfs16 KO without having to engage in the costly experiments to regenerate the parasite and work on it side-by-side, especially given the limited resolution it would give towards the overall goal of the work in terms of defining the effect and likely target of this drug class on parasites.
Addressing the second comment, we are happy to alter areas of the paper that may have over-stated the conclusions of the work including the abstract/introduction and discussion.
CROSS-CONSULTATION COMMENTS I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper
Reviewer #1 (Significance (Required)):
The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.
The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.
We thank the reviewer for this positive assessment of our work. Mirroring comments above, our challenge with Pfs16 knockout or mutation is that if we ablate Pfs16 function we cannot assess the effect of drug action. Definition of a mutant that would demonstrate precisely the drug mode of action would require structural resolution of drug bound to target (i.e. to identify which residues to target) – this is a major goal for our research group moving forwards, but likely many years’ work. In general, our core approach here has been one of chemo-proteomic based methods and phenotypic investigation of the novel antimalarial. Further evidence might be forthcoming from molecular genetics/structural biology, but we believe these are beyond the scope of the current work (and our available resources at present). We state future directions in the discussion and can add more to this in any revised manuscript.
This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.
The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.
Major comments: 1. The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway.
Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).
We don’t disagree with reviewer 2’s comments that ETRAMP10.3 could be an additional target. Although not traditionally related there is some similarity between these proteins and it may be that at the macroscopic level there is a structural homology between them. As stated elsewhere we are happy to tone down the assertion that Pfs16 is the only drug target candidate, leaving open the possibility of future follow up work that may yet reveal additional targets. This cannot be explored much further without extensive experimentation, which is beyond our current capacity. Given the strong phenotypic effect on gametocytes, whilst ETRAMP may be upregulated, this paper naturally focused its core attention on Pfs16 as a candidate target. We certainly subscribe to the view that absence of evidence is not evidence of absence.
Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.
We don’t necessarily agree that the evidence is not strong (three methods pointing to the same target is by many accounts solid evidence). Additionally, whilst it is true that the N-4HCS photoaffinity probes likely interact with PVM proteins in first instance, it is also worth noting that this doesn’t necessarily deduct from their likelihood to be true targets, but instead fits with the N-4HCS phenotype. We observe the compounds to inhibit microgametogenesis without any prior incubation and to retain this activity even beyond activation of microgametogenesis, specifically during the window in which the PVM remains associated with the parasite. Our phenotypic observations therefore fit with the notion that the molecules target proteins that lie within the PVM and interact with the molecules at first instance. Whilst we understand the concern that PVM proteins may be likely to be enriched given their abundance and localisation, we believe this to support our phenotypic findings.
The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.
As stated, we are happy to tone down conclusions in this direction. We also note comments above about Pfs16 disruption.
Minor comments: 4. From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.
The modification space of the compounds is explored extensively in previous work from our group, which we feel more than adequately addresses this question. See Rueda-Zubiaurre et al (2020) J Med Chem.
Line 67: references are superscript.
We can change this
Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.
We can change this
Line 116: consider removing 'interdisciplinary'.
We can change this
Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target
We can change this along with the general “tone” of the manuscript.
Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.
We can add this to the final version. The actual IC50 for this compound was 1.7uM. For the SAR study we grouped compounds with IC50 >1uM into discrete groups based on rough IC50 (>1uM, >10uM etc.) hence this fell in the intermediate group.
Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2
This can be addressed in a revised manuscript.
Line 194: was the data deposited in a database?
The proteomics data has not been deposited in a database but is accessible in the extended SI.
Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.
We can embellish the description here.
Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.
We can amend this and label each hit within the scatter plot.
Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).
We can discuss this to a limited extent for future exploration of Etramp10.3.
Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).
We can change this
Line 233 and elsewhere: define 'reversibility' (binding? activity?).
We can change this
Line 240: clarify what is in the cited paper (see major points).
We can clarify this
Line 297: We utilised in-lysate...... clunky sentence, please rephrase.
We can change this
Line 325: reference is missing the year.
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Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?
We believe that Pfs16 is functionally different at different stages of gametocyte development, this is either in terms of its presentation (e.g. perhaps due to complex formation, though this remains elusive) or the functionality of different domains, as per the effect of different truncation mutants. We can address some of these concerns in a revised manuscript.
Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.
We can change this
Line 405: remove the 'to'
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Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.
We can state this
Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.
We can make mention of this
Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?
The reviewer is astute to point this out. Limitations with access to the EM facility meant that whilst IFAs were completed for pre-activation treated samples, the post-activation EM became impossible as the EM facility closed during the COVID lockdown. Thus, we do not have a complete set here. However, we do not feel this takes away from the EM observations presented. We can clarify this incompleteness in the revised manuscript.
Line 450: is this really CytB, or was it CytD?
We did indeed used Cytochalasin B here, which whilst less potent than D does still target microfilament formation.
Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.
We can change this
Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.
We can attempt to clarify this in a revised discussion.
Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).
We can clarify this
Line 534: consider replacing 'highly plausible' with something more cautious.
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Line 550: Given this discussion how stable are N- 4HCS compounds?
We can clarify this.
Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.
We can change this
Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.
We can change this
Figure 3d: Unclear as to why a difference temperature range is displayed here.
We can clarify this
Figure 3e: Unclear % Inhibition compared to what.
We can clarify this
Figure 5G: What is the white arrow pointing to?
We can clarify this
Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.
We are not sure what the comment relates to here but we can endeavour to clarify this
Figure 6: Erythrocyte membrane colour not stated in legend.
We can change this
Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.
We can endeavour to clarify this
Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.
We can attempt to clarify this
Reviewer #2 (Significance (Required)):
Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.
Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.
Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.
Your expertise: malaria, P. falciparum, biology of apicomplexans
Reviewer #3 (Evidence, reproducibility and clarity (Required)): The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.
Major Concerns: The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.
As discussed, we are happy to tone down the conclusions about Pfs16 being an exclusive target for the N-4HCS drug class, however, we feel the reviewer is being unnecessarily negative. There are myriad papers in the literature based on singular proteomics experiments (given their cost, complexity and time -consuming nature) that then facilitate downstream experiments that support findings. We have endeavoured to be as thorough as we could in the work and believe, like others, three replicates of a massive experimental pipeline should be sufficient to make a defined conclusion – whether the additional downstream evidence we have then leaned on is supportive of this (as we judge it to be) is another matter. We agree, proteomics often suffers with low protein abundance. The complexity of growing large quantities of gametocytes is familiar to anyone who has struggled to grow these finicky parasites at a larger scale than 10-25mL dishes. Given the scales we have reached, we believe these might in fact be some of the most comprehensive proteomics studies to date!
Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.
Whilst we understand the concerns with insignificant enrichment in the competition labelling, we believe the enrichment in the presence of photoaffinity probe 2 over background (i.e. DMSO vs. probe experiments) to be of more value given the design of the experiment. The competition experiments were performed by co-treating gametocytes with photoaffinity probe 2 and parent molecule 1 prior to UV irradiation, to enable irreversible conjugation to protein target(s). However, given that both compounds, probe and parent, theoretically bind to Pfs16 at the PVM in a reversible manner (i.e. losing interaction with even gentle washing), UV irradiation is likely to favour probe-binding irrespective of competition with a marginally more potent parent molecule (in this case, parent molecule 1). This is especially true as treated parasites were very thoroughly washed after irradiation, so should the parent molecule have bound the target protein(s), these drug-target interactions were likely lost during stringent washing. The drug-target interactions with parent molecule 1 wouldn’t have been aided by UV irradiation, as the molecule lacks the functional group required for bioconjugation. So, even if parent molecule-target interactions were more abundant than probe-target interactions, interactions between parent molecule 1 were most likely lost and proteins bound by probe were enriched.
This would have been true with more potent N-4HCS derivatives such and DDD01028076 and (-)-DDD01028076 (where potency is tested in the DGFA, independent of bioconjugation), and here we opted for a structurally similar compound of similar potency to not skew competition solely based on potency.
We can embellish on this in the revised manuscript to make our conclusions from this part clear.
A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions: - Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed.
It is certainly the case that Pfs16 is abundant in gametocytes, a reason behind its early discovery. Thus it is challenging to remove it from background. We still believe the enrichment to be specific, highlighting the comparative work with Pfg377 in Figure 2. Further repetitions with more stringent washing might resolve the background, however, this is beyond our current resources to repeat.
-- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.
We don’t disagree with this though this would involve an entire re-running of the experimental workflow which is not possible.
- Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.
Again, the presence of Pfs16 in the flow-through is unsurprising, given its abundance in stage V gametocytes. The relative abundance in the eluate is not an indication that the binding and subsequent enrichment is not specific, rather this shows the compound does not necessarily bind each and every protein – which is not unexpected. The crucial conclusion to be drawn here is the concentration-dependent enrichment of Pfs16 in the eluate in the presence of probe.
- Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.
As we have noted above, it is not unsurprising that modification of the N-4HCS scaffold to yield this probe may introduce a level of irradiation-independent binding, which explains the presence of signal in the UV-independent sample.
- Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.
These experiments are extremely challenging (something that is perhaps beyond the expertise of the reviewer) and what is presented is the result of substantial optimisation. Loss of AzTB fluorescence in the gel which is subsequently analysed by western blot explains this.
- Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.
We do not concur with the reviewer here and their dismissal of what was extremely thorough and well-executed experimtns. These are not like traditional western blots and require substantial optimisation. We refer them to our previous point in reference to the UV controls. With regards to the Pfg377 variability, the experiment itself is inherently variable with such large volumes of parasites. In many cases, for example, the male:female ratio within a mature gametocyte culture can vary and this can contribute to the variability in 377 abundance between replicates.
The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies. - The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?
The individual western blot replicates can be provided in a revised manuscript if judged important.
- The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.
Given all proteins within a lysate will aggregate with thermal treatment, antibody loading controls are not feasible with these experiments. Each sample is normalised prior to thermal stabilisation (ensuring the same protein quantity is treated in both DMSO and drug, at each temperature) and any protein that is not aggregated is loaded – the nature of CETSA itself is to compare the stabilisation between DMSO and drug.
- The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?
We agree that this temperature of stabilisation is unusually high and may require further biochemical validation. Without further investigation we cannot say definitively why the melting temperature of Pfs16 is so high, but suspect its size and membrane localisation may play a role.
- The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).
Please we have already addressed this in the text – refer to line 312 and beyond.
- The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.
Whilst further replicates with different conditions might be preferable, as discussed extensively here, this would be beyond the scope of what we are able to achieve for a revision.
- The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.
We would have to agree to disagree on this point.
- Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.
We don’t agree with this statement, since the drug is binding the protein in native lysate – this may be a multi-meric complex (homo or hetero) which only exists at certain stages. As such we disagree with the reviewer that this argues against Pfs16 being the target.
In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.
We have discussed this in the manuscript and strongly feel the reviewer is being unnecessarily dismissive of a body of work that is coherent. We are happy to tone down the narrative of the paper with Pfs16 being the exclusive target. Structural homology of P. berghei Pfs16 orthologues has never been done but it would not be unprecedented if another target was functionally homologous (an idea we are currently pursuing). Stage specificity is also possible given the nature of Pfs16 (e.g. if it is in a complex). The reviewer appears fixated on a singular entity and unable to imagine a complex scenario where structure or protein-protein interactions might affect drug binding (as it does with other proteins present in complexes, e.g. proteasomal targeting drugs).
Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies.
Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).
We strongly disagree with this reviewer’s entire dismissal of an extensive body of work. In line with other reviewers comments we accept a need to tone down our conclusions, but do not consent to dropping the majority of the paper in favour of a phenotypic descriptive work.
MINOR COMMENTS The manuscript is very well-written and presented.
Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).
We can address the overstatement of conclusions in a revised manuscript.
I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).
This is simple to address – to minimise confusion for readers, we can simply state where photoaffinity labelling and bioconjugation were performed (and not refer to the latter as crosslinking).
The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?
We can simply provide the provide IC50s for compounds of greater potency. We are also happy to provide the curves but with such a large body of work already, this might be unnecessary.
Reviewer #3 (Significance (Required)):
The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.
If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Yahiya et al describes an extensive investigation of the mode of action of DDD01028076, which specifically inhibits microgametogenesis in Plasmodium falciparum. The phenotypic characterisation of the MOA uses some very nice imaging to demonstrate the point at which this compound inhibits microgametogenesis. The authors have also attempted to identify the molecular target using chemoproteomics and label-free CETSA techniques. The photoaffinity labelling and pull-down approach suggested the Pfs16 may be preferentially enriched by a PAL probe that is representative of this series. However, the data supporting the validation of this target is not very conclusive, and in some cases argues against Pfs16 being a specific target of DDD01028076. Whilst the presented data makes a significant contribution to the literature regarding a novel drug candidate that targets microgametogenesis, it does not support the author's claims that Pfs16 is the target.
Major Concerns:
The strongest evidence for Pfs16 being the target comes from the chemoproteomics pull-down study that found Pfs16 to be the most significantly enriched protein by compound 2 vs DMSO. However, this should be interpreted with caution as it is based on only 3 replicates and omics studies are prone to false-positives. That only 125 proteins were detected also raises questions about the coverage of the proteomics, it is quite possible that the actual target is not detectable using this method, and the Pfs16 appears because it is one of the more abundant proteins during this stage of the lifecycle.
Somewhat concerningly, the control with 1 as the competitor did not show significant enrichment of Pfs16, although a trend was observed. More concerning, was the lack of enrichment when using DDD01028076 as the competitor. This result essentially proves that Pfs16 is not the specific target (and the argument about reversibility is unlikely since most drugs are reversible binders, but many have worked with this type of approach). It is surprising that DDD01028076 (ideally the (-) form) wasn't used as the competitor for the proteomics study. This compound has ~100-fold better potency than the probe 2, which should provide much better competition that 1. It would also be more specific than 1, which is an important control considering that (-)-DDD01028076 has activity in the low nanomolar range, whereas 2 acts in the micromolar range. Non-specific interactions are an important consideration to exclude, and whilst 1 is structurally similar, it is not very potent and therefore not the best control to find the target associated with activity.
A closer look at the gels in the supplementary data raises many questions that undermine the authors conclusions:
- Fig S1a - The lane without probe (2) still identifies Pfs16 (or a protein at that MW) as the most abundant protein. Also, as the Pfs16 band increases, you can see that most other proteins also increase in abundance, so either the loading is inconsistent, or the probe actually causes non-specific enrichment of many proteins. This figure also indicates that the washing protocol is not sufficient to remove non-specific binders. Given the covalent nature of the PAL approach I would think a very thorough washing protocol could be employed. -- Running another negative control in the proteomics using one of the inactive controls from table 1 might help to disambiguate specificity.
- Fig S2a - The anti-Pfs16 Western blots show that this protein is actually enriched more in the flow-through than the eluates. This shows that this protein is not specifically enriched by the PAL-CuAAC pull-down, it is just more abundant in the treated samples.
- Fig S2b - The darkest Pfs16 spot is actually the sample with no UV treatment. This is a negative control, so should not enrich the target protein. This sample also has significant signal in replicates A and C.
- Fig S2c - This blot is very messy and difficult to read, but in general the Pfs16 spots in the IGF don't correlate with the intensities in the anti-Pfs16 western.
- Fig S2 - This data, and the main figures based on this data, generally don't support the hypothesis that Pfs16 is the specific target. The controls are not as would be expected, and there are no loading controls. Looking at the flow-throughs suggests that there was just more Pfs16 (and possibly total protein) in the treated samples before the enrichment step. The Pfg377 also appears quite variable in the different samples, with replicates B and C not consistent with A.
The other major concern is with the CETSA analysis, which appears to show very minor stabilisation of Pfs16, but the specificity of this target is questionable, and the data has the following inconsistencies.
- The supplementary data only shows n=1, yet there are error bars in the main figures. Where did these come from?
- The samples with apparent destabilisation are all near the edge of large western blots, which often doesn't run straight and has no loading controls. We need to see the loading controls.
- The melting temperature of Pfs16 is extremely high at around 85 degrees C. Most plasmodium proteins melt at around 50-60 degrees (Dzekian et al, 2019). Even the cited work on membrane proteins didn't go to those temperatures (Kawatkar et al, 2019) Can this high temperature be explained, and has the CETSA approach been validated at such high temperatures where additional physical and chemical processes may be occurring in the sample?
- The lack of difference between + and - isomers suggests that the very small stabilisation observed here is not specific to drug activity, but is more likely a non-specific binding effect. Additional negative control compounds might help here, but the + isomer is probably the best negative control (albeit the concentrations were not ideal in the presented data).
- The very high concentration (100uM) increases the chances of non-specific effects being observed here (especially since the authors claim to see stabilisation at about 10nM). The study should be repeated at lower concentrations (with negative controls) in order to confirm a specific binding effect.
- The concentration-ranging study was performed at 78.4 degrees, at which temperature very little denaturation of Pfs16 occurs fig S4a (and Fig 3b-c). Therefore, you would not expect to see any drug-induced stabilisation, and it is not plausible that significant stabilisation could occur at this temperature. Therefore, the apparent destabilisation at sub-10nM drug concentrations is highly questionable.
- Stabilisation of Pfs16 did not occur in lysates from younger gametocytes (fig s4g-h), but this is a biophysical assay, so regardless of the function of this protein at different stages, the biophysical interaction between the drug and the protein should be the same regardless of the source of the protein. This data argues against Pfs16 being a specific binding target of Pfs16.
In addition to the above concerns, the fact that this compound doesn't inhibit the earlier functions of Pfs16 in gametocytogenesis, and that it doesn't inhibit P. berghei, also argue against this being the specific target of this drug. Whilst the authors have a valid argument that these findings don't exclude the possibility of stage-specific targeting of Pfs16, we could also argue that all the phenotypic data in figures 4-6 is merely correlative of a drug that acts at the same point in the lifecycle as Pfs16.
Overall, I believe that significant additional studies would be required to identify the target of this compound. Either by repeating the included studies with additional controls and conditions, or by follow-up studies such as genetic manipulation (knock-down or overexpression) or heterologous expression and biophysical binding studies. Alternatively, the manuscript could be restructured as primarily a report on the phenotypic effect of this compound on microgametogenesis, with the target identification work reported as a hypothesis-generating chemoproteomics study that provides some ideas about possible targets, but requires substantial follow-up to confirm the target (which may be beyond the scope of this report?).
Minor comments
The manuscript is very well-written and presented.
Several of the conclusions are overstated (as detailed above) and several statements should be tempered based on this data (e.g. statements linking DDD01028076 effects to Pfs16 function).
I find the term 'crosslinking' confusing for the photo-affinity labelling, as crosslinking in proteomics often refers to crosslinking between proteins (not between protein and drug).
The data and terminology around activity (IC50) for compounds in table 1 is a little confusing. Some IC50 values are reported as >1000, while others have precise mean values reported over 1000, and others are >10,000 or >25,000. This is especially confusing where 9 is claimed to have retained activity, but is >1000. If consistent thresholds are not appropriate then perhaps including dose response curves in the supp data might be necessary to explain these?
Significance
The work is potentially interesting to Plasmodium biology and drug discovery researchers. The concept of a transmission-blocking drug is quite attractive to this community, so the topic is highly relevant. Keeping in mind that this compound was reported previously, the main novelty is in defining it's window of activity during the microgametogenesis process, and differentiating this from other drugs/compounds that inhibit this process. There is clearly an advance in knowledge presented here.
If Pfs16 were to be confirmed as the target of this series then I think that this study would have much greater impact and attract interest from a broad audience. However, at this stage I don't see strong evidence for this hypothesis, and some of this data casts significant doubt on the likelihood that Pfs16 is the direct target.
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Referee #2
Evidence, reproducibility and clarity
Transmission blocking drugs are of high interest as a strategy to combat malaria but they are difficult to study. For instance it is problematic to raise resistant parasites to find mode of action of transmission blocking drugs and to identify their targets in the cell. In this manuscript Yahiya et al. build on previous work which identified the N-4HCS scaffold, of which DDD01035881 is the lead compound, as an inhibitor P. falciparum male gametocytes. Using PAL to enrich for target proteins Pfs16 was identified and validated as a possible target of DDD01035881. Binding was validated through CESTA. Determination of the phenotype following DDD01035881 treatment was found to partially match the previously published Pfs16 KO phenotype. However curiously no impact was seen in gametocytogenesis despite published evidence of Pfs16 being involved in sexual conversion. The authors speculate as to reasons but a direct experimental comparison with Pfs16 mutant parasites (which likely would have been revealing) is not provided. On the positive side, this analysis of the stage-specific effect of the drug pinpoint the stage inhibited during microgamete development which is a very interesting part of the manuscript.
This work deepens the understanding of a novel class of transmission blocking drugs with reasonable potency (foremost (-)-DDD01028076, which has low nanomolar activity, the modified versions considerably less). Question on how to achieve serum concentrations for sufficient potency aside, these compounds will in the very least provide experimental tools to study their mode of action and might reveal interesting biology. This work is therefore of interest to the malaria field.
The experimental methodology seems excellent but some of the results raise questions that make definite conclusions difficult and this should be addressed. Overall, this is very solid work but leaves some doubts whether Pfs16 is indeed the (only) target of this class of compounds.
Major comments:
- The reasons for excluding Etramp10.3 are not convincing. In fact it could be argued it is nearly as good a candidate as Pfs16. Contrary to the author's statements in the results section, etramp10.3 transcription is highly upregulated in gametocytes (see e.g. PMID: 22129310) with a generally very low transcription in asexual stages. It is argued that Etramp10.3 is essential in blood stages because MacKellar et al failed to disrupt the gene and because the PiggyBAC screen predicted it to be essential. However, if this is an argument for exclusion then this would also apply to Pfs16 which is also predicted by the PiggyBAC screen to be essential (likely both are non-essential in blood stages as they are barely expressed but Pfs16 and Etramp10.3 might by chance have not received an insertion in the PiggyBAC screen due to their very small size which may also explain failure of disrupting integration in MacKellar). Given the finding that the drug binds Pfs16 only in late gams it might also be argued that an essential function in asexuals might not be affected if they behave similarly to young gams and hence this criterion is not valid anyway. Further following this line of thought that ETRAMP10.3 could be a hit equivalent to Pfs16, Figure 2D shows a band below the band considered to be Pfs16. It would not be all surprising if this were ETRAMP10.3 (the size would fit).
- Both, Pfs16 and ETRAMP10.3 can be expected to be very abundant proteins in the parasite periphery in gams. Can the authors exclude that these simply are the first to encounter the N-4HCS photoaffinity probe and that this may have led to their enrichment in the target identification experiments. The biochemical data argues for a specific interaction with Pfs16, but by itself is not that strong. Given the discrepancies of the phenotype with the Pfs16 disruption and the peculiar finding that the drug binds Pfs16 only in later stage gametocytes, it might be a good idea to further caution the conclusion of Pfs16 as the inhibited target.
- The phenocopy evidence of the NH compounds with the Pfs16 disruption is based on comparison with published evidence. It would have been much preferred to have a side-by-side comparison with the (or an) actual Pfs16 disruption parasite line. Although the authors stress that the phenotype with DD01035881 fits the phenotype of the targeted gene disruption in the results, this only partially matches the cited publication (PMID: 14698439) which concludes there is an effect on the number of gametocytes produced. The exflagellation phenotype in that publication was classified as preliminary. Although this is discussed, the main results text should be adapted to reflect this and the conclusion that Pfs16 may be the target should be further cautioned.
Minor comments:
- From the modifications of the compounds it seems the chemical space for further modification to achieve higher potency is limited with this scaffold. Maybe the authors can comment whether they envisage this to be a potential obstacle.
- Line 67: references are superscript.
- Line 77: I would recommend replacing 'quiescence' here, a cell that matures is not quiescent.
- Line 116: consider removing 'interdisciplinary'.
- Line 120: I would caution here (see major comments) and recommend a less definite proclamation of Pfs16 as a promising new drug target
- Page 7: compounds 9 is still considered active ("retained micromolar activity"), but in Table 1 this is given as >1000nM. Please add the actual IC50.
- Line 138- 173: The order in which this is discussed makes it unclear that the work described was done prior to, and guided, the synthesis of compound 1 and probe 2
- Line 194: was the data deposited in a database?
- Line 202: introduction as to the benefits of using a competition + probe condition here could aid reader understanding. The interpretation of this data is complicated by the covalent and reversible binding of the two compounds and the weight of this control is therefore difficult to gage.
- Table 2 and Extended Data Table 1 show different p values and enrichments for the same hits. This is confusing. It would also be useful to label the hits in the scatter plots in Figure 2 for easy identification and comparison to the tables.
- Line 215-218, please correct the data on Etramp10.3 (see major points) and put in perspective to Pfs16 (Etramp10.3 is similarly upregulated in gams where it is highly expressed; PiggyBAC predicts essentiality for Pfs16 and Etramp10.3 in blood stages).
- Line 221: the results from the PiggyBAC screen are stated as fact, but what the screen provides is a prediction of the probability of importance for parasite growth. I would replace 'is' with 'is predicted' (even though in the case of Rab1b it seems likely the prediction is correct).
- Line 233 and elsewhere: define 'reversibility' (binding? activity?).
- Line 240: clarify what is in the cited paper (see major points).
- Line 297: We utilised in-lysate...... clunky sentence, please rephrase.
- Line 325: reference is missing the year.
- Line 343: It is utterly puzzling that binding is specific to Pfs16 in mature gametocytes and I do not find the explanation in the discussion convincing (see point 28 below). Do the authors have another explanation? Could Pfs16 be modified in later gams (or vice versa)?
- Line 388: Justification seems odd as a PV protein would be unlikely to directly impact DNA replication. Please rephrase the sentence.
- Line 405: remove the 'to'
- Line 411: it would be useful to the reader to state at what IC-value the drug was used in these experiments.
- Line 431: While the alpha-tubulin staining indicates exflagellation and is similar to the DMSO only control, the staining for the RBC membrane (Glycophorin A) and DNA (DAPI) appear different, yet this is ignored. One interpretation of this could be that while late treatment doesn't block exflagellation, it still impacts other aspects of microgamete development.
- Line 436: IFA work was done with drug treatment post activation while EM was done post activation but drug treatment prior to activation. Is there a reason for this?
- Line 450: is this really CytB, or was it CytD?
- Line 465: Pfs16 localised to vesicles: there is no data showing the dots in the micrograph are vesicles, please rephrase.
- Page 19 and 20, discussion on stage-specific differences of Pfs16 during gametocytogenesis to explain the difference in binding: without experimental data using H-4HCS in the parasites of the publication cited to explain this (PMID: 21498641), this is very speculative. The cited work used episomal expression of Pfs16 tagged with fluorescent proteins. This would be the first integral PVM protein that is actually inserted into the PV membrane when tagged in that way (usually this results in a PV location), casting some doubt on the findings in that paper. All in all the provided explanation is not very convincing.
- Line 519: if with the conserved part the N-terminus is meant, then this has for other PVM proteins already been shown to be PVM internal, not facing the erythrocyte (show in very early work; PMID: 1852170 but also multiple times after that).
- Line 534: consider replacing 'highly plausible' with something more cautious.
- Line 550: Given this discussion how stable are N- 4HCS compounds?
- Table 1: Having all chemical structures in same orientation would be nicer visually. I assume blue indicates modification but this is not stated.
- Figure 1: Please use different colours or symbols. The dark green crosses and the blue Pfs16 cross are hard to distinguish.
- Figure 3d: Unclear as to why a difference temperature range is displayed here.
- Figure 3e: Unclear % Inhibition compared to what.
- Figure 5G: What is the white arrow pointing to?
- Figure 5j: Given how the explanation is written this would make more sense between current image 5G and 5J.
- Figure 6: Erythrocyte membrane colour not stated in legend.
- Figure 6A: were the exposure times similar? How can so little be left after ~4-5.5 minutes but at later time points there seems to be much more Pfs16 signal left? Maybe amount of signal should be taken into consideration to establish the fate of Pfs16 in the process.
- Figure 6B: is the second phenotype (successful but aberrant egress) shown? The only image where WGA is not circular around the parasite is an exact match of Pfs16 which is in dots (image at 7.5-8.5 minutes). The imaging data for this phenotype should be presented more clearly.
Significance
Nature and significance: a lot of weight has been placed on transmission blocking drugs although there are also a number of problems associated with them (ethics for testing and use etc; drugs acting on asexuals and transmission stages alike might be even more useful). Transmission blocking drugs are difficult to study and this work is therefore important. The experiments are well done, but the conclusions are not fully convincing, leaving some doubts in regard to Pfs16 being the actual target of the class of drugs studied.
Compare to existing published evidence: it is a logic continuation of previous work and this is appropriately highlighted in the manuscript.
Audience: medium interest for malaria researchers; high interest for researchers working on transmission blocking drugs and those studying microgametes.
Your expertise: malaria, P. falciparum, biology of apicomplexans
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Referee #1
Evidence, reproducibility and clarity
The authors develop a previously identified lead compound for the blocking of malaria transmission from humans to mosquitoes further and identified a protein target of the chemical. The protein target, Pfs16 is long known to be upregulated in gametocytes and has been speculated to be a target for small molecules. The work is well (if at time maybe too well/too detailed) described and potential shortfalls are highlighted.
My major comment is that without a deletion mutation of Pfs16, the paper will remain somewhat preliminary. I would strongly encourage the authors to generate such a mutant and compare it to the parasites treated with their drug candidate. I feel the text can be much shortened and a lot of information moved to the materials and methods. The conclusions should be toned down on several occasions (abstract, introduction, discussion). Avoid adjectives, e.g. what is a 'powerful starting point' (abstract) or 'compelling interdisciplinary evidence' but hot air?
Referees cross-commenting
I think these three reviews are pretty much in line with their overall assessment. I am happy if send as is to authors as it will help them shape a much better paper
Significance
The paper shows that very likely a new chemical with some potential for transmission inhibition of malaria parasites for mosquitoes binds to a Plasmodium protein that is specifically expressed in the sexual stages of the parasite.
The paper compares to good papers published in journals like ACS Infectious Diseases or Antimicrobial Agents and Chemotherapy, but I am not sure which of the Review Commons sister journals it would fit to. I am a molecular parasitologist.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: * Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.*
Major comments:
- The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed.*
Response: We thank the reviewer for appreciating our work and we have tried to address concerns of the reviewers to the maximal possible extent with the hope to strengthen our claims further.
One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.
Response____: We acknowledge the concern raised by reviewer and to address it, we evaluated different allelic combination of Cup to rule out issues with background mutation. We evaluated the Delta count, NICD and border cell numbers in a different allelic background of cup8/ cup01355. Satisfyingly we observed similar results like that observed for cup01355/ cup01355 homozygotes. This result is included as (Fig S1E-G)
In addition, we have specifically downregulated Cup function in the germline employing the RNAi approach and validated the non-cell autonomous effect of Cup function in border cell fate specification. This result is included in (Fig 1M-O)
A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.
Response: As per the suggestion of the reviewer, we did attempt to over express Delta in the germline of cup mutants egg chambers. Unfortunately, we couldn’t record any Delta overexpression as the available vector (UASt- Delta) can drive stable expression only in the somatic cells but not in the germline cells. However, to check out the possibility if Delta was being directly regulated by Cup, we compared the levels of proteins between wild type and Cup mutant egg chambers (Figure 4E-G). Unlike our expectation we didn’t observe any significant differences in the levels of Delta in Cup compared to the control. This kind of supports our belief that Cup may not be directly regulating the levels of Delta in the germline.
Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects?
Response: We appreciate the reviewer for raising this concern as it kind of intrigued us to examine if the overexpression Rab11CA was rescuing the cytoskeleton too. Interestingly, we observed that Rab11CA overexpression restored the actin filament in Cup mutant germline(figure S6H-K). This result is in line with report that Rab11 effector Nuf can modulate actin polymerization (Jian Cao et al.,2008).
Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.
Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function.
Minor comments:
It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.
Response: We have modified the introduction to highlight the role of Notch/ Delta signaling in early oogenesis.
It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn’t seem assess overall numbers.
Response: As per the suggestion of the reviewer, we assessed the total number of follicle cell nuclei in stage 8 egg chambers. This analysis was done each confocal z slide of the egg chamber taking care that each nuclei (DAPI) was counted only once. Satisfyingly we didn’t observe any significant difference in the number of follicle cell nuclei between wild type and cup mutant egg chambers supporting our earlier claims with pH3 and Cut antibody that cell proliferation is not responsible for the excessive border cell fate in Cup mutants. This result in included in (Fig S2O-Q)
Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects.
Response: As per the suggestion of the reviewer, we have included the data for statp1681/+ control in figure 2L.
In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.
Response: We have removed the data on Kuzbanian and have added data that suggests that Notch activation in the follicle cells downstream of Cup facilitates specification of appropriate number of migratory border cells (Fig 3K-N).
Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)? * Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?*
Response: We believe that dramatic increase in the volume of the border cell cluster I (>2x) than the number of cells (30% more) is due the loose arrangement of the cells in the border cell cluster. Interestingly, the cup mutant border cell clusters do exhibit migration defect that we are examine as part of separate study.
Several of the figure legend titles state conclusions that are over interpretations of the data shown:
- Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.
Response: We have modified the Figure legend in line with the data presented.
- For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.
__Response: __We have included the data that suggests that cytoskeleton organization is critical for Delta trafficking. Specifically we demonstrate that treatment of egg chambers with Cytochalasin D exhibits accumulation of Delta in the nurse cell cytoplasm (Fig S5D-F).
- The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6. * Some figure legends are quite brief and could benefit from a little more detail on what is being shown*.
__Response: __We have modified the title of the Figure legends with respect to data presented.
Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.
Response: We have reorganized the figures and worked on the graph text for easy read.
Not all papers cited in the text are in the reference list.
Responses: We have modified the title of the figure legends and cross checked our reference list with the papers mentioned in the main text.
CROSS-CONSULTATION COMMENTS
I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.
Reviewer #1 (Significance (Required)):
My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC
fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of
the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.
Mayor points:
In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:
o The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dl mut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells. o The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.
o In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.
Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function. This has been acknowledged in the main text and it now reads as “We did note that irrespective of partial rescue in the levels of NRE-GFP and Delta puncta count, a complete reversion to wild type border cell numbers was observed when Rab11CA was overexpressed in the cup mutant germline. This may suggest either that border cell fate specification is quite robust beyond a certain base level of signaling or Cup may affect other aspects of egg chamber development independent of Rab11 function.”
One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation.
Response: To further support the non-autonomous role of Cup in border cell fate specification, we down regulated Cup function in germline nurse cells employing Mat-alpha GAL4 and Cup RNAi. Since Mat-alpha GAL4 driver has weak expression in the nurse cells of early stage chambers, it enabled us to evaluate Cup function during mid oogenesis. Consistent with our expectation, we observed higher number number of border cells in the migratory cluster compared to the control supporting our conclusion that germline Cup modulates the number of adjacent anterior follicle cells that acquire migratory border cell fate. The above results are included in (Fig 1M-O). In addition over expression if Cup cDNA in the anterior follicle cells failed to the rescue the excessive border cells observed in the Cup mutant egg chambers supporting the germline role of Cup further. This result in included in (Fig S1L-O).
- The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup
phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NDECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype.
Response: Following the suggestion of the reviewer, we attempted over expression of NICD in the follicle using driver slbo-GAL4 in the cup mutant background. Gratifyingly, we observed rescue in the border cell fate of Cup mutant egg chambers. However, we didn’t observe any rescue in the morphology of nurse cell nuclei of Cup mutants. This supports our conclusion that increase in Notch function in the FCs rescues the cup phenotype with respect to the border cell fate only. (Fig 3K-N).
- The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically:
o They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers * o They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking. * Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section.
Response: As Rab11GTPase over expression rescued the excessive border cell fate in the cup mutants, to test the specificity we downregulated Rab11 function in the germline itself to check Delta trafficking and border cell fate specification. We employed a late expressing GAL4 driver in the germline and observed that down regulation of Rab11 function resulted in more number of follicle cell acquiring border cell fate and decrease in the number of Delta puncta at the interface of Anterior follicle cells and nurse cells. This phenotype is reminiscent of the Cup mutants suggesting that perturbing the recycling component of endocytosis perse affects border cell fate and Delta trafficking. This result in included in (Fig 6D-I)
- Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments:
- o First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.*
- o Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005)*
Response: We have perturbed Rab11 function during mid oogenesis which is quite distant from early stage egg chambers examined by Windler & Bilder. We observed that down regulation of Rab11 activity in germline affects both border cell fate in the AFCs and Delta trafficking in the germline itself. Protein Trap analysis of Rab11 in wild type and Cup mutant background suggests Rab11 is enriched in the trans-golgi network where the activity of Rab11 is modulated through nucleotide exchange. Over all our results suggest that Rab11 activity is diminished in the cup01355 egg chambers and thus stimulating the recycling endocytosis restores Notch signalling in the AFCs, limiting JAK-STAT activation and restricting BC cell fate specification.
- The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.* Response: The border cell fate is linked to activation of JAK-STAT signaling in the anterior follicle cells. As we have already exhausted the STAT antibody, it will difficult to access the levels of STAT perse.
Minor points:
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The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.*
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o First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.*
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o Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.*
Response: The follicle cell exhibits a switch from mitotic to endocycle phase at a particular stage of oogenesis (Sun & Deng’ 2005). Our premise is that incase this switch is delayed, will the extra proliferation can account for the excessive border cell fate? In this context we have modified the text to render clarity to this section.
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The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)mb-CD2 or E(spl)m7. Response: We believe that our new set of data with NICD over expression (in the AFCs) rescuing border cell fate in Cup mutants coupled with NRE-GFP, NICD, NECD data now lends stronger support to our claim that Notch signaling in the follicle cells is indeed downstream of Cup function in developing egg chambers.
- In M&M the authors explain that NRE-GFP levels were expressed in Fold change. However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please,*
check this small inconsistency
Response: We have modified this as per reviewer’s suggestion.
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In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution. Response: We have modified the figure and included quantitation of actin fibre length in Supplementary figure 6H- K.
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In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly. Response: We have modified the figure to render clarity
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In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells. Response: Delta, NICD, NECD proteins marks the apical surface of the follicle cells. We used this as a reference to segregate nurse cell puncta with respect to follicle cells. This has been elaborated in the Material & Method section.
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In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.
Response: We have crosschecked the images and the YFP signaling is from nurse cell periphery which gives the wrong impression that it is from stretched follicle cells.
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In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial. *Responses: To avoid cramming the Figures, we have including some of the p values in the Figure legends. *
- Line 174: should say "mutant egg chambers".*
- Line 281: There is a reference that is missing from reference list: Liu et al., 2010;*
- Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.*
- In line 462 of the manuscript you have a reference that is missing from your reference list.*
- In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.*
Response: We have modified the text as per the all the suggestions above Reviewer #2 (Significance (Required)):
The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.
My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:
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- The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?*
Response: We are using the border cell number as readout for the output of JAK-STAT signaling. Though the difference in numbers may appear to be subtle, we believe our data clearly demonstrates that Cup non cell autonomously regulates border cell fate by modulating Notch signaling in the follicle cells*. *
- Wang, et al. (PMID 17010965) have described previously that Notch signaling, and*
Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.
Response: We provide genetic evidence to support our claims that Cup in the germline modulates Notch activation in the anterior follicle cells thus limiting border cell fate specification to a few. The overlap in the expression of Notch reporter m7-lacz and STAT in the follicle cells and border cells is interesting and will need further investigation in real time to decipher any comparison between the two studies.
- Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.*
Response: We have tried to edit the manuscript with the aim to improve on the language, grammar and punctuations.
Reviewer #3 (Significance (Required)):
Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.
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Referee #3
Evidence, reproducibility and clarity
This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:
- The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?
- Wang, et al. (PMID 17010965) have described previously that Notch signaling, and Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.
- Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.
Significance
Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.
Major points:
- In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:
- The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dlmut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells.
- The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.
- In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.
One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation. - The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype. - The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically: - They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers - They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking. Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section. - Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments: - First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.<br /> - Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005) - The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.
Minor points:
- The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.
- First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.
- Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.
- The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)m-CD2 or E(spl)m7.
- In M&M the authors explain that NRE-GFP levels were expressed in Fold change. However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please, check this small inconsistency
- In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution.
- In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly.
- In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells.
- In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.
- In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial.
- Line 174: should say "mutant egg chambers".
- Line 281: There is a reference that is missing from reference list: Liu et al., 2010;
- Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.
- In line 462 of the manuscript you have a reference that is missing from your reference list.
- In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.
Significance
The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.
My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.
- In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:
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Referee #1
Evidence, reproducibility and clarity
Summary:
Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.
Major comments:
The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed. One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.
A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.
Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects? Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.
Minor comments:
It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.
It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn't seem assess overall numbers. Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects. In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.
Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)?
Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?
Several of the figure legend titles state conclusions that are over interpretations of the data shown:
- Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.
- For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.
- The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6. Some figure legends are quite brief and could benefit from a little more detail on what is being shown.
Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.
Not all papers cited in the text are in the reference list.
Referees cross-commenting
I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.
Significance
My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary:
In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.
Major comments:
The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.
1.- In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?
Response: The Caulobacter ΔpseI mutant accumulates low levels of flagellin in the supernatant. TEM analysis reveals that the flagellar filament is not assembled and only the hook structure is visible (PMID: 33108275). Brevundimonas subvibrioides ΔlegI or ΔflmG cells feature a shorter filament compared to WT by TEM. In all these analyses, TEM was performed on cells grown in broth to exponential growth phase as detailed in the Experimental procedures section. These mutant cells do not swim when analyzed by phase contrast microscopy. While is not known if swimming on semi-solid medium would further destabilize the flagellar structures seen in liquid cultures by TEM, there is more residual motility in B. subvibrioides mutants that make a short filament compared to C. crescentus mutants that lack the flagellar filament. Thus, our analyses point to a positive correlation between the residual motility and residual filament length when comparing the B. subvibrioides and C. crescentus mutants.
2.- In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches. Thanks. Corrected. 3.- In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.
We thank the reviewer for raising this point. We have now clarified this question in the updated Experimental procedures section. Our immunoblots harbor the same number of cells harvested in exponential phase (OD=0.4). One mL of cells was harvested from cultures by centrifugation at full speed. The supernatant that was used for the immunodetection corresponds to the supernatant after the centrifugation. The supernatant fraction contains flagella that have been shed during the cell cycle at the swarmer cell to stalked cell (G1-S) transition of C. crescentus and B. subvibrioides.
Thus, it is clear that the majority of flagellins detected by immunoblotting are in fact cell associated and specifically the intracellular flagellins. The evidence for this is that the levels are comparable between WT and ΔflmG mutant cells, even though the latter has shorter or no flagellar filaments. Moreover, while C. crescentus cells are not constantly flagellated during the cell cycle, flagellins are detectable on cell-associated samples by immunoblotting even when cells do not yet or no longer have a flagellar filament. Based on these two points, we conclude that the total flagellin levels associated with cells do not reflect the levels of flagellin assembled into a flagellar filament, but rather the flagellin bulk present in the cytoplasm.
Consistent with this view, we previously reported that C. crescentus ΔpseI cells have the same amount of flagellins in cell lysates compared to the WT strain (PMID: 33108275), even though the mutant cells lack a flagellar filament. Thus, the results obtained here are consistent with previous observations and indicate that B. subvibrioides flagellin glycosylation mutants also still produce comparable amounts of flagellins intracellularly like the WT strain, despite the absence of flagellin glycosylation and inefficient assembly into a flagellar filament.
Concerning the potential role of LegI and FlmG in flagellin stability after export, we were referring to protein stability (half-life), not filament stability. Glycosylation may impact the half-life of extracellular flagellins since glycosylation can protect from proteolytic degradation of proteins, possibly in this case by different proteases that may accumulate in the supernatant. Thus, non-glycosylated flagellins could be more easily degraded by extracellular proteases once they are exported, ultimately resulting in a lower amount in the supernatant.
Addressing the final question about the specificity of the anti-FljKCc antiserum: we used this anti-serum because it detects the B. subvibrioides flagellins owing to the high sequence similarity between B. subvibrioides flagellins and C. crescentus flagellins. We previously showed that the anti-FljKCc anti-serum detects all six flagellins from C. crescentus, as determined by individually expressing each flagellin in a strain deleted for all six flagellin genes (Δfljx6) (PMID: 33108275). FljKCc (against which the antibody was raised) is 65% similar to the most distant C. crescentus flagellin, FljJ. As the similarity of FljKCc to the three B. subvibrioides flagellins ranges from 74% -67% sequence similarity, they should be even better recognized by the anti- FljKCc antibody than C. crescentus FljJ. However, on immunoblots we cannot attribute the signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE and therefore all flagellins might reside in the same immunoblot band. However, we can clearly demonstrate that the immunoblot band corresponds to flagellins: a B. subvibrioides ΔflaF mutant (see below) that we constructed revealed that the flagellin signal is lost, as is the case for a C. crescentus ΔflaF mutant (PMID: 33113346). In the case of C. crescentus, the FlaF secretion chaperone is required for flagellin translation (synthesis) and we suspect that this also the case for B. subvibrioides FlaF. This experiment provides additional evidence that the B. subvibrioides flagellins are recognized by the anti-FljK (C. crescentus) anti-serum.
4.- Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background. The answer to the first question is “No” or “very inefficiently” as determined from immunoblot analyses of B. subvibrioides ΔflmG cells expressing the chimeric FlmG_Cc-Bs protein that we now show in Fig S2B.
Expression of the different FlmG (FlmG_Cc, FlmG_Bs, FlmG_Cc-Bs) in C. crescentus cells producing Pse or Leg revealed that FlmG_Bs does not support efficient flagellin modification with Pse in C. crescentus, likely because FlmG_Bs interacts poorly with the C. crescentus flagellins. By using the FlmG_Cc-Bs chimera we hoped to overcome this interaction problem with the C. crescentus flagellins (because the FlmG chimera harbors the C. crescentus TPR to bind the C. crescentus flagellins), however glycosyltransfer still does not occur efficiently because the GT domain from FlmG_Bs does not function with Pse. However, FlmG_Cc-Bs can modify the C. crescentus flagellins once C. crescentus is genetically modified to produce CMP-Leg (instead of CMP-Pse). This confirms that the FlmG TPR from C. crescentus is important for flagellin modification through the FlmG/flagellin interaction and that GT_B type glycosyltransferase only transfers Leg. In addition, we have now added as Fig S2B an immunoblot and as Fig S2C a motility test of B. subvibrioides ΔflmG cells expressing the FlmG_Cc-Bs chimeric protein in which we only observed little modification of B. subvibrioides flagellins and a poor motility, respectively. We extended our discussion of these results.
5.- Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.
To investigate why the C. crescentus cells that produce Leg and express the chimeric FlmGCc-Bs glycosyltransferase are non-motile (Figure S5B) despite flagellin modification (by immunoblotting, Figure 7C), we employed two strategies. First, we performed immunoblot analyses on the supernatant fraction from these cells to determine if flagellins accumulate extracellularly. As now showed in Figure S5A, only low amounts of C. crescentus flagellins modified by Leg are present in the SN fraction. Second, we conducted TEM analyses of cells grown to exponential growth phase in broth. As shown in Figure S5C, the C. crescentus cells producing Leg and expressing FlmG_Cc-Bs glycosyltransferase harbor a shorter flagellum compared to those expressing the FlmG_Cc in which C. crescentus flagellins are modified by Pse. Altogether these results explain why these cells are non-motile both on soft agar plate and in liquid.
Minor comments: 1.- Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...." 2.- Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend. 3.- Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)." 4.- Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.
5.- Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence. 6.- Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......." 7.- In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.
8.- Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.
Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....." 10.- Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..." 11.- Introduce a line break between line 503 and 504. 12.- Page 14 line 543, please change "XbaI" to "XbaI" Thanks for the careful editing. We changed the text as suggested by the reviewer. We also added a scheme showing the genetic organization of the genes involved in Leg production and present as Figure 1B. When this study was initiated, the pMT335 plasmid with a Pvan promoter was used before we switched to using the pSRK plasmid with Plac promoter for better induction. Note that the results with Pvan or Plac are comparable regarding the PseI synthases interchangeability. Color code is now homogenous through the manuscript.
Reviewer #1 (Significance (Required)):
This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species. Major comments: The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium.
We have now added the glycopeptide analyses as requested. They are described in the last experimental section and confirm our results.
The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.
*The motility pictures are representative of three independent experiments and the immunoblots are representative of at least two independent experiments. This has now been mentioned in the Experimental procedures. The raw unprocessed blots have now been added as supporting info. *
Minor comments: There are a few textual errors that the authors should fix: -page 2, line 70: change "used" to "use" -page 11, line 407: add the word "are" after Pse On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected. We corrected the text as the reviewer suggested. We changed the sentence in the introduction to be more nuanced. The drawing of the monosaccharide has been improved.
Reviewer #2 (Significance (Required)):
The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented. These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.
Major comments: 1. Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg". Putative PseI and LegI were proposed based on BlastP analyses in which the protein sequences of interest were aligned to the three experimentally validated synthases from C. jejuni 11168: PseI, LegI, NeuB as well as PseI from C. crescentus, as indicated in Table S1. While, the assignment of the donor sugar is based only on the sequence identity and similarity to LegI or PseI, this assignment corresponds well according to the restoration of the motility of the C. crescentus ΔpseI mutant upon expression of PseI ortholog and B. subvibrioides ΔlegI mutant with heterologous LegI expression.
It is true that for Shewanella oneidensis the assignment as PseI or LegI is ambiguous, exhibiting nearly identical similarity, but it is quite distinct from NeuB. This actually makes the S. oneidensis synthase a very interesting case to explore the enzymology of its Pse/LegI ortholog, knowing that it has been previously shown that this bacterium glycosylates its flagellins with Pse derivatives (PMID: 24039942). The results from our genetic complementation analysis are however very clear (PseI ortholog) and very consistent with the functional analysis in S. oneidensis.
Concerning the different derivatives of Pse or Leg: McDonald and Boyd (PMID 32950378) recently published a review giving some examples of Bacteria/Archaea experimentally shown to contain Pse/Leg-derivatives: C. jejuni 11168 modifies its flagellin with 5,7-N-acetyl Pse, Sinorhizobium fredii NGR234 (not used in this study but in our previous work PMID 33113346 and showed to restore the motility of C. crescentus ΔpseI cells) modifies its capsule with 5-acetamido-7-3-hydroxybutyramido-Pse), Treponema denticola modifies its flagellin with 7-(2-metoxy-4,5,6-trihydroxy-hexanoyl-Pse, A. baumannii LAC-4 produces 5,7-N-acetyl-8-epi-Leg to decorate the capsule, Halorubrum sp. PV6 modifies the LPS with N-formylated Leg and L. pneumophila produces 5-acetamidino-Leg.
The reviewer is right in that we do not know the exact version of Pse or Leg produced in C. crescentus and B. subvibrioides, HOWEVER, the fact that complementation works with the majority of the orthologs of PseI and LegI including many from bacteria that are known to produce modified Pse derivatives for example in Shewanella oneidensis and Treponema denticola, the most likely explanation is that derivatization occurs after the PseI or LegI step, but we concede that the results are also compatible with a promiscuous enzyme that can accept different Pse derivatives or different Leg derivatives.
- Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included. We agree that for many PseI or LegI tested, there is no provided biochemical evidence. HOWEVER, this is not the case for some of them including the PseI, LegI and NeuB from Campylobacter jejuni (PMID 19282391), some A. baumannii strains (α-epi-legionaminic acid for A. baumannii LAC-4 PMID 24690675), Shewanella oneidensis (Pseudaminic acid with methylation PMID 23543712), Legionella pneumophila (Legionaminic acid PMID 18275154) or Halorubrum sp. PV6 (N-formylated legionaminic acid PMID 30245679). Thus, we maintain the two conclusions: the PseI and LegI synthases are generally interchangeable and the complementation assays can enable to identify and assign PseI and LegI function. BLAST2P was used to compare the protein sequences of the tested NeuB-like synthases with NeuB1, LegI (NeuB2) and PseI (NeuB3) from Campylobacter jejuni but also with PseI from C. crescentus. BLOSUM62 matrix was used as well as a word of size 3 for the comparison. We have now added this procedure in the legend of the Table S1.
- It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants? The number of flagellin subunits that are assembled into a full-length flagellar filament is unknown in C. crescentus and in B. subvibrioides. There are 3 different flagellin genes that are now presented schematically in Figure 1C. No redundancy has been found for LegI or FlmG. It is possible that the B. subvibrioides is better in exporting non-glycosylated flagellin or that the capping proteins can function better with sugar modification or that the filament of B. subvibrioides mutants is less fragile when it is non-glycosylated or that its flagellins “stick” better. It is also possible that short filaments are not actually containing flagellins mounted on the hook but another protein that polymerizes aberrantly in the absence of Leg or FlmG. This remains to be investigated and compared to the situation of Pse and FlmG mutants of C. crescentus.
B. subvibrioides possesses an ortholog of the C. crescentus flagellin secretion chaperon FlaF (PMID 33113346). As observed in C. crescentus, FlaF likely has a role in flagellin translation as its inactivation totally prevents flagellins production (see answer to reviewer #1). For C. crescentus, bacterial two hybrid experiments revealed that FlaF can interact with non-glycosylated flagellins in E. coli. Thus, it is strongly possible that FlaF/flagellins interaction is not dependent on the flagellins glycosylation state. In addition, the short flagellum filament observed in B. subvibrioides ΔlegI or ΔflmG mutants argues that at least some flagellins are secreted while not glycosylated.TEM pictures have been performed in liquid medium from exponential growth phase. In this condition, no fragment of flagella was observed in the culture medium by TEM but only small flagella with a hook structure attached. Also, flagella breaks might result in more random length of flagellum.
Three flagellins are in B. subvibrioides (Bresu_2403 is 59% identical with FljLCc, Bresu_2638 is 57% identical with FljKCc and Bresu_2636 is 62% identical with FljJCc). We now show this genetic organization of the flagellins in Fig. 1C. The three flagellins are all detected by the anti-FljKCc anti serum (see answer and figure to reviewer #1). We cannot attribute the immunoblot signal to any individual B. subvibrioides flagellin as they could all co-migrate on SDS-PAGE. However, the signal often looks like a doublet (as shown in Figure 4B for example) suggesting that at least two flagellins are detected and this doublet is always found to migrate faster in absence of glycosylation that could indicate that all B. subvibrioides flagellins (or at least 2) are modified.
Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody? Antibodies raised to FljKCc were raised against His6-FljK produced in E. coli (previously published in Ardissone et al, 2020). This serum recognizes the 6 flagellins from C. crescentus (PMID: 33108275). It recognized the three flagellins from B.s. (see answer to reviewer #1).
It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.
C. crescentus cells that produce Pse (endogenously) or Leg (reconstituted pathway) and BsFlmG and BsFljK (formerly Figure 5C and now as Figure 7C) are indeed not motile as shown by the motility tests presented in Figure S5B. Motility assays with cells used in the former Figure 5B (now Figure 7B) have also been done and are now presented Figure S4B. These cells are non-motile because BsFljK is not efficiently secreted (or unstable after secretion) as shown on the immunoblot of the supernatant fraction in Figure S4A lower panel. As a result, flagellar filament is not properly assembled as only a short flagellum was observed by TEM in such cells compared to the WT C. crescentus (Figure S4C and S4D).
Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.
*__ Immunoblots of the supernatants for Figure 5B (now Figure 7B) have been done and been added (Figure S4A lower panel). BsFljK is barely detected in the supernatant whatever its glycosylation state (with or without Leg). Note that in the supporting info where the raw unprocessed blot used for this panel is shown, a positive control of blotting (C. crescentus Δfljx6 mutant expressing CcFljK from pMT463) has been used. Immunoblots of the supernatant from Figure 5C (now 7C) have been done and been added in figure S5A. The CcFljK modified with Leg is poorly secreted (or unstable after secretion). As a result, these cells only harbor a short flagellum compared to those that are able to modify CcFljK with Pse (Figure S5C).
HPLC of the acid extracts from flagellins have been performed on purified flagella obtained by ultracentrifugation. As C. crescentus cells expressing BsFlmG and Cc-BsFlmG harbor no or short flagellar filament, the purification by ultracentrifugation is limited. Thus, to further confirm that CcFlmG has transferred Pse and Cc-BsFlmG (and BsFlmG) has transferred Leg on CcFljK (former Figure 5C and now Figure 7C), we performed immunoblots on the cell extracts of C. crescentus ΔflmG ΔpseI cells that cannot produce Pse but able to produce Leg (reconstituted pathway). These experiments, now presented in Figure 7C (lower panel) confirmed that no modification of CcFljK was observed in C. crescentus cells expressing CcFlmG whereas CcFljK is modified in C. crescentus expressing Cc-BsFlmG, confirming that Cc-BsFlmG has transferred Leg (the only NulO produced in this condition).__*
Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.
GlmU is a potential bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase that can be part of both Pse and Leg pathway (PMID 19282391). Accordingly, a GlmU ortholog is found in C. crescentus and B. subvibrioides that we showed are producing Pse and Leg, respectively. Thus, GlmU cannot be attributed to a Leg pathway signature. On the other hand, PtmE is barely found in the organisms from which PseI orthologs restore the motility of C. crescentus ΔpseI cells.
PtmF, PtmA, PgmL and GlmS are proposed to act upstream of the production of GlcN-1-P that is a precursor of both UDP-GlcNAc and GDP-GlcNAc, the precursors of Pse and Leg respectively. In addition, orthologs of these genes are not prevalent in the Leg synthetizing organisms present in Table S2 using BlastP analyses with C. jejuni proteins as templates.PtmE ortholog is found in most of the Leg synthetizing organisms as shown in Table S2 and often genetically linked with other genes coding for proteins involved in Leg production (shown with the asterisk * in table S2). Of note, PtmE is found not only in organisms that modify flagellin(s) with Leg but also in organisms that add Leg on capsule such as A. baumannii LAC-4.
It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of? The motility pictures are representative of three independent experiments. The immunoblots are representative of at least two independent experiments. This information is now added in the Experimental procedures section.
Minor comments:
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The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.
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Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy. Done
- Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy Corrected
- Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives" corrected
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Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.
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Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI. Indeed, we modified the sentence.
- Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors" Corrected
- Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"? Corrected
- Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate. Correct. Because we cannot test all enzymes we must keep the statement non-committing.
“Most (if not all)” refers to the latter case i.e. the modification occurs after PseI synthase. In this context, PseI should do the same reaction, however, there might be some exceptions.
There is, to our knowledge, no reports showing that modifications occur before the PseI synthase. The glyco-profiling experiments all suggest that modification occurs after Pse production based on our motility readout. It is possible that PseI enzymes that condense a modified precursor would not be functional in our motility assay.
Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome? We now have modified the sentence to make it clearer.
At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text. Thanks for raising this issue. We have now tried to address this concern by re-organizing the order of occurrence of the figures. Notably we have now exchanged Figure 5 (on Leg pathway reconstitution and FlmG rewiring) with Figure 7 (on LegB and LegH). We modified the text accordingly. We hope that it makes the manuscript and corresponding figures easier to follow.
Reviewer #3 (Significance (Required)):
The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases
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Referee #3
Evidence, reproducibility and clarity
Summary of the findings and key conclusions (including methodology and model system(s) where appropriate): Kint et al describe a neat study of bacterial flagellin glycosylation by a recently identified class of protein glycosyltransferases called FlmG. The experiments are well designed, the data presented is convincing and the conclusions drawn are mostly in line with the experimental evidence presented.<br /> These are the key findings. Kint et al show that genetic tools and motility can be used as a readout to probe the sugar biosynthesis pathway in bacteria. Using the recently characterized system of Caulobacter crescentus, they have performed a survey of different PseI/LegI/NeuB genes from various bacteria, checking whether they could rescue the motility defect in C. crescentus ΔpseI cells. They found that those genes that did confer motility also had higher sequence similarity to C. jejuni PseI than to C. jejuni LegI or C. jejuni NeuB. They also found that these genes also restored flagellin glycosylation as checked by mobility shift on gel electrophoresis with immunoblotting to anti-FljK antibody. This survey brought up an interesting finding that the PseI/LegI/NeuB orthologs of the closely related Brevundimonas species were unable to confer motility to C. crescentus ΔpseI cells, and were more similar to C. jejuni LegI than to C. jejuni PseI or C. jejuni NeuB. They also performed similar glycoprofiling experiments using B. subvibrioides ΔlegIBs cells and various PseI/LegI/NeuB orthologs from different bacteria, which indicated the restoration of motility by putative LegI synthases. Kint et al demonstrate flagellin glycosylation in B. subvibrioides by performing in-frame deletions of FlmG, and LegI genes in B. subvibrioides and checking for motility, presence of flagella, and flagellin glycosylation by motility shift on gel electrophoresis. Further, they confirm the critical nature of GDP-GlcNAc for Leg biosynthesis by assessing flagellin glycosylation and motility in B. subvibrioides with an in-frame deletion in PtmE/LegX and by performing heterologous complementation with an M. humiferra PtmE ortholog. They also reconstitute the legionaminic acid biosynthesis pathway in C. crescentus cells that lack flagellins, PseI and FlmG, and show that the heterologously expressed B. subvibrioides flagellin is glycosylated by heterologously expressed B. subvibrioides FlmG. Finally, they also show that whereas the CcFlmG cannot substitute for BsFlmG and vice versa, a chimeric FlmG bearing the TPR domain from C. crescentus FlmG (that recognizes C. crescentus FljK) and the GT domain from B. subvibrioides FlmG (that transfers CMP-Leg) modifies CcFljK in C. crescentus cells that lack CcFlmG but express both Pse (endogenously) and Leg (from the reconstituted pathway). This demonstrates the modularity of the FlmG glycosyltransferases. Kint et al provide the chemical nature of C. crescentus flagellin glycosylation. Kint et al have analyzed the glycans released from the flagellin by acid hydrolysis and clearly shown the nature of the glycan in C. crescentus flagellin to be Pse4Ac5Ac7Ac by use of Pse standards. The glycan from B. subvibrioides was distinct from the Leg standard used, and could be a Leg derivative distinct from Leg5Ac7Ac.
Major comments:
- Table 1 and Text in Results, lines 116-119, "In support of the notion that derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg, our glyco profiling analysis revealed that putative PseI proteins (identified by sequence comparisons to C. jejuni 11168, Table S1) conferred motility to C. crescentus ΔpseI cells, whereas putative LegI synthases did not." Not clear how putative PseI and LegI synthases were identified. Table 1 only lists overall percent sequence identity and similarity to Cj PseI, LegI and NeuB, and percent identities and similarities of the various nonulosonic synthases to these proteins are in the similar range, as expected. In the absence of sequence alignments indicating the presence of conserved residues, particularly related to the substrate binding region, that are distinct in these paralogs, calling out the type of synthase based on the highest percent identity (to Cj PseI, LegI or NeuB) is speculative. Also, Shewanella oneidensis does not follow the pattern of highest similarity to NeuB3. Second, in the absence of data showing that the Leg and Pse found in these different organisms actually are different derivatives, this does not support that "derivatization occurs after the PEP-dependent condensation reaction to form Pse or Leg".
- Related to (1), Text in Results, lines 130-131, "We conclude from our survey that (heterologous) PseI synthase activity generally confers motility to C. crescentus ΔpseI cells, whereas LegI-type (or NeuB-type) synthases are unable to do so." There is no a priori evidence provided indicating that these were PseI or LegI type synthases. So the conclusion really is that assuming only PseI type synthases would be able to rescue the motility defect in C. crescentus ΔpseI cells, this glyco-profiling motility assay now provides the first biochemical evidence telling us which synthases are Pse-type, and which are Neu/Leg-type. And in my view, this is the conclusion of greater significance in the field - to be able to now identify which is a PseI and which a LegI based on these complementation assays. However, if the authors still wish to retain their original conclusion, they could cite or provide evidence (either biochemical evidence in this work or reported literature regarding the sugar synthesized or bioninformatics analysis regarding the presence of distinct genes such as the Ptm genes for legionaminic acid biosynthesis pathway or genes that differ in their enzyme activities and overall fold such as PseB/LegB or PseG/LegG in the gene neighborhood) indicating or suggesting the PseI/LegI/NeuB nature of the different synthases. Also, methods for the bioinformatics analysis (eg. BLASTp settings used, dates of searches, whether regular BLAST or PSI-BLAST was used, etc.) are missing in the manuscript, and need to be included.
- It is interesting that there is still a signification amount of flagellin secretion/assembly in the B. subvibrioides LegI and FlmG mutants. It will be good to see a discussion about whether this is likely from due to low level of function despite the in-frame deletion of genes; how many flagellin subunits are likely to have managed secretion and assembly in these short flagella; whether there is any redundancy of LegI / FlmG (perhaps with lower levels of expression); considering Parker and Shaw's findings of glycosylation being required for flagellin binding to the chaperone and subsequence secretion in A. caviae whether there is a FlaJ homolog in B. subvibrioides. Also, can the authors rule out the possibility that absence of glycosylation does not affect flagellin assembly but makes the flagellum prone to shear/breaks in B. subvibriodes, resulting in smaller flagella? How many flagellins are there in B. subvibrioides? Is it possible that one is glycosylated but another/others are not, and that is the reason for the small flagellum in these mutants?
- Text in Results, lines 170-171, "We then probed the resulting ΔlegIBs and ΔflmGBs single mutants for motility defects in soft agar and analyzed flagellin glycosylation by immunoblotting using antibodies to FljKCc". Was the antibody to FljKCc determined to also specifically bind to FljKBs? Also, how many flagellins are there in B. subvibrioides? Are all detected with this antibody?
- It is interesting that C. cresentus cells expressing Pse (endogenously) and Leg (reconstituted pathway), and BsFlmG and BsFljK (corresponding to Figure 5C) are not motile. Was the motility assay done for the experiment of figure 5B as well? Are the C. crescentus cells lacking Pse and FlmG but with heterologous expression of Leg and BsFljK and BsFlmG also non-motile? Also, it will be good to see the TEM images for these cells.
- Immunoblotting of the supernatants should be shown (in addition to the cell extracts) for Figures 5B and 5C so that the reader can appreciate whether glycosylation has taken place but secretion/assembly has not. Further, HPLC of the acid extracts from flagellin could be done to unambiguously show whether the CcFlmG has transferred Pse and the BsFlmG and Cc-BsFlmG have transferred Leg on to the CcFljK in Figure 5c, and the identity of the sugar, if any, transferred by CcFlmG in the absence of Pse, and BsLeg genes or BsLegX gene in figure 5B.
- Text in discussion, lines 334-338, "By extension, having recognized the LegX/PtmE enzyme as a critical element in the Leg-specific enzymatic biosynthesis step (Figure 6) likewise offers another functional, but also a novel bioinformatic, criterion for the correct assignment and discrimination of predicted stereoisomer biosynthesis routes residing in ever-expanding genome databases" It will be nice to see a discussion on the prevalence of PtmE versus GlmU (or equivalent gene), PtmF, PtmA, PgmL in the Leg synthesizing organisms. Is the PtmE but not the other genes found in all cases, which makes it better as a molecular determinant for bioinformatics predictions of the type of pathway? Also, on whether PtmE has any homology to genes in other pathways (not associated with flagellin glycosylation) and how reliable a marker it is to differentiate Leg biosynthesis from Neu5Ac biosynthesis pathways.
- It is not clear from the methods or the figure legends how many times the immunoblotting, motility experiments were done; how many experiments/trials are the images representative of?
Minor comments:
- The gene for GlcN-1-P guanylyltransferase in the Leg-specific enzymatic biosynthesis step is already known as PtmE from the work of Schoenhofen's group. For the sake of consistency, it would be better to retain the nomenclature as PtmE throughout the manuscript instead of introducing the name LegX, which makes it sound like it is a previously unknown gene.
- Text in abstract, lines 15-17: "Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages" The glycosyl donor is the nucleotide sugar and not the nonulosonic acid or sialic acid... rephrasing required for accuracy.
- Text in abstract, lines 18: "a new class of FlmG protein glycosyltransferases that modify flagellin" The authors are presumably referring to FlmG as the new class of protein glycosyltransferases... rephrasing required for accuracy
- Text in introduction, lines 41-42 "Pse and Leg derivatives synthesized in vitro can be added exogenously in metabolic labeling experiments" It should be "derivatives of Pse and Leg precursors" and not "Pse and Leg derivatives"
- Text in introduction, line 46 "Pse- or Leg-decorated flagella may also be immunogenic." This sentence is not referenced and it is not clear why it is written here.
- Text in introduction, lines 63-66 "The synthesis of CMP-Pse or CMP-Leg proceeds enzymatically by series of steps [20-22], ultimately ending with the condensation of an activated 6-carbon monosaccharide (typically N-acetyl glucosamine, GlcNAc) with 3-carbon pyruvate (such as phosphoenolpyruvate, PEP) by Pse or Leg synthase paralogs, PseI or LegI, respectively" The synthesis begins with activated GlcNAc. The substrate for condensation is not activated GlcNAc. It is 2,4-diacetamido-2,4,6-trideoxy-D-mannopyranose in case of LegI and 2,4-diacetamido-2,4,6-trideoxy-b-L-altropyranose in case of PseI.
- Text in introduction, line 70 "for used as glycosyl donors" Typographical error, "for use as glycosyl donors"
- Text in Results, line 102, "C. crescentus only encodes only PseI" Do the authors mean "only one PseI"?
- Text in Results, lines 108 and 109, "Such modifications could occur before the PseI synthase acts or afterwards. In the latter case, most (if not all) synthases would be predicted to produce the same Pse molecule," Do the authors know of any reports of modifications occurring before the PseI synthase? Please cite references, if known. Why "most (if not all)"? If the former case is true, the PseI synthase might not be able to accept the substrate.
- Text in Results, lines 141-143, "our bioinformatic searches using C. jejuni 11168 as reference genome identified all six putative enzymes in the B. subvibrioides ATCC15264 genome (CP002102.1) predicted to execute the synthesis of Leg from GDP-GlcNAc" Not clear how this was done. Do the authors mean that they used the genes from C. jejuni 11168 as the query genes to identify homologs in B. subvibrioides ATCC15264 genome (CP002102.1)? Or did they use putative genes from B. subvibrioides ATCC15264 genome (CP002102.1) and pull out homologs from C. jejuni 11168 by using C. jejuni 11168 as the reference genome?
- At first reading, the flow of the manuscript is difficult to follow due to the figures not appearing in full in order of their occurrence. For instance, Figures 5B and 5C are discussed only in the end of the manuscript after the results of Figures 6 and 7. Other instances also exist. The authors may consider re-ordering the figure parts if possible so that all parts of each figure appear in order of occurrence in the manuscript text.
Significance
The nonulosonic acids, Pseudaminic acid and Legionaminic acid, are abundant in bacterial systems in the capsular and lipopolysaccharides as well as in glycoprotein glycans. The Ser/Thr-O-nonulosonic acid glycosylation of flagellins has been studied with respect to the system of Maf glycosyltransferases in Campylobacter jejuni, C. coli, Helicobacter pylori, Aeromonas caviae, Magnetospirillum magneticum, Clostridium botulinum and Geobacillus kaustophilus, and recently with respect to the system of FlmG glycosyltransferases by Viollier's group in Caulobacter crescentus. However, the determinants that govern the glycosyltransferase function are not still well known. Kint et al have performed excellent work using bacterial genetics tools to (1) highlight the "functional insulation" of the Leg and Pse biosynthesis pathways, (2) demonstrate the modularity of the FlmG glycosyltransferase proteins with respect to the flagellin binding and glycosyltransferase domains. This work makes a significant advance in the field with respect to (1) understanding flagellin glycosylation by FlmG, (2) making designer protein Ser/Thr-O-glycosyltransferases, and (3) bioinformatics analysis of genomes with respect to the Pse/Leg/Neu nonulosonic acid biosynthetic potential encoded. The findings will be of great interest to scientific audiences working in the areas of glycobiology and bacteriology. My area of expertise: Maf flagellin glycosyltransferases
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Referee #2
Evidence, reproducibility and clarity
Summary:
Viollier and co-workers present a study in which they preform an elegant and rigorous genetic profiling of the the legionaminic and pseudaminic acid biosynthesis and flagellar glycosylation pathways in C. crescentus (native Pse) and B. subvibrioides (native Leg). They use motility as a representative readout for functional flagellar glycosylation with these microbial sialic acids. They discover orthologous Pse synthase genes can replace the function of the native synthase in C. crescentus and orthologous legionaminic acid synthase genes can achieve the same in B. subvibrioides. However, not vice versa indicating a strong preference for each microbial sialic acid stereoisomer in these species. For the Leg biosynthesis pathway, which requires GDP-GlcNAc, the authors also identify LegX as an essential component to synthesize this sugar nucleotide and thus a marker for Leg biosynthesis pathways. Upstream in theses pathways, they also identify a new class of FlmG flagellar protein glycosyltransferases. Importantly, through heterologous reconstitution experiments to uncovered that these glycosyltransferases possess two distinct domains, a transferase domain the determines specificity for either CMP-Leg or CMP-Pse, and a flagellin-binding domain to achieve selectivity for the substrate. Interestingly, by creating chimeric FlmG for these two domains between C. crescentus and B. subvibrioides they show that these two modular parts can be interchanged to adapt flagellin glycosyltransferase specificity in these species.
Major comments:
The key conclusions of the manuscript by Viollier and co-workers are convincing and well supported by their experiments and used methods, with respect to the insulation of the Leg and Pse biosynthetic pathways, they key role of LegX in launching the Leg pathway and the successful reconstitution of Leg glycosylation in a previously Pse-producing C. crescentus strain. Finally, they convincingly show that a chimeric version of the involved glycosyltransferases is functional, which besides intriguing future glycoengineering possibilities also emphasizes the two discrete domains in these transferases that dictate their sugar nucleotide and acceptor specificity. There is one additional experiment I would suggest with relation to the detection and confirmation of Pse and Leg on flagella of respectively, C. crescentus and B. subvibrioides. In the case of C. crescentus the detected DMB derivatized monosaccharide co-elutes with a validated standard of tri-acetylated Pse, which is convincing evidence of its identity. However, for B. subvibrioides. Their DMB derivatized monosaccharides from its flagella, results in a peak the does not co-elute with the only Leg standard (Leg5Ac7Ac) they have, it does elute at the same time as their Pse standard. Although it cannot of course be Pse as B. subvibrioides. Does not possess a Pse biosynthesis pathway, it also does not provide enough evidence to conclude that it is a Leg derivative. An MS(-MS) measurement of the eluted signal would not be a big investment in time and resources and would provide additional evidence to at least assign this peak to microbial sialic acid related to the present Leg biosynthesis pathway. It the identified mass would lead to identification of the derivative, it would also add to the proper characterization of the flagella glycosylation in the bacterium. The data and the methods presented in this study are presented with sufficient detail so that they can be reproduced? However, I would suggest as is common nowadays in most journals that the authors include images of the raw unprocessed blot in de supporting info.
Minor comments:
There are a few textual errors that the authors should fix:
- page 2, line 70: change "used" to "use"
- page 11, line 407: add the word "are" after Pse
- On page 2, line 36, the authors state that "most eubacteria and the archaea typically decorate their cell surface structures with (5-, 7-)diacetamido derivatives, either pseudaminic acid (Pse) and/or its stereoisomer legionaminic acid (Leg,". This should be nuanced as to my knowledge it is not most eubacteria, but more a subset as identified by Varki in his seminal PNAS paper. The authors clearly present their data and conclusions in the figures of this manuscript. However, I would recommend the take a critical look at the drawing of their monosaccharide chair conformations and the positioning of the axial and equatorial groups on these chairs in Figure 1 and 5, as these are in most cases drawn a bit crooked, which can easily be corrected.
Significance
The family of carbohydrates called sialic acids was long thought to exclusively occur in glycoproteins and glycolipids of vertebrates, but has since also been found in specific microbes. Especially symbiotic and pathogenic microbes associated with the humans express a wide array of unique microbial sialic acids for which their functional roles are not well understood and the associated glycosylhydrolase and glycosyltransferase have in most cases not been identified yet. The authors present an impressive insight into flagellar glycosylation with Pseudaminic and Legionaminic acid in two bacterial species, using genomic analysis, rewiring, immunoblots and motility assays as their main tools. They provide compelling evidence on the insulation of the Pse of Leg pathway in these species, the flexibility in exchanging between biosynthetic enzymes from the same pathway between various species. Crucially, most glycosyltransferases that add the Pse or Leg glycoform onto various acceptor sites in bacteria, have up to this point remained elusive in most cases. It is therefore very valuable information that the authors here provide on the involved glycosyltransferases. Especially, on the two domains that govern their sugar nucleotide and acceptor specificity, and that these can be reengineered as chimeric glycosyltransferases. To me as a chemical glycobiologist this provides compelling possibilities for glycoengineering possibilities in future studies in the field to elucidate the functional roles of Pse and Leg glycosylation.
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Referee #1
Evidence, reproducibility and clarity
Summary:
In this manuscript, authors establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. They used B. subvibroides and C. crescentus specific mutants in pseI and legI genes involved in the Pse and Leg biosynthesis, respectively, and cross-complementation assays with orthologous genes from different bacterial species, analysing motility and flagellin glycosylation. These assays show that Pse and Leg biosynthetic pathways are genetically different and recognize the LegX enzyme as a critical element in the Leg-specific enzymatic biosynthesis. Since that legX orthologous were only identified in the genome of bacteria with Leg biosynthetic pathways, it becomes a good marker to distinguish Leg from Pse biosynthesis pathways and a novel bioinformatic criterion for the assignment and discrimination of these two pathways. Reconstitution of Leg biosynthetic pathway of B. subvibroides in the C. crescentus mutant that lack flagellins, PseI and FlmG, complemented with both flagellin and FlmG of B. subvibroides, identified a new class of FlmG protein glycosyltransferases that modify flagellin with legionaminic acid. Furthermore, the construction of a chimeric FlmG through domain substitutions, allowed to reprogram a Pse-dependent FlmG into a Leg-dependent enzyme and reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.
Major comments:
The conclusions obtained are convincing and well-supported. However, I think some points should be specify or clarify.
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- In the mutants (pseI, legI, flmG,...) the non-glycosylated flagellin are exported and assembled in a flagellum filament shorter than the WT strain. However, motility in plates is absent or very reduced. This might be produced by instability of the flagellum filament when rotating in a semi-solid surface. MET was performed from plates or liquid cultures? Do the author analyses motility in liquid media? If they did, changes in motility were observed?
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- In page 5, lines 158-163, the analysis, by HPLC, of derivatized nonulosonic acid from B. subvibroides flagella, shows a major peak at 9.8 minutes retention and a minor peak at 15.3 minutes. Since that Pse-standard have retentions peaks at 9.7 and 13 minutes, and Leg-standard at 12.3 minutes, the authors cannot infer, only with these data, the flagella sugar is a legionaminic acid derivative. In my opinion, should be included that inference comes from the data obtained by HPLC analysis and genetic approaches.
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- In page 5, line 173-175. Authors indicate, "While no difference in the abundance of flagellin was observed in extracts from mutant versus WT cells, flagellin was barely detectable in the supernatants of mutant cultures, suggesting flagellar filament formation is defective in these mutants". MET images show that the flagellum filament length is shorter in the mutants than in the WT strain. Therefore, if the same number of mutants and WT cells has been used in the immunodetection assays, there should be more flagellin monomers in the WT samples than in the mutants ones and flagellin bands should be less intense in mutant samples corresponding to the anchored flagellum. Why bands corresponding to flagellin in mutants and WT show similar intensity in the immunodetection assays (Figure 3C and D)? Furthermore, in lane 177-178, authors suggest that LegI and FlmG govern flagellin glycosylation and export (or stability after export). However, if filament stability is affected, the amount of flagellin monomers in the supernatant of mutants should be higher than in the WT. However, immunodetection assays show less abundance of flagelin monomers in the supernatant of mutants. Please, can you clarify this? In relation to this point, I suggest that authors include, in the experimental procedures, how they obtained the supernatants to flagellin immunodetection, as well as why they used anti- FljKCc anti-serum to detect the B. subvibroides flagellin.
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- Authors demonstrate the specificity of the GT-B domain of FlmG, using a chimeric FlmGCc-Bs in a mutant of C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides. However, since that TPR comes from C. crescentus, this chimeric protein, could be transfer the legionaminic acid to the flagellin of B. subvibroides? Furthermore, the complementation of this mutant with the FlmGBs did not support efficient flagellin modification and this might be related to the TPRCc domain. Therefore, in my opinion, the chimeric protein should be introduced in the B. subvibroides∆flmG background.
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- Page 8, line 299-301. Authors point out that C. crescentus that lacks FlmG and harbour the Leg biosynthetic pathway of B. subvibroides and the chimeric FlmGCc-Bs, although it has a glycosylated flagellin, whose mobility in SDS-PAGE is like the WT strain, is non-motile. They suggest that additional factors exist in the flagellation pathway that exhibit specificity towards the glycosyl group that is joined to flagellins. However, would be interesting to see if the flagellum filament has similar length to the WT strain or at least, it has increased in relation to the flagella length of the mutant. If flagella length has not increased, it could suggest that changes in the glycan type might affects the flagellin assembly or the stability of the flagellum filament. Therefore, would be also important to analyse its motility in liquid media.
Minor comments:
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- Pag 3 line102. Please change ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes only PseI...." to ".....two predicted synthases, a PseI and LegI homolog, and C. crescentus only encodes a PseI...."
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- Figure 2 A. Plasmid nomenclature (Plac-neuB) is confusing because C.c. ΔpseI cells express predicted LegI or PseI synthases. Please change to Plac, as in Figure 2B and 4. Figure 2A and 2B do not contain any complementation with Bacillus subtilis (Basu), however two complementation are labelled as Bs in Figure 2A and 2B. Furthermore, no Bs are present in the Figure 2 legend.
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- Legend of figure 3 should include B. subvibrioides abreviation Bs. Line 774: Please change ".......glycosylation and secretion in B. subvibrioides." to ".......glycosylation and secretion in B. subvibrioides (Bs)."
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- Figure 3. In order to keep a similar nomenclature in all plasmids, plasmid Plac-legI syn and Plac-flmG should be labelled as Plac-legIBs syn and Plac-flmGBs.
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- Legend of figure 4 should include B. subvibrioides abreviation Bs. Line 791: Please change "....... complementation of the B.subvibrioides ΔlegI mutant with ...." to "....... complementation of the B.subvibrioides (Bs)ΔlegI mutant with ...." Furthermore, Legend of figure 4 indicate in line 795, that immunoblots reveal the intracellular levels of flagellin, however figure 2 and 3 show immunoblot of cell extracts. Please, correct this sentence.
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- Legend of figure 5, 6 and 7 should include B. subvibrioides abreviation Bs. Line 808: Please change "Predicted Leg biosynthetic pathway in B. subvibrioides " to"Predicted Leg biosynthetic pathway in B. subvibrioides (Bs)" Line 834: Please change "....affects motility, flagellin glycosylation and secretion in B. subvibrioides."to "....affects motility, flagellin glycosylation and secretion in B. subvibrioides (Bs).Line 852: Please change "...acetyltransferase in flagellar motility of B. subvibrioides cells." to ""...acetyltransferase in flagellar motility of B. subvibrioides (Bs) cells." Furthermore, figure 5 should include C. crescentus abbreviation. Line 815: Please change "....whole cell lysates from C. crescentus mutant cultures......." to "....whole cell lysates from C. crescentus (Cc) mutant cultures......."
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- In my opinion it would be useful to include a scheme of the gene organization involved in Leg biosynthesis in B. subvibrioides.
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- Legend of figure S1 should include B. subvibrioides (Bs) and C. crescentus (Cc) abbreviations. Line 888-867: Please change "...C. crescentus ΔpseI cells and B. subvibrioides ΔlegI cells with plasmids expressing..." to "...C. crescentus (Cc) ΔpseI cells and B. subvibrioides (Bs) ΔlegI cells with plasmids expressing..." Furthermore, the name and abbreviations (Mm, So, Ku, Pi, Dv) of the species used should be included in the legend. Why the authors used a plasmid with a Pvan promoter in these assays? Why the authors changed the code color of pseI and legI orthologous genes? It would be more useful and understandable follow the code color used in figure 2 and 4.
- Page 6 line 200, Please change ".....complementing synthases exhibit greater overall sequence similarity to LegI than Pse of C. jejuni. 22268,....." to ".....complementing synthases exhibit greater overall sequence similarity to LegI than PseI of C. jejuni. 22268,....."
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- Page 7 line 231, Please change ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and P. sp. Irchel 3E13..." to ".....negative bacteria A. baumannii LAC-4 (GCA_000786735.1)[38] and Pseudomonas sp. Irchel 3E13..."
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- Introduce a line break between line 503 and 504.
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- Page 14 line 543, please change "XbaI" to "XbaI"
Significance
This is an interesting manuscript that contributes to the knowledge of the legionaminic biosynthetic pathway and establish a glyco-profiling platform for the functional analysis of genes involved in pseudaminic (Pse) and legionaminic (Leg) acid biosynthetic pathways. The analysis of Leg patway allowed to identify a gene (legX) that can be used to distinguish Leg from Pse biosynthesis pathways, becoming a bioinformatic tool for the assignment and discrimination of these two pathways. Furthermore, a new class of FlmG protein glycosyltransferases, able to transfer Leg to the flagellin, has been identified and its analysis reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse, and a specialized flagellin-binding domain to identify the substrate.
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Reply to the reviewers
the authors do not wish to provide a response at this time.
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Referee #3
Evidence, reproducibility and clarity
The manuscript by Wong et al., provides the first report of a homozygous loss-of-function mutation of the RAF1 gene in humans. The mutation (T543M) was found in two siblings of a consanguineous family in association with perinatal death and multiple developmental abnormalities. These abnormalities show strong similarities with a rare congenital malformation syndrome with unknown aetiology named Acro-cardio-facial syndrome (ACFS, MIM600460). Conversely, reported abnormalities are different from those observed in RASopathies, congenital diseases caused by gain-of-function mutations in either the RAF gene or genes implicated in the same signaling pathway (MAPK) and that induce its ectopic/over-activation. By performing functional experiments in cellular systems (cell line where the mutated RAF was either overexpressed or knocked-in) and in Xenopus Laevis embryos, the authors demonstrate that the reason for the phenotypic differences compared to RASopathies is that the RAF1T543M variant impairs the signaling activity of RAF and blunts MAPK pathway activation. In particular, the RAF1T543M variant: 1) is not actively phosphorylated at key activating residues, 2) is unable to transduce MAPK signaling towards MEK/ERK substrates, 3) is inherently unstable and prone to proteasome-mediated degradation and 4) is unable to block stress-induced apoptosis. On the basis of increased apoptosis detected in cellular systems and some morphological defects observed in the probands, the author classify this novel syndrome as a segmental progeroid syndrome.
Major comments:
The authors perform a thorough analysis of the structural and functional defects of the RAF1T543M protein, using in silico analyses and in vitro systems. The data are corroborated by an elegant and clear-cut experiment in Xenopus embryos that demonstrates that the mutated RAF is not able to transduce MAPK signaling when overexpressed in an in vivo model. The lack of patients' material prevents a validation in human cells, but I think the evidence collected in the manuscript is supportive of the loss-of-function mutation in RAF as the causative mutation of the observed phenotype.
The concept illustrated in the last sentence of the discussion, i.e. that different mutations in the same genes can either cause cancer (when overactivating) or premature aging (when blunting the activity of the enzyme) is fascinating. I think discussing this concept in the context of this manuscript is appropriate. However, the authors classify the syndrome caused by the RAF1T543M variant as a "novel segmental progeroid syndrome" in the title, abstract and first sentence of the discussion. I don't think that presented data are convincing for this classification. Indeed, two affected siblings die at very early post-natal stages (7 and 50 days, respectively) likely because of malformations that are not compatible with life and that are due to altered in uthero development. Progeroid syndromes are a heterogeneous group of syndromes, a minority of which is characterized by malformations at birth. The more general concept is that physical abnormalities are progressively acquired during post-natal life, in specific tissues that show typical features associated with aging, including senescence, decline of the stem cell compartment, increased inflammation. In the case of individuals carrying the RAF1T543M variant, none of these tissue abnormalities have been reported (due to the lack of material from patients) and the classification as a progeroid syndrome is based on external inspection of organs. Reported phenotypes are not specific and a failure of in uthero development of heart, limbs and other organs seems like the absolutely predominant trait. This in uthero phenotype is perfectly consistent with the physiological role of the MAPK pathway downstream multiple RTKs that transduce morphogenetic, in addition to mitogenic, signals. As for the authors words: "endogenous FGF/FGFR signaling is required for proper mesoderm and neural induction" and supports the author claim that the analysis of individuals carrying the RAF1T543M variant "underscores the importance of RTK signaling during human development". I think that the "progeroid" phenotypes are less clear. It is still important to present the phenotypes reminiscent of progeroid syndromes and discuss them and perhaps more clearly explain the logical connection with the increased apoptosis observed in in vitro experiments.
In regard to the apoptosis experiment in figure 4, it supports the claim that RAF1 activity is necessary for protection from stress-induced apoptosis. However, the cartoon presented in figure 4C also shows that this process is mediated by increased ASK1/MST2 signaling. This part of the cartoon is based on the literature and has not been formally demonstrated. The authors can try to rescue the apoptotic status by silencing ASK1 in the RAF1T543M cells, similar to what has been done in Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943. Literature also suggests that RAF1-mediated inhibition of apoptosis is kinase-independent. This part is particularly interesting since the variant produces a protein that is both kinase-inactive and unstable. It would be a nice addition to the matuscript if the authors could clarify, at least in their cellular model, whether the increased apoptosis is due to the loss of either the protein or the kinase activity.
Minor comments:
A minor comment to the Xenopus ISH: the number of embryos that have been analyzed per condition is not reported anywhere. Figure legend explains that presented images are "representative pictures", but knowing the number of tested embryos and the penetrance of the phenotype would help undertsand the relevance of the RAF mutation in the signaling pathway under investigation.
Significance
This work by Wong and colleagues provides conceptual advance and will be of interest for researchers dealing with RTK/MAPK signaling in multiple contexts including oncology, developmental biology and cardiomyopathy.
The gene under investigation is widely studied in cancer, where gain-of-function, oncogenic mutations are common. The role of RAF1 during embryonic development is less known. A couple of studies have investigated the developmental phenotype of mouse models carrying either a knock-out allele (Mikula, M, et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J. 2001. 20:1952-1962) or an allele producing a truncated protein (Wojnowski L et al., (1998) Craf-1 protein kinase is essential for mouse development. Mech Dev, 76, 141-149). A few studies have investigated the conditional inactivation in specific tissues, such as the cardiac muscle (Yamaguchi, O., 2004, Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J. Clin. Invest. 114, 937-943.). And one study has found heterozygous carriers of a loss-of-function mutation among cohorts of children affected by dilated cardiomyopathy (Dhandapany, P.S., et al. (2014). RAF1 mutations in childhood-onset dilated cardiomyopathy. Nat. Genet. 46, 635-639). The manuscript reports the spectrum of defects acquired during embryonic development by carriers of a pathogenic mutation in the RAF1 gene. The mutation impairs both stability and kinase activity of the protein. The manuscript points out the non-redundant role of RAF1-mediated signaling in specific organs during embryonic development.
The person who is reviewing the manuscript has expertise in cellular signaling, mouse embryonic development and human aging.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors describe a homozygous missense variant in RAF1 identified in a child with a lethal malformation syndrome. The T543M variant not only blocks the kinase activity, but also destabilizes the RAF1 protein. In combination, this leads not only to a loss of MAPK signaling, but also to elevated apoptosis upon cell stress, which both together very likely explain the dramatic phenotype, which may correspond to the rarely described acro-cardio-facial syndrome.
Major comments:
The results of the functional analysis presented by the authors are highly convincing and clearly demonstrate a LOF effect of the identified variant. The experiments are described in sufficient detail and the statistical analysis is sound. Depending on the target journal the clinical information might be a bit scarce. There are neither clinical pictures nor molecular data from the first affected sibling. For a more clinically oriented journal, a summary of clinical features in form of a table and a comparison to ACFS will be a prerequisite and facilitates appreciation of this very special phenotype. The affected child seems much older than his chronological age, which seems due to the hypoplastic viscerocranium and the loss of subcutaneous fat tissue. The heart and limb defects have no link to chronological aging. Whether such phenotypes should be really called progeroid can be debated, but it is common practice (and always a good selling point). The authors do not discuss their findings in the light of chronological aging. The hypothesis that the RAF1 LOF liberates ASK1 leading to increased apoptosis is very attractive. It would be very nice to show some experimental data proving this, but such data is not pivotal for the paper.
Minor comments:
The facial and overall progeroid phenotype of the affected child is quite different from most of the described ACFS patients. In contrast, the prominent neurocranium with low hairline and the hypoplastic viscerocranium remind this reviewer of Gorlin-Chaudhry-Moss/Fontaine-Petty syndrome. This differential diagnosis is also interesting since the disorder is caused by GOF variants in a mitochondrial ATP transporter increasing the sensitivity for apoptosis. This underlines the suggested link between RAF1 and the apoptosis inducer ASK1. There is probably no cellular function that has not been linked to the MAPK pathway. One of these connections is to cellular aging. Strongly activating variants in pathway members lead to oncogene-induced cellular senescence. Here, we have a progeroid phenotype, but a variant with LOF effect. This might be worthwhile to discuss. The discussion could also use some sentences on the SHSF mechanism. FGFs produced by the AER are important for proliferation of the limb mesenchyme. What is the connection between MAPK and p63?
Significance
This is a central signaling pathway and a prominent and historically outstanding oncogene. Up to now all variants in the MAPK pathway have been described as GOF and this is the first phenotype related to a LOF. Thus, this is a landmark paper widening the view on the pathway. The paper is interesting for clinical and molecular geneticists, tumor biologists, and cell biologists. This reviewer is a biochemist and clinical geneticist with research focus on mechanisms of skeletal and connective tissue disorders.
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Referee #1
Evidence, reproducibility and clarity
In the present manuscript, Wong et al describe for the first time the human phenotype resulting from the bi-allelic germline T543M mutation in the RAF1 proto-oncogene. Although somatic RAF1 mutations are outnumbered by BRAF mutations, they also occur in human cancer, but so far germline RAF1 mutations, usually dominant-acting ones abrogating RAF1 autoinhibition, have been mainly associated with RASopathies, in particular with Noonan syndrome. Here, Wong et al describe for the first time that the two individuals with a homozygous T543M mutation is associated with a neonatal lethal progeroid syndrome presenting with cutaneous, craniofacial, cardiac and limb anomalies. Moreover, the affected residue, despite its conservation across RAF1 orthologues and in the ARAF and BRAF paralogues, has neither been described as a target of somatic and germ-line mutation in cancer and RASopathies, respectively. The authors then use a combination of ectopic expression experiments in HEK293T cells and Xenopus embryos as well as CRISPR/Cas9 genome editing and structural bioinformatics to provide several lines of evidence that RAF1 T543M represents a hypomorph suppressing ERK activation. In summary, this is a very interesting and well-written manuscript with a stimulating discussion of the novel and convincing findings and the mechanisms driving the described pathology. The suggestions below might further strengthen this otherwise already very advanced manuscript.
Major:
- The data presented in this manuscript imply that RAF1 T543M represents a hypomorph suppressing ERK pathway activity. Nevertheless, it remains unclear whether this mutation reduces/abolishes the intrinsic activity of RAF1 or acts by a dominant-negative mechanism, e.g. by sequestrating critical components of RAF complexes, such as KSR proteins, or MEKs. To distinguish between both possibilities, it would be helpful, if the authors were able to document the intrinsic kinase activity of immunoprecipitated RAF1 T543M (and appropriate controls such as wildtype RAF1, the S257L gain-of-function allele and a truly kinase-dead variant, e.g. by mutating the aspartate of the DFG motif) towards recombinant and commercially available GST-MEK1.
- RAF kinases engage in complex homo- and heterodimerization events. Does RAF1 T543M form homo- and heterodimers, e.g. with BRAF or ARAF, to a similar or different extent than wildtype RAF1?
Minor:
- Abstract. To my knowledge, RAS but not RAF was the first human oncogene identified, albeit the discovery of RAF and its viral counterparts also took place very early in the oncogene discovery era.
- Introduction, p. 2 "...while Braf-/- mice are embryonic lethal due to vascular defects.7" For a more balanced review, I would suggest citing additional and more recent papers describing the complex phenotypes of Braf deficient mice, e.g. PMIDs: 18332218 and PMID: 16432225
- Figure 1D. Here, CRAF is used instead of RAF1. As the latter is the official gene/protein name RAF1 should be used to avoid confusion to readers outside of the RAF field.
Significance
Very interesting novel findings to researchers being active in the signalling, cancer and developmental biology fiels as well as to human geneticists and pediatricians.
My expertise: MAPK signalling, functional characterization of oncogenic mutations
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Reply to the reviewers
In this paper we exploit the model system of the Y loops in Drosophila spermatocytes to provide a super-resolution view of the topology of active transcription. We are pleased that Reviewer 1 assesses the work as “highly significant”. They also mention the caveat that findings may be “specific to the model system”. Reviewer 2 provides a counter to this caveat by pointing out that our findings are in line with some previous reports in mammalian cells. The interpretation in previous studies has however been hindered by the density of chromatin in the nucleus making it difficult to clearly identify individual fibres and Reviewer 2 points out that our work provides a significant advance as in our system we are able to “visualize the organization of chromatin within an individual, isolated loop”.
*Reviewer #1 *
∙ Have you assessed if you are under-labeling the nucleosomes? or RNA Polymerase?
Yes, we tried a range of antibody concentrations which did not change the observations. We have added a statement in the M&M; “To control for under-labelling of structures, we tested a range of antibody concentrations and found no significant difference in the observed nucleosome or RNAPol distribution.”
*∙ Can you stimulate and inhibit transcription to see how changes in RNA Polymerase and nucleosome organisation occur? *
We are not aware of an approach to further stimulate transcription in this system, however we have some preliminary analyses of the effects of transcription inhibition that we plan to firm up within weeks. We agree with the reviewer that it may potentially be interesting and informative to include such studies but we do not consider this as central to the significance of the paper. So our plan is to attempt to quickly gather data on this for potential inclusion but we would plan to only include this material if we can rapidly achieve a clear understanding of the effects of transcription inhibition on nucleosome cluster organisation.
∙ The 300 msec exposure time is very long for typical STORM experiments - blinking would usually be lost at this time scale?
Apologies, this was a typo. Now corrected to 30ms.
∙ Please quote laser power in absolute values rather than percentage
Done.
∙ Collecting variable number of frames (20K - 50K) can impact the number of detections/clusters. How can you account for this variation in your analysis?
Although data was collected with variable number of frames (20K-50K), in the original paper we only used data from 50K samples for quantitative analysis. In response to reviewer #2, we have now performed the analysis on a larger data set with variable frame number. Analysis of the two data sets controls for any impact of frame number variation and makes very little difference to the results of the analysis e.g the median cluster FWHM remains at 52 nm.
Reviewer #2
∙ Pages 2/3, the authors estimate the number of nucleosomes per cluster identified in the Y loops. They do so "based on a simple volume calculation" but the quantification method and reference values used to reach the estimate of a "maximum of 158 nucleosomes" is not reported in the methods. Also, the authors should discuss how do they account for multiple blinking effect in the estimate of cluster measures. Methods based on normalization curves with nucleosome arrays (Ricci et al Cell 2015) and on DNA origami (Cella Zanacchi et al Nat Meth 2017) among others were previously used to address similar questions. The authors should consider applying a normalization approach to achieve a more accurate estimate or at least discuss the caveats of their strategy.
We have now specified the details of our estimation of the number of nucleosomes per cluster in the Materials and Methods. Techniques to quantify clusters based on localisation number have been used in the past on SMLM data, but the normalisations are difficult to validate as there is uncertainty around numbers of antibodies binding, differences in frame numbers affecting blink number, as well as unknown binding efficiencies of antibodies. Our cluster quantification instead is based on the size of the clusters (cluster width), as this does not vary significantly across frame number once signal saturation has been achieved and avoids the difficulties associated with normalisation of localisation number. As we have mentioned in the Discussion, our “simple volume calculation” does have some caveats arising from the STORM precision, the labelling method and the EM-derived estimate of packing density; nevertheless, as we say in the Discussion we consider it provides a useful “starting point for interpretation of the observed labelling in terms of underlying chromatin structure.”
∙ Methods, STORM imaging: the authors should provide the imaging buffer composition as the papers they refer to make use of an array of buffers. If possible, the authors should include laser powers in standard units (kW/cm^2) to increase clarity and reproducibility.
We have provided the imaging buffer composition and the laser power in standard units.
∙ Methods, Cluster analysis: the authors should specify the values for the parameters used in "First a description of the average cluster was established using spatial statistics, then a clustering algorithm using the average parameters provided by the initial spatial statistics further refined the description"
As detailed in response to the next comment, we have clarified the text in the Materials and Methods to specify the parameters and workflow of the analysis.
∙ Given the availability of broadly used methods for clustering such as DBScan (Ester, et al. A density-based algorithm for discovering clusters in large spatial databases with noise. Kdd, 1996), Bayesian methods (Rubin-Delanchy et al. Bayesian cluster identification in single-molecule localization microscopy data. Nature methods, 2015), (Ricci et al. Cell 2015), Voronoi based tessellation (Levet et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nature methods, 2015), and more recently machine learning approaches (Williamson et al. Machine learning for cluster analysis of localization microscopy data. Nature communications, 2020) the clustering method of choice is peculiar. It makes more difficult the direct comparisons with previous work. The authors could justify the choice or compare results using other more broadly used methods.
We have added text to justify our selected analysis method and to clarify the analysis workflow. We do not consider our approach “peculiar” as very similar approaches have been employed previously (e.g. Sengupta et al Nat. Methods 8, 969–975 (2011), Veatch et al PLoS ONE 7, e31457 (2012) and Cisse Science 341:664-667 (2013)). In selecting this approach we compared various approaches and we found Meanshift to perform best with respect to identifying clusters closely positioned along a fibre. (Example data comparing the performance of Meanshift with DBscan in cluster identification with varying radius input parameter was provided to reviewers). We concluded that MeanShift provides a more robust cluster identification and that the radius value of 2Xsigma derived from the PCF is appropriate.
∙ No statistical tests were performed in the study, although no comparisons between experimental conditions are shown. The authors mention the number of replicates performed only on Fig 3 ("13 regions of interest (ROI) were selected from 3 cells"), which is a low number of cells analyzed. The authors should enlarge the analyzed datasets and mention how many replicates were used throughout the work.
As mentioned above, the quantitative analysis originally presented used the data with the constant, highest frame number (3 cells, 895 clusters). In the revised paper we present the analysis of the wider data set (12 cells, 2473 clusters). In practice this makes very little difference to the results, e.g the median cluster FWHM remains at 52 nm.
∙It is unclear why the authors did not perform STORM on EU-labeled samples immunolabeled with Pol2 antibodies. In principle, they could have applied the same imaging protocol used in previous figures gaining spatial resolution to better characterize Y nascent transcripts and how Pol2 is arranged with respect to nascent RNA.
There are indeed a number of questions that can be approached by super-resolution imaging of nascent transcripts in association with co-labelling for RPol. We are exploring this interesting direction but this is a whole separate project and we propose to report our findings on this in a subsequent publication.
∙ The text is well organized and clear as well as the figures but the authors should revise the text in the section "linking RNA polymerase distribution and Y loop transcription" to include missing references to Figure 10. For example, the statement "This distribution of nascent transcript along the Y loops fits with the distribution of RNA-PSer2," refers to results shown in Fig 10A.
We are pleased the reviewer found the text and figures well organised and clear. We have added/modified figure references in this section to clarify the relationship between text and figures.
∙ At end of page 5 the authors describe the location of RNA Pol2 pSer2 with respect to nucleosomes/RNA. As these results are related to findings reported by Castells-Garcia Nucl Acids Res 2022, cited elsewhere in the manuscript, the authors could cite the work here.
Done.
∙ It is unclear for which experiments goat anti-mouse Ig-Alexa Fluor 405 has been used, remove from antibody list if not relevant.
This antibody has been removed.
∙ The use of the 488 laser as the activatory laser is peculiar, can the authors better explain this choice?
Empirically we found the 488 laser performed better as an activatory laser- as it gave a higher signal to noise than the more commonly used 405 laser. We have added this statement to the M&M.
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors investigate the nucleosome and RNA Pol2 arrangement along the Y loop of Drosophila primary spermatocytes with super-resolution (STORM) and confocal microscopy. Given the peculiarities of the model system (extremely long transcriptional unit, transcriptional expression restricted to spermatogenesis, well-characterized nuclear location), Y loops are an interesting choice to investigate the organization of chromatin loops as they allow to physically isolate one specific DNA filament from the rest of the genome. Ball and colleagues found that, although this loop undergoes transcription and is under a decondensed state, chromatin is arranged as a chain of nucleosome clusters of variable sizes and not as a 10 nm fiber. They also show that RNA Pol 2 pSer2 is interspersed along the loop, preferentially at the periphery of nucleosome clusters and at linker sites and do not form transcription factories. RNA Pol 2 pSer5 instead is less associated with the loop and is preferentially found close to the nucleolus consistent with its role in transcriptional initiation. Given the relative scarcity of RNA Pol 2 with respect to nucleosomes along the Y loop, the authors conclude that nucleosome arrangement in chains of clusters is not determined by the transcriptional activity.
Major comments
- Pages 2/3, the authors estimate the number of nucleosomes per cluster identified in the Y loops. They do so "based on a simple volume calculation" but the quantification method and reference values used to reach the estimate of a "maximum of 158 nucleosomes" is not reported in the methods. Also, the authors should discuss how do they account for multiple blinking effect in the estimate of cluster measures. Methods based on normalization curves with nucleosome arrays (Ricci et al Cell 2015) and on DNA origami (Cella Zanacchi et al Nat Meth 2017) among others were previously used to address similar questions. The authors should consider applying a normalization approach to achieve a more accurate estimate or at least discuss the caveats of their strategy.
- Methods, STORM imaging: the authors should provide the imaging buffer composition as the papers they refer to make use of an array of buffers. If possible, the authors should include laser powers in standard units (kW/cm^2) to increase clarity and reproducibility.
- Methods, Cluster analysis: the authors should specify the values for the parameters used in "First a description of the average cluster was established using spatial statistics, then a clustering algorithm using the average parameters provided by the initial spatial statistics further refined the description"
- Given the availability of broadly used methods for clustering such as DBScan (Ester, et al. A density-based algorithm for discovering clusters in large spatial databases with noise. Kdd, 1996), Bayesian methods (Rubin-Delanchy et al. Bayesian cluster identification in single-molecule localization microscopy data. Nature methods, 2015), (Ricci et al. Cell 2015), Voronoi based tessellation (Levet et al. SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nature methods, 2015), and more recently machine learning approaches (Williamson et al. Machine learning for cluster analysis of localization microscopy data. Nature communications, 2020) the clustering method of choice is peculiar. It makes more difficult the direct comparisons with previous work. The authors could justify the choice or compare results using other more broadly used methods.
- No statistical tests were performed in the study, although no comparisons between experimental conditions are shown. The authors mention the number of replicates performed only on Fig 3 ("13 regions of interest (ROI) were selected from 3 cells"), which is a low number of cells analyzed. The authors should enlarge the analyzed datasets and mention how many replicates were used throughout the work.
- It is unclear why the authors did not perform STORM on EU-labeled samples immunolabeled with Pol2 antibodies. In principle, they could have applied the same imaging protocol used in previous figures gaining spatial resolution to better characterize Y nascent transcripts and how Pol2 is arranged with respect to nascent RNA.
Minor comments
- The text is well organized and clear as well as the figures but the authors should revise the text in the section "linking RNA polymerase distribution and Y loop transcription" to include missing references to Figure 10. For example, the statement "This distribution of nascent transcript along the Y loops fits with the distribution of RNA-PSer2," refers to results shown in Fig 10A.
- At end of page 5 the authors describe the location of RNA Pol2 pSer2 with respect to nucleosomes/RNA. As these results are related to findings reported by Castells-Garcia Nucl Acids Res 2022, cited elsewhere in the manuscript, the authors could cite the work here.
- It is unclear for which experiments goat anti-mouse Ig-Alexa Fluor 405 has been used, remove from antibody list if not relevant.
- The use of the 488 laser as the activatory laser is peculiar, can the authors better explain this choice?
Referees cross-commenting
I believe the comments of the other reviewer are very good and in line with the comments I made. I do not have much to add.
Significance
The conclusions reached by the authors are in line with previous reports (Ricci et al. Cell 2015, Ou et al. Science 2017, Castells-Garcia et al Nucl Acids Res 2022, among others). With different SR techniques, these works show that chromatin in mammalian cells is arranged in heterogeneous groups of nucleosomes and that RNA Pol 2 is not organized in transcription factories but rather binds the chromatin fiber forming small clusters at the periphery of nucleosomes. The use of Y loops as a paradigm of transcriptionally active allows the authors to confirm previous findings on another organism, Drosophila Melanogaster, and to visualize the organization of chromatin within an individual, isolated loop, without the need for FISH-like labeling. The work is potentially interesting to the field of genome and chromatin organization and SR microscopy, it is also relevant to scientists working on transcriptional regulation.
Field of expertise: Chromatin organization - SR microscopy - transcription.
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Referee #1
Evidence, reproducibility and clarity
The authors use STORM imaging to investigate transcription/spatial organisation of Drosophila Y loops. They find that the Y loop is organised in to small sections of clustered nucleosomes. Active RNAP is then found on the periphery of these clusters - furthermore, RNAP is distributed along the fibre rather than in to distinct "factory" units. The RNAP foci were less frequent than the nucleosome clusters therefore RNAP is not directing organising of the chromatin at these sites.
Comments:
- Have you assessed if you are under-labeling the nucleosomes? or RNA Polymerase?
- Can you stimulate and inhibit transcription to see how changes in RNA Polymerase and nucleosome organisation occur?
- The 300 msec exposure time is very long for typical STORM experiments - blinking would usually be lost at this time scale?
- Please quote laser power in absolute values rather than percentage.
- Collecting variable number of frames (20K - 50K) can impact the number of detections/clusters. How can you account for this variation in your analysis?
Referees cross-commenting
I agree with the other reviewer statements/comments.
Significance
Highly significant work to visualise active transcription. It provides an interesting finding that transcription is not acting to determine chromatin organisation. Nevertheless, this may be specific to the model system.
Field of expertise: Transcription, Super resolution imaging, single molecule imaging, spatial organisation.
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Reply to the reviewers
The reviews are on balance an accurate, thoughtful, thorough assessment of the manuscript. We appreciate the careful engagement with the B cell differentiation aspect of our work. We identify 2 major critiques from the reviews:
- The manuscript should make stronger connections with existing literature on ____in-vitro _and _in-vivo ____B cell differentiation. We agree the manuscript should be revised to interact more holistically and carefully with relevant B cell differentiation research. In this respect, the reviewers both help by pointing to high-quality and relevant literature that will be discussed and cited.
The cytokine mixture we used on the B cells was not defined / described in the manuscript. This fact hinders the interpretation of the data because B cells will respond to diverse stimuli in quite different ways.
We agree this hinders interpretation of the data, and the reviewers bring up astute points about different types of stimuli (TD vs. TI vs. TLR vs. BCR). Unfortunately, the manufacturer of the product, Stem Cell Technologies, will not disclose exactly what is in the product. Given we are in strong agreement with the reviewers on this point, we analyzed the cytokine contents of the cocktail and our cell culture supernatants using a luminex cytokine panel. We present a discussion of our findings on this data in a supplementary note and figure. We acknowledge this analysis is non-exhaustive, because it does not include possible additions of non-cytokine stimulants. However, we maintain it adds much clarity to the interpretation of the data.
We note that the contents of the stimulation cocktail are knowable and well-defined. These attributes are in contrast to almost all B cell stimulation protocols of which are aware. Typical stimulation protocols use various types of feeder cells, cytokines, and FBS (Fetal Bovine Serum). In particular, the feeder cells and FBS, are highly variable between labs, lots, and even experiments. FBS has a myriad of issues which are described here (____https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349753/____). Major variability, from genomic to phenotypic, has been described in laboratory cell lines like the ones used as feeder cells. With respect to B cells specifically, large differences in B cell activation programs are observed between lots of FBS, as described here (____https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854248/#r5____). Additionally, we have observed the presence of bovine viruses and other contaminants in FBS (unpublished data). Thus, the stimulation protocol we used is reproducible and robust in ways generally unseen by us in B cell stimulation literature. In summary, we view this cocktail as useful in a similar way to how FBS is useful to biologists – a major difference being that this cocktail is better defined and controlled. We provide similar thoughts in our supplementary note.
A final general point we will make is about the significance of our work, which appears to be lost on Reviewer #1. Similar technical and conceptual advances by our lab have been cited 1000s of times. Thus, we think the impact of our scientific approach speaks for itself. Many of our results confirm and expand on previous literature about B cells. We deliberately chose to make this novel technical and conceptual advance in the well-studied system of B cell differentiation. This allows us to integrate our findings with prior literature and helps validate the general approach. Reviewer #1 has performed a scholarly service by independently verifying our findings are coherent with existing literature, and we thank them for that.
In response to the reviews, we have edited the manuscript to reference even more of the papers in the field which report similar findings. Thus, our concordance with prior literature should be viewed as a strength of the manuscript. It shows readers of the manuscript the conceptual framework we use here is valid and can generate similar insights in less well-studied systems. For example, the approach developed here could be used in non-B cells, non-human immune systems, or even non-model organisms. In response to the reviewers critique, we modified the discussion of our work in multiple places to emphasize these points.
Description of the planned revisions
Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.
1) Which B cell activation protocol was used? No information is provided in the main text or supplementary information. Yet, this information is key to fully understand many of the conclusions of this work (e.g., ... memory B cells are intrinsically two-fold more persistent in vitro (2A)), which largely depend on the nature of the stimuli used in the in vitro B cell culture.
We used the B cell activation protocol developed by StemCell Technologies as described in our methods section. We agree the reader and scientific community would benefit from additional information about this cocktail. To this end, we added a discussion of the cocktail to the supplementary information. We also used a cytokine analysis panel to analyze the cocktail, which provided detailed although non-exhaustive information about what is in the cocktail.
2) It would be informative to use more than one B cell activation program, e.g., CD40L with or without a cytokine as well as CD40L vs. CpG-DNA. Authors make broad statements about B cell fates without discussing the impact of a given signal on a given B cell fate. For instance, do memory B cells follow the same differentiation program upon stimulation with CD40L, IL-4 or a combination of CD40L and IL-4? How about differences between a TD signaling program such as that provided by CD40L and IL-10 and TI signaling program such as that provided by CpG-DNA and IL-10?
This is a good point. We agree stimulation using a panel of different agents would be a worthwhile experiment. It stands as a goalpost for future studies. Currently, performing single cell RNA sequencing on so many samples is both beyond the scope of this manuscript and very resource intensive. ____
3) Page 3, first line: After low quality and non-B cells (Fig S1A & B). What does this statement mean? The sentence seems incomplete.
Thank you for catching this typo. It is now clarified in the manuscript that we removed these cells bioinformatically.
4) First, we noted that non-B cells present in the input rapidly became undetectable by day 4, which shows the specificity of the cytokines for B cell expansion. Which cytokines are we talking about? No detail is provided.
We now provide our analysis of the stimulation cocktail, in Supplementary note 1 and Supplementary figure 1A. We still believe it is an interesting observation that this cocktail specifically stimulates B cells because many cytokines are not specifically B cell division signals, there were some impurities in the input population, and many cytokines are produced by the cultured cells themselves.
5) Plasmablasts were not distinguished from plasma cells.
We agree this is an interesting and important distinction to make. We have now distinguished between these classes of cells.
6) Critically, we observed no appreciable evidence of hypermutation in vitro (S2C), consistent with prior literature (Bergthorsdottir et al. 2001). This statement is vague, misleading and likely inaccurate for the following reasons. (a) The B cell culture conditions used by the authors are completely unknown. (b) It was shown that SHM can be achieved under specific in vitro B cell culture conditions that include the presence of activated CD4+ T cells (PMID: 9052835; PMID: 10092799; PMID: 10878357; PMID: 12145648). Did authors try to recapitulate those culture conditions?
We see how this statement could be misunderstood. We only claim not to observe evidence of hypermutation in our specific culture conditions, which is important for the inferences we make. We added language to make this more clear.
We did not try to recapitulate the conditions in the references supplied by the reviewer. We note that these references use cell lines and not B cells. While there is immensely valuable work done on cell lines, they behave very differently from actual cells and these findings may not be relevant to our human B cells.
7) Some of the reported findings are repetitive of previously published results and provide no additional new information. For example:
- a) "Interestingly, we found mutated B cells were far more likely to express genes involved in T cell interaction (2B), suggesting Memory B cells are intrinsically licensed to enter an inflammatory state which activates T cells". This evidence is already published (PMID: 7535180 among many other published studies).
We will cite this paper, which is a landmark study. We don’t claim we are the first to discover a propensity of mutated B cells to present help to T cells, but note that we were able to observe this fact via lineage tracing in a single experiment, which is a conceptual and technical advance. Additionally, we report an entire transcriptional module of genes which are upregulated in memory B cells vs. Naive B cells exposed to the same stimulus. This adds to the systematic understanding of the Memory B Cell activation program.
- b) "Instead, Naive B cells were biased toward expressing lectins and CCR7, suggesting Naive B cells are intrinsically primed to home into the lymphatic system and germinal centers (2B)". This evidence is already published (PMID: 9585422 among many other published studies).
While this is an interesting and important paper referenced by the reviewer, we are unable to find anything similar to our claim about naive B cells in the reference provided. The investigators do not discuss intrinsic differences between memory and naive subsets when responding to the same stimulus.
8) We quantified the in vitro dynamics of CSR through the lens of mutation status, which revealed strongly different fate biases between germline and mutated cells (2D). Most strikingly, B cells which switched to IGHE were almost exclusively derived from germline progenitors: the ratio of germline IGHE cells to mutated IGHE cells was (8-fold - inf, 95 % CI). Also this evidence is not novel (PMID: 34050324 among other published studies) and, again, must reflect the presence of specific culture conditions that remain completely undisclosed. This is incredibly confusing.
Thank you for providing this reference, we were not aware of this interesting study. These studies are quite different and complementary. Differences between these studies likely reflect the fact that their B cells are isolated from a niche, rather than generated ____in-vitro_. Most of the tissue-resident cells in their study are quite mutated, and thus are not the Naive B cells we are making a claim abountj. In fact, despite their claim of low mutational load, these cells would fall into the “mutated” or even “heavily mutated” categories we defined in our paper. Cells with mutation levels of 5% are not thought to be Naive in any classification scheme. Our study showed that, _in vitro____, IGHE B cells effectively came exclusively from germline progenitors, their study shows no such result. The novelty of this finding was appreciated by reviewer 2.
9) Authors should mention that non-switched memory B cells include IgDlowIgM+CD27+ and IgD-IgM+CD27+ memory B cells. Some authors define these distinct memory B cell fractions as marginal zone (MZ) or MZ-like B cells (please, notice that splenic MZ B cells recirculate in humans) and IgM-only B cells, respectively (PMID: 28709802; PMID: 9028952; PMID: 10820234; PMID: 11158612; PMID: 26355154; PMID: 15191950; and PMID: 24733829 among many other published studies).
We appreciate these points. We attempted to classify our B cells within this taxonomy and found no such separation clearly exists in single-cell RNA-seq profiles. Instead, we opted to re-classify our data with a state-of-the-art algorithm called celltypist (DOI: 10.1126/science.abl5197____ )____ which harmonizes cell annotations across a growing number of single-cell RNA sequencing studies. While this classification system is not currently mutually exclusive / completely exhaustive, we believe using this system provides standardization and data availability that are key for sharing results. As single-cell RNA-seq and flow/mass cytometry harmonize their classification systems, anyone should be able to transfer their preferred classification scheme to the cells profiled here.
10) Thus, CSR from IGHM cells did not meaningfully contribute to the abundance of IGHA+ cells in the population. Also this conclusion may be misleading and/or inaccurate. Indeed, an efficient class switching to IgA requires the exposure of naïve B cells to the cytokine TFG-beta in addition to a robust TD (CD40L) or TI (CpG DNA or BAFF or APRIL) co-signal. Was TGF-beta present in this culture?
This is a good point about TGF-beta and switching to IgA. Here is a clear example of the novelty and power of our approach, as well as the benefits of using a well-characterized system such as B cell differentiation. Lineage tracing clarifies between two explanations for why there are IgA cells in the output population. One explanation is that non-IgA B cells in the input switch to IgA, driven by TGF-beta. Another explanation is that IgA cells in the input expand modestly and account for IgA cells in the output. Lineage tracing offers clear evidence that the latter explanation is true. Following from this, our approach allows us to make a strong inference that TGF-beta is not present in the incompletely determined cytokine mixture. We are not sure how this conclusion may be misleading or inaccurate, as it is a clear and simple description of our data, not a claim about what factors are necessary for switching.
11) In contrast, we noted that many intraclonal class-switching events appeared to be directly from IGHM to IGHE. Explanations involving unobserved cells with intermediate isotypes notwithstanding, these data illustrate the relative ease with which B cells can switch directly to IGHE. It is very difficult to interpret this statement, as no information regarding the B cell-stimulating conditions used is provided. In addition, relevant literature is not quoted (e.g., PMID: 34050324).
We clarify our discussion here to claim the ease with which peripheral blood IGHM B cells switch to IGHE. Again, lineage tracing has allowed us to distinguish between two very different population-level phenomena. One explanation is that undetected IGHE+ progenitors in the input population expanded rapidly and account for the IGHE+ cells. Another explanation is that cells class-switch to IGHE. Our data are consistent with the latter. We note that this validates the conceptual use of lineage tracing to understand rapid population dynamics in immune responses and cell differentiation protocols. This is a strength of our manuscript. We appreciate the the reviewer has furnished relevant studies, which we will cite.
12) Our data for IGHE cells contrasts with in vivo data which show IgE B cells to be: (1) very rare, (2) apparently derived from sequential switching (e.g. from IgG1 to IgE) (Horns et al., 2016; Looney et al., 2016), and (3) often heavily hypermutated (Croote et al., 2018).
While this reviewer agrees with the first comment (switching to IgE is relatively rare in vivo, at least in healthy individuals), the other statements are quite inaccurate. Indeed, unmutated extrafollicular naïve B cells from tonsils and possibly other mucosal districts directly class switch from IgM to IgE in healthy individuals, thereby generating a low-affinity IgE repertoire. In principle, low-affinity IgE antibodies may protect against allergy by competing with high-affinity IgE specificities. In allergic patients, high-affinity IgE clones emerge from class-switched and hypermutated memory B cells that sequentially switch from IgG1 or IgA1 to IgE as a result of specific environmental conditions, including an altered skin barrier (PMID: 22249450; PMID: 30814336; PMID: 32139586).
Moreover, in contrast to what stated by authors, sequential IgG1/IgA1-to-IgE class switching mostly occurs in allergic patients but is less frequent in healthy individuals, where IgE specificities are less mutated (PMID: 30814336). Along the same lines, IgE is heavily mutated only in allergic individuals with significant molecular evidence of sequential IgG1/IgA1-to-IgE class switching (PMID: 30814336; PMID: 32139586). Overall, the data provided by Swift M. et al. are largely confirmatory of previously published evidence.
We appreciate the clarification of this complex field and will cite the relevant literature. We also agree with the reviewers assessment that our data are validated by other approaches and groups. We see that our discussion of IgE B cells should have included that caveat that we are discussing IgE B cells detected in the peripheral blood. We have restricted claim to the suggestion that if our conditions mimic such niches where B cells switch to IgE, there are clearly efficient mechanisms which limit the amount of circulating IGHE B cells mechanisms in comparison to other isotypes.____
Taken together, these data suggest that while direct switching to IGHE from Naive progenitors is trivial in vitro, niche factors or intrinsic death programs efficiently limit their generation or lifetime in vivo." I cannot understand this conclusion, which seems to contradict earlier statements.
We hope we have clarified via the above comment.
13) I am not sure I learned much regarding the "cell-intrinsic" fate bias and transcriptional memory of B cells after reading this elegantly presented but confusing and superficially discussed manuscript (please, see also comments 15-23).
We understand the reviewer is confused about various aspects of our manuscript and appreciate the opportunities to clarify. We show cells with broad identities (such as germline vs. mutated or naive vs. memory) respond differently to the same stimulus. These are cell intrinsic fate biases. We quantify them and provide statistical bounds on the effect sizes of these differences, which to our knowledge has not been done. We agree with the reviewer that in the case of memory and naive B cells, much is already known about their biases. We recapitulate some of this knowledge, while adding a quantitative and an unbiased transcriptomic lens with which to view the biases. However, our analysis moves beyond cell types broadly defined, and focuses on the concept that each clone is a cell state or identity, where some of the identity may be faithfully propagated over generations and other information may not be. To this end, we tracked the transcriptome of clones during differentiation. We show that B cell clones share highly similar cell fates, implicating cell-intrinsic heterogeneity as a major contribution to diversity in immune responses. We note this reviewer did not critique this aspect of our work. The review also did not critique Figure 3 or 4, in which we present a quantitative analysis of which transcriptional programs are maintained by B cells and contribute to their clonal identity. Finally, via our analysis of human long-lived plasma cells, we report these transcriptional identities are observed in-vivo, over long time scales. This type of cell-intrinsic bias has not been studied or described to our knowledge. These findings were of particular interest to reviewer 2 and other readers of the manuscript.
MINOR COMMENTS
Thank you for reading the manuscript carefully and providing these comments and observations. We have fixed all clerical errors that were pointed out. We also responded to some of these minor comments here, and made changes to the manuscript to clarify.
1) Figures 1B, S1C and S1D are not referred to in the text. x
2) 2B in the Text is 2D in the Figure. x
3) 2D in the Text is 2E in the Figure. x
4) Figure 2D seems to show only 10 genes. Please, clarify.
We clarify in the manuscript that we present the top differentially expressed genes
5) 2E in the text is 2F in the Figure. x
6) Figure S3B is not indicated in the text. x
7) Figure 3E is not indicated in the text. x
8) Figure S4A is not indicated in the text. x
9) In some sections of the text, Figure panels are not sequentially discussed, which makes the text very difficult to follow. x
Reviewer 2:
Major comments:
On p.3 the authors assume that a B cell with an unmutated BCR in the time course arose from a naive B cell progenitor. However, it is also possible that it arose from a IgM memory B cells since they also contain a non-negligible proportion of cells with 0 mutations. This was initially seen already in the Klein, J Exp Med, 1998 paper and later confirmed by e.g. Weller et al, J Exp Med, 2008 and Wu et al, Front Immunol, 2011. And since the authors herein and others have demonstrated that IgM memory B cells have a high proliferative capacity it is possible that IgM memory B cells are overrepresented among those unmutated BCRs seen in the cultures.
The finding that IgM memory B cells are highly proliferative is not novel. It has been demonstrated by other groups before and one good example is Seifert et al, PNAS, 2015 where IgM memory B cells proliferated significantly more to BCR stimulation than naive or IgG memory B cells. However, it is also shown that IgG memory B cells are more responsive to TLR9 stimulation than IgM memory B cells as demonstrated by e.g. Marasco et al, Eur J Immunol, 2017. This is not discussed by the authors and should be added into the discussion for context of their finding by scRNAseq methods.
These are astute points. We incorporated a more nuanced discussion of the prior literature about highly proliferative IgM memory B cells, which have been reported before. We also added a figure which identifies the genes associated with proliferative clones in Figure 3d, which adds to our understanding of the gene regulatory networks which govern IgM memory B cell behavior. We appreciate the reference to the Seifert et al paper, which is relevant and high quality work. We concur that a discuss of Marasco would be helpful, especially because it is unknown if a TLR9 agonist is in the stimulation cocktail, but their data would suggest there is not.
The notion that a memory transcriptional program can be induced without SHM is not novel and this should be brought up in the discussion. One paper showing a memory transcriptional program in unmutated memory B cells is Kibler et al, Front Immunol, 2022.
We were not aware of this literature and have now cited it in our discussion of this finding.
The observation that memory B cells are more likely to enter an inflammatory state and support T cells has been suggested by other groups (Seifert et al, PNAS, 2015; Magri et al, Immunity, 2017, Grimsholm et al, Cell Reports, 2020).
We have now cited and discussed a number of papers which contain similar findings. We note that we add to the holistic understanding of this phenomenon via our single cell transcriptomic approach.
Please provide the age distribution of the peripheral blood samples as well.
We have now provided the age distribution of the peripheral blood samples
Please show flow cytometry analysis of the cultures to assist in assessing subset distribution, viability and plasma cell differentiation for each time point. This can be provided as supplementary information.
We did not use flow cytometry for subset distribution and measurements of differentiation per se, only to exclude non-viable cells and we have now made this clearer in the methods section. We also now include representative plots show our sorting strategy.
The stimulation cocktail used for this study, what does it contain? This needs to be specified in the manuscript and not only referring to the manufacturer. This has major impact on the results since different stimulatory agents will induce different pathways.
This is a valid point that we addressed in our response to reviewer 1. See supplementary note 1 and Figure S1A for our analysis of the stimulation cocktail.
Minor comments:
Please avoid the term plasma B cells, does it refer to plasmablasts and/or plasma cells?
Thank you for the suggestion, we have modified our language to refer to plasmablasts and plasma cells separately.
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Referee #2
Evidence, reproducibility and clarity
I have with great interest read the manuscript "Fate bias and transcriptional memory of human B cells" by Swift et al where they use an in vitro approach and single cell RNA sequencing to elucidate fate decisions of human B cells. The authors demonstrate that genes related to cell fate determination in human B cells are persistent both in their in vitro culture and in ex vivo plasma cells from bone marrow. This is a very important finding in this manuscript and the technical execution of the paper is overall of high quality. The finding of switching to IgE in vitro from unmutated precursor is very interesting and does not necessarily contradict existing data in the field but rather adds new important information. However, some of the findings are not totally novel and need to be put in context of earlier findings (outlined below). The discussion does not acknowledge literature on memory B cell transcriptional programs and mutation status in a balanced manner. Important literature by leading groups in the field are left out.
Major comments:
On p.3 the authors assume that a B cell with an unmutated BCR in the time course arose from a naive B cell progenitor. However, it is also possible that it arose from a IgM memory B cells since they also contain a non-negligible proportion of cells with 0 mutations. This was initially seen already in the Klein, J Exp Med, 1998 paper and later confirmed by e.g. Weller et al, J Exp Med, 2008 and Wu et al, Front Immunol, 2011. And since the authors herein and others have demonstrated that IgM memory B cells have a high proliferative capacity it is possible that IgM memory B cells are overrepresented among those unmutated BCRs seen in the cultures.
The finding that IgM memory B cells are highly proliferative is not novel. It has been demonstrated by other groups before and one good example is Seifert et al, PNAS, 2015 where IgM memory B cells proliferated significantly more to BCR stimulation than naive or IgG memory B cells. However, it is also shown that IgG memory B cells are more responsive to TLR9 stimulation than IgM memory B cells as demonstrated by e.g. Marasco et al, Eur J Immunol, 2017. This is not discussed by the authors and should be added into the discussion for context of their finding by scRNAseq methods. The notion that a memory transcriptional program can be induced without SHM is not novel and this should be brought up in the discussion. One paper showing a memory transcriptional program in unmutated memory B cells is Kibler et al, Front Immunol, 2022.
The observation that memory B cells are more likely to enter an inflammatory state and support T cells has been suggested by other groups (Seifert et al, PNAS, 2015; Magri et al, Immunity, 2017, Grimsholm et al, Cell Reports, 2020). Please provide the age distribution of the peripheral blood samples as well.
Please show flow cytometry analysis of the cultures to assist in assessing subset distribution, viability and plasma cell differentiation for each time point. This can be provided as supplementary information.
The stimulation cocktail used for this study, what does it contain? This need to be specified in the manuscript and not only referring to the manufacturer. This has major impact on the results since different stimulatory agents will induce different pathways.
Minor comments:
Please avoid the term plasma B cells, does it refer to plasmablasts and/or plasma cells?
Referees cross-commenting
Comments on reviewer 1 report: The reviewer raised the same significant objection to the manuscript as it stands today i.e. the content of the stimulation cocktail is not described. Without this information the manuscript is hard to put into the perspective of the existing literature.
Significance
See above comments.
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Referee #1
Evidence, reproducibility and clarity
Summary
Swift M. et al. explored how gene expression responds to extracellular stimuli by dissecting the transcriptional programs that underlie cell-intrinsic clonal fate bias. They also verified how extrinsic signals, intrinsic state, and clonal population structure interacted to determine the dynamics of the B cell immune response. To address these questions, lineage and single-cell RNA sequencing measurements were performed in human B cells stimulated in vitro through an undisclosed protocol. The B cell receptor (BCR) gene was used as a genetic lineage marker and this information was paired with that gained from transcriptomics, which provided an additional readout of cellular identity. Authors concluded that B cells have intrinsic biases towards specific cell fates and identified specific transcriptional memory states.
Major comments
While this is an elegantly presented manuscript that presents a wealth of data obtained from state-of-the-art transcriptomics and bioinformatics, some issues considerably weaken the main conclusions. These weaknesses entail an almost complete absence of details as to the stimuli being used for the in vitro activation of B cells (e.g., were they exposed to IL-4, TGF-beta and/or CD40L?). In the absence of this information, the discussion of intrinsic biases towards specific cell fates cannot be fully interpreted and understood, as different stimuli induce different B cell fates. In addition, there seems to be some misunderstanding about IgE class switching and somatic hypermutation, which differ in healthy and allergic individuals. Also, the novelty of this work is reduced by the fact that some of its conclusions are either largely expected or already known from studies published previously. Altogether, these issues make some statements and conclusions confusing and, sometimes, misleading. The following additional major comments are provided.
- Which B cell activation protocol was used? No information is provided in the main text or supplementary information. Yet, this information is key to fully understand many of the conclusions of this work (e.g., ... memory B cells are intrinsically two-fold more persistent in vitro (2A)), which largely depend on the nature of the stimuli used in the in vitro B cell culture.
- It would be informative to use more than one B cell activation program, e.g., CD40L with or without a cytokine as well as CD40L vs. CpG-DNA. Authors make broad statements about B cell fates without discussing the impact of a given signal on a given B cell fate. For instance, do memory B cells follow the same differentiation program upon stimulation with CD40L, IL-4 or a combination of CD40L and IL-4? How about differences between a TD signaling program such as that provided by CD40L and IL-10 and TI signaling program such as that provided by CpG-DNA and IL-10?
- Page 3, first line: After low quality and non-B cells (Fig S1A & B). What does this statement mean? The sentence seems incomplete.
- First, we noted that non-B cells present in the input rapidly became undetectable by day 4, which shows the specificity of the cytokines for B cell expansion. Which cytokines are we talking about? No detail is provided.
- Plasmablasts were not distinguished from plasma cells.
- Critically, we observed no appreciable evidence of hypermutation in vitro (S2C), consistent with prior literature (Bergthorsdottir et al. 2001). This statement is vague, misleading and likely inaccurate for the following reasons. (a) The B cell culture conditions used by the authors are completely unknown. (b) It was shown that SHM can be achieved under specific in vitro B cell culture conditions that include the presence of activated CD4+ T cells (PMID: 9052835; PMID: 10092799; PMID: 10878357; PMID: 12145648). Did authors try to recapitulate those culture conditions?
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Some of the reported findings are repetitive of previously published results and provide no additional new information. For example:
- a) "Interestingly, we found mutated B cells were far more likely to express genes involved in T cell interaction (2B), suggesting Memory B cells are intrinsically licensed to enter an inflammatory state which activates T cells". This evidence is already published (PMID: 7535180 among many other published studies).
- b) "Instead, Naive B cells were biased toward expressing lectins and CCR7, suggesting Naive B cells are intrinsically primed to home into the lymphatic system and germinal centers (2B)".
This evidence is already published (PMID: 9585422 among many other published studies). 8. We quantified the in vitro dynamics of CSR through the lens of mutation status, which revealed strongly different fate biases between germline and mutated cells (2D). Most strikingly, B cells which switched to IGHE were almost exclusively derived from germline progenitors: the ratio of germline IGHE cells to mutated IGHE cells was (8-fold - inf, 95 % CI). Also this evidence is not novel (PMID: 34050324 among other published studies) and, again, must reflect the presence of specific culture conditions that remain completely undisclosed. This is incredibly confusing. 9. Authors should mention that non-switched memory B cells include IgDlowIgM+CD27+ and IgD-IgM+CD27+ memory B cells. Some authors define these distinct memory B cell fractions as marginal zone (MZ) or MZ-like B cells (please, notice that splenic MZ B cells recirculate in humans) and IgM-only B cells, respectively (PMID: 28709802; PMID: 9028952; PMID: 10820234; PMID: 11158612; PMID: 26355154; PMID: 15191950; and PMID: 24733829 among many other published studies). 10. Thus, CSR from IGHM cells did not meaningfully contribute to the abundance of IGHA+ cells in the population. Also this conclusion may be misleading and/or inaccurate. Indeed, an efficient class switching to IgA requires the exposure of naïve B cells to the cytokine TFG-beta in addition to a robust TD (CD40L) or TI (CpG DNA or BAFF or APRIL) co-signal. Was TGF-beta present in this culture? 11. In contrast, we noted that many intraclonal class-switching events appeared to be directly from IGHM to IGHE. Explanations involving unobserved cells with intermediate isotypes notwithstanding, these data illustrate the relative ease with which B cells can switch directly to IGHE. It is very difficult to interpret this statement, as no information regarding the B cell-stimulating conditions used is provided. In addition, relevant literature is not quoted (e.g., PMID: 34050324). 12. Our data for IGHE cells contrasts with in vivo data which show IgE B cells to be: (1) very rare, (2) apparently derived from sequential switching (e.g. from IgG1 to IgE) (Horns et al., 2016; Looney et al., 2016), and (3) often heavily hypermutated (Croote et al., 2018). While this reviewer agrees with the first comment (switching to IgE is relatively rare in vivo, at least in healthy individuals), the other statements are quite inaccurate. Indeed, unmutated extrafollicular naïve B cells from tonsils and possibly other mucosal districts directly class switch from IgM to IgE in healthy individuals, thereby generating a low-affinity IgE repertoire. In principle, low-affinity IgE antibodies may protect against allergy by competing with high-affinity IgE specificities. In allergic patients, high-affinity IgE clones emerge from class-switched and hypermutated memory B cells that sequentially switch from IgG1 or IgA1 to IgE as a result of specific environmental conditions, including an altered skin barrier (PMID: 22249450; PMID: 30814336; PMID: 32139586). Moreover, in contrast to what stated by authors, sequential IgG1/IgA1-to-IgE class switching mostly occurs in allergic patients but is less frequent in healthy individuals, where IgE specificities are less mutated (PMID: 30814336). Along the same lines, IgE is heavily mutated only in allergic individuals with significant molecular evidence of sequential IgG1/IgA1-to-IgE class switching (PMID: 30814336; PMID: 32139586). Overall, the data provided by Swift M. et al. are largely confirmatory of previously published evidence.
Taken together, these data suggest that while direct switching to IGHE from Naive progenitors is trivial in vitro, niche factors or intrinsic death programs efficiently limit their generation or lifetime in vivo." I cannot understand this conclusion, which seems to contradict earlier statements. 13. I am not sure I learned much regarding the "cell-intrinsic" fate bias and transcriptional memory of B cells after reading this elegantly presented but confusing and superficially discussed manuscript (please, see also comments 15-23).
Minor comments
- Figures 1B, S1C and S1D are not referred to in the text.
- 2B in the Text is 2D in the Figure.
- 2D in the Text is 2E in the Figure.
- Figure 2D seems to show only 10 genes. Please, clarify.
- 2E in the text is 2F in the Figure.
- Figure S3B is not indicated in the text.
- Figure 3E is not indicated in the text.
- Figure S4A is not indicated in the text.
- In some sections of the text, Figure panels are not sequentially discussed, which makes the text very difficult to follow.
Significance
NATURE AND SIGNIFICNCE OF THE ADVANCE
I could not grasp a significant advance in this study as provided.
COMPARE TO EXISTING PUBLISHED KNOWLEDGE
As detailed in my specific comments, many of the conclusions of this work are largely expected or already published. It is difficult to evaluate the substance and originality of other conclusions in manuscript as provided.
AUDIENCE
If adequately and extensively revised, this work could be interesting to a broad audience.
MY EXPERTISE
B cell biology; B cell subsets; regulation of antibody production, including class switching; signals skewing antibody responses to a specific isotype.
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Reply to the reviewers
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Reply to the Reviewers
I thank the Referees for their...
Referee #1
- The authors should provide more information when...
Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...
Response: We expanded the comparison
Minor comments:
- The text contains several...
Response: We added...
Referee #2
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Referee #3
Evidence, reproducibility and clarity
This study addresses the newly appreciated role of E-cadherin in tumor cell invasion. A strength of this study is their inducible RasV12 Drosophila model of transformation, which allows them to follow cell dissemination at the basal midgut. The authors demonstrate, convincingly, that depletion of E-cadherin in this model impairs cell dissemination. However, their efforts to demonstrate a role for two Ca2+ signaling pathways mediated by IP3R and Piezo channels are not convincing given the use of blunt and non-specific pharmacological tools, lack of Ca2+ monitoring, and generally descriptive observations.
A significant weakness in this study is the sole and unsubstantiated use of Arm (the Drosophila b-catenin ortholog) as a proxy for e-cadherin localization and abundance. This leads to unsubstantiated statements such as, "disassembly of adherens junctions rather than E-cad levels" is involved in dissemination of cells. The role of E-cad is complicated by the observation that both knockdown and overexpression of E-cad perturb cell dissemination in their model.
In Figure 4, the role of Piezo in Arm distribution is presented in confocal images of a few cells. Is this statistically significant? This experiment needs to be quantified with more cells to substantiate the claim that Arm signals redistribute from cell boundary to cytosol in Piezo and Calpain knockdowns. Piezo channels are non-selective for cations. This means that the results of the knockdown cannot be assigned to changes in cytosolic Ca2+. At the least, cytosolic Ca2+ levels should be monitored. The authors suggest that calpains work with Piezo through Ca2+ signaling on the basis of a single experiment in which calpain knockdown has a similar effect on Arm localization to Piezo knockout. This conclusion seems speculative and is not adequately supported.
Thapsigargin is a blunt tool that causes a large, non-physiological and irreversible increase in cytosolic Ca2+. The observation that high Ca2+, resulting from Tg treatment, mediates disassembly of cadherins junction is not new, and indeed has been known for at least 20 years. Therefore, the effect of Tg in causing relocation of Arm signals from the adherens junctions is not insightful.
Use of GdCl3 is a non-specific inhibitor of many ion channels, including voltage gated Ca2+ channels, TRP channels, leak K+ channels, etc. It cannot be used to infer the role of Piezo when used as described in this study. The observation that Tg + GdCl3 somewhat preserves basal/junction ratios of Arm, relative to Tg alone, is interesting but uninterpretable without additional experiments, including measurements of cytoplasmic Ca2+ levels. None of the findings related to Figure 5 are mechanistically insightful and the imputed role of Piezo is not convincing.
Significance
Overall, although this study addresses a topic of high significance and interest, the limitations in experimental approach do not allow mechanistic insights that advance our understanding of the role of E-cadherin in tumor invasion.
Referee Cross-commenting
Dear fellow reviewers,
My expertise is in calcium signaling and ion transporters/channels. Although I was excited to review this work based on the abstract, I do not think that the link to calcium signaling (figs 4-5) is insightful or mechanistic, as I explain in my review. I realize that experimental approaches may be limited by the fly model, but the conclusions made by the authors would not be persuasive in other models.
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Referee #2
Evidence, reproducibility and clarity
In this study, Cabrera et. al. examine the role of DE-cadherin in cell dissemination, using a model of RasV12-transformed cells in the Drosophila hindgut. They show that E-cad relocates in the transformed cells from cell-cell junctions to basal invasive protrusions, and this relocation depends on calcium release from the ER by the PLC-IP3R-CAMK pathway. DE-cad is not required for the formation of actin-rich protrusions. However, upon RNAi-mediated DE-cad depletion (or depletion of the PLC-IP3R pathway), the basement membrane is not degraded and cell dissemination is inhibited. Furthermore, piezo and calpain are also shown to be required for DE-cad to assemble at invasive protrusions.
Major comments:
The data in figures 1-3 is overall convincing, although some of the points are missing quantification. The data supporting a role for piezo in regulating DE-cad assembly at adherens junctions is weaker. The data derived from ex-vivo pharmacological treatments (figure 5) is difficult to interpret or draw conclusions from.
Specific comments:
- The data on DE-cad overexpression is puzzling. When DE-Cad is overexpressed in Ras-transformed cells it relocates from AJ to invadopodia and the ECM is degraded just like in Ras-transformed cells, yet cell dissemination is reduced by 50%. Why? The authors don't offer any explanation and in the absence of one I don't see how the DE-cad overexpression data adds to the story.
- DE-cad is knocked down by RNA interference using two different sequences. Admittedly, I am not a fly person, but isn't there a way for fly geneticists to show how well the KD worked? What percentage of WT DE-cad is left? This same question can be applied to all the RNAi experiments.
- Arm redistribution and co-localization with F-actin should be quantified. While the "representative" images are of high quality and their presentation in XY and XZ or basal, middle, ortho views is very helpful, the data from multiple cells, from multiple flies, from at least 3 different experiments must be quantified to show by how much does the level of E-cad decrease at AJ and by how much does it increase at the basal protrusions. Also, the degree of co-localization of F-actin and E-cad at invadopodia should be quantified. Perhaps F-actin is not the best marker for invadopodia since it is also present in other structures.
- The data presented in Figure 3c-h also needs to be properly quantified for level of DE-cad at lateral sides of cells and on basal side from multiple cells in multiple animals and at least 3 experiments.
- The connection made between piezo and DE-cadherin (figure 4) is tenuous. Piezo is known (based on the authors previous work as well as others) to be upstream of invadopodia formation, so it is not surprising that E-cad does not localize to invadopodia when they don't exist. The authors do not provide any evidence to directly link piezo activity or calcium entry to DE-cadherin localization at invadopodia or at AJ and therefore their claim that "calcium signaling mediated by the Piezo-calpain pathway plays a distinct role in the DE-cad/Arm remodeling process" is purely speculative. The images of fuzzy DE-cadherin in figure 4b are hardly proof.
- The experiment in fig 5a,b showing that calcium release from the ER leads to AJ disassembly, nicely complements the in vivo data in fig 3c-h. However, the remaining of figure 5, dealing with piezo, is entirely unconvincing. The images in 5f don't resemble invadopodia and have nothing to do with them. The data in 5i regarding apical delamination is interesting, but does not support their point about piezo and AJ. They don't even show DE-cadherin.
Minor comments:
Have a figure "for the reviewer" should be avoided. The authors must write the paper and include diagrams or images so that any reader is able to understand the model system just as well as the reviewer.
Significance
In my assessment of significance I am only taking into account the conclusions that I feel are well supported. Depending on when the preprint they cite (ref. 52) will be published, this could be the first report of E-cad localizing in invadopodia and, importantly, being essential for ECM degradation and cell dissemination. Also of significance is the identification of intracellular calcium release as a signal for AJ disassembly and relocation of E-cad to invasive protrusions. While the molecular mechanism through which E-cad regulates ECM degradation is not elucidated, the paper still represents a significant advance for the fields of cancer cell biology and cell and developmental biology and would be of interest to a wide audience.
I am a cell and developmental biologist with expertise in cell adhesion and morphogenesis. I am not an expert on Drosophila.
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Referee #1
Evidence, reproducibility and clarity
Summary:
This interesting manuscript examines how intracellular calcium signaling and cation channels (piezo) may affect the remodelling of adherens junctions in tumor cells. The authors use the Drosophila midgut model to examine the dissemination of RasV12 cells. Authors show that at day 2 of Ras expression, DE-cadherin/b-catenin complex redistributes from adherens junction to basal invasive protrusions. Depletion of DE-cadherin attenuates the dissemination and local invasion of RasV12 cells, as manifest in damage to underlying visceral muscles. The authors attribute disassembly of cadherin from cell-cell junctions to IP3R-dependent release of calcium from ER stores and subsequent activation of CaMKs. Surprisingly, the authors found an apparently separate role for Piezo (potentially mediating calcium signaling) to support the assembly of DE-cad at the invasive protrusions.
Overall, this is a well-presented report that outlines the basic features of the phenomena with data of good quality. Over the years, there have been intermittent efforts to characterize the impact of calcium signaling on cell-cell junctions but much remains to be learnt. This manuscript is informative for focusing attention on a model of cadherin downregulation (and redistribution) in early tumors. However, it is somewhat descriptive and lacks depth of mechanistic analysis. For example, how might the cadherin in invasive protrusions be contribution to matrix degradation? What are the targets of either the IP3/CamK pathway or piezo/calpain signaling? Clearly, these foundational observations could be taken in many different directions. But some development would enrich the current package. Some suggestions follow.
Major comments:
- What is the role of E-cadherin in invasive protrusions? The authors use DE-cadherin depletion to support a role for this relocated pool of cadherin in tumor dissemination. However, this doesn't distinguish between a direct action of the cadherin complex at the protrusions from some indirect effect. Further analysis of the "protrusion" cadherin could be informative. For example, one possibility is that E-cadherin is essential to localise other proteins to the invasive protrusion. Authors have previously reported (Lee et al. 2020) that cortactin is essential for dissemination of the RasV12 cells. Considering that the phenotype of cortactin KD (presented in Lee et al. 2020) and DE-cad KD mostly overlap, and literature suggests that E-cadherin and cortactin colocalise and can influence each others' localisation in cultured cells (e.g. Ren et al. 2009; PMCID: PMC2707247). Could KD of DE-cad perturb cortactin localisation at the invasive protrusions? This could be doable in a reasonable time, as according to Lee et al. 2020 authors already have a relevant Drosophila strain, that lets visualisation of cortactin.
- Another route to explore in greater depth is the impact of DE-cadherin depletion on the protrusion. The authors show that upon DE-cad KD, some actin-rich puncta are still able to form at the basal surface of the Ras cells. This should be characterised in more details to assess whether DE-cad KD affects the number, size and dynamics of forming protrusions. At current stage, it's not obvious why DE-cad depleted Ras cells do not disseminate, even though they are able to form the invasive protrusions. As authors may potentially use movies/images they already have, this should be doable within reasonable time frames.
- Authors could expand the discussion section to address the issue of the unusual DE-cad localisation. The open, basic question that the authors haven't addressed at any stage is "what does the DE-cad bind to at the invasive protrusions?". Do authors speculate that DE-cad is able to interact with some components of ECM/VM (as would be suggested by the cartoon Fig S5)? Have authors considered that E-cadherin transinteractions forming eg. between small adjacent protrusions may serve to stabilise the initial structures and let them develop into more prominent invasive protrusions?
- Given the rapid intracellular diffusivity of calcium, it is interesting that two different sources of calcium have such different effects on the cadherin pool. Why does calcium released from ER by IP3R is enough to disassemble the cadherin complex at AJs, but signaling from Piezo is needed to regulate assembly of the cadherin complex at the invasive protrusions. Can authors comment on why calcium released by ER can't control both processes? Is it possible to visualize the dynamic subcellular distribution of calcium in these cells to test if there are different pools involved?
- Further, the discrepancy between the influence of the piezo1 KD and GdCl3 treatment on the localisation of the DE-cad/Arm is quite striking. As literature suggests an existence of a physical interaction between Piezo channel and E-cadherin, blocking Piezo vs depleting it may potentially affect E-cadherin differently. To address this, can authors e.g. add GdCl3 treatment (covering same timeframes as the Piezo KD) to data presented in Fig 4b (this would also eliminate the ambiguity caused by possible differences between in vivo vs ex vivo conditions - Fig 4 vs Fig 5). Also, is the phenotype of loss of junctional DE-cad/Arm upon Piezo KD limited to the Ras cells or does it also happen in GFP control cells?
- Is the observed phenotype (dissemination and involvement of DE-cad) limited to RasV12 oncogene? Do authors have any experience with other oncogenes (e.g. Src)? If an adequate Drospihila strain is available to authors, it would be worth confirming that the presented findings are not limited to only one oncogene.
- Finally, the paper feels like a small, follow up story that is hugely dependent on author's 2020 paper (Lee et al. 2020). It may be quite hard to follow if the reader is not familiar with the 2020 paper, as eg. the current paper does not describe what the "dissemination process" actually is, instead it directs the reader to the 2020 paper (similarly the figure for reviewers consists of images taken from the 2020 paper, rather than the current one).
Minor comments:
- How was junctional intensity of proteins quantified in samples that don't have obvious junctional staining (eg. Fig5b images and quantification for TG/ TG+GdCl3 in c and d or Image 5f and quantification in h). Do authors use an additional junctional marker to define where the junction is? If not, how is the position of junctions determined when the measured protein is dispersed from junctions?
- Is the calculation for IP3R-iGD1676 in Fig S3a correct? It doesn't match representative images presented in Fig3a or data in Fig3b, where IP3R-iGD1676 is not any different from other IR3R knock downs. If the quantification in Fig S3a is correct, the image presented in Fig3a is not representative of the majority of phenotype in this condition.
- Can delamination of Ras cells (Fig S1b and S3b) be quantified? It's quite challenging to assess the number of delaminated cells (especially in S3b).
- It would be good to quantitate the change in Arm (Fig 3d-h), in addition to showing representative images.
- Can authors describe the names used for labelling graphs in the figure captions? Some of the names used are not obvious for readers not already familiar with them eg. labels presented FigS3c - it's not obvious what norpA is, and the figure captions don't help at all.
- Is the antibody used for DE-cad able to distinguish between DE-cad and other cadherins (especially N-cadherin, which is commonly upregulated during EMT).
Significance
We believe that after addressing the above concerns, the paper may provide an informative advance to current knowledge about dissemination of early cancer cells leading to cancer metastasis. As authors mentioned, literature is not clear about involvement of E-cadherin in cancer metastasis. It was first suggested that loss of E-cadherin increases the metastatic potential of tumourigenic cells (Berx et al. 1995; Bogenrieder et al. 2003), however, more recent work indicated that the loss of E-cadherin, while increasing invasiveness, decreases metastatic potential, as well as cell proliferation and survival of circulating tumour cells (Padamanaban et al. 2019), indicating that the role of E-cadherin in cancer is much more complicated than previously appreciated. Thus, assessing the role of E-cadherin during cancer cell delamination in vivo may be a powerful and informative tool to bring some clarity to the cancer biology field.
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Reply to the reviewers
Reply to the reviewers
We sincerely thank the reviewers for their thoughtful and helpful input. Below were have included our response to the points raised by our reviewers.
*Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: Reichling et al. used flow-injection time-of-flight mass spectrometry (FIA-MS), a chromatography-free high-throughput method of untargeted metabolomics, to assess the temporary changes in the concentrations of polar metabolites within budding yeast exposed to the TORC1 (target of rapamycin complex 1) inhibitor rapamycin. The water-soluble metabolomes of the rapamycin-treated wild-type (WT) strain, many mutants in the non-essential genes lacking known protein components of the TORC1-upstream and -downstream signaling and numerous gene-deletion mutants missing the redundant small-molecule protein receptors that might participate in TORC1 signaling were analyzed by FIA-MS. The quality of the quantitative metabolome profiling performed by the FIA-MS method was validated with the help of a time-consuming liquid chromatography/mass spectrometry-based metabolomics of WT cells. *
* Using the FIA-MS-based metabolome profiling, the authors revealed that rapamycin treatment upregulates or downregulates the polar metabolomes specific to a distinct set of cellular processes. Similar patterns of the temporal dynamics of rapamycin-induced changes in the metabolomes characteristic of these cellular processes were observed in WT cells and mutants deficient in known protein components of TORC1 signaling. Remarkably, the authors found that three mutants (i.e., BCK1, CLA4 and CFF1) impaired in the small-molecule protein receptors that were unknown for their roles in TORC1 signaling exhibit the temporal dynamics of rapamycin-induced metabolome changes comparable to the mutants deficient in the known positive protein regulators of TORC1 signaling. Furthermore, the comparative metabolome-based analyses of relationships between rapamycin-treated cells defective in the many known and several unknown protein regulators of TORC1 signaling allowed authors to conclude that such analyses objectively reflect the functional connectivity of these protein regulators. Moreover, the authors provided evidence that comparing the metabolome profiles in rapamycin-treated WT and TORC1 signaling mutant cells enables the identification of new metabolic reactions and pathways affected by and/or contributing to the TORC1-dependent nutrient signaling network. *
* In this proof-of-principle study, the authors also employed the liquid chromatography-tandem mass spectrometry for a quantitative proteomic comparison of rapamycin-treated WT cells and mutants impaired in the known and previously unknown protein components of TORC1 signaling. They found that rapamycin induces comparable changes in the proteomes of all these cells. This proteomic analysis confirmed that the small-molecule protein receptors previously unknown as TORC1 signaling components and identified as such components only with the help of dynamic metabolome are integrated into the TORC1 signaling network. The authors further confirmed the essential contribution of the novel protein components of the TORC1 signaling to this type of nutrient signaling in the experiments on measuring growth rate changes following a nitrogen source upshift and assessing metabolome and proteome alterations after a nitrogen source downshift.
Comments: The manuscript is clearly written and of high technical quality. All claims are convincing, fully supported by the experimental data and appropriately discussed in the context of previous literature. The authors have been fair in their treatment of previous literature. They provided the methodological detail sufficient for others to reproduce the experiments. No additional experiments are needed to support the claims made in the manuscript. The experiments are adequately replicated and statistical analysis is appropriately performed. *
* Reviewer #1 (Significance (Required)):
This pioneering study represents a major conceptual advancement in the fields of using condition-specific, dynamic metabolome profiling for the deep understanding of relationships between genes integrated into a signaling pathway, discovering novel genes incorporated into a cellular signaling network and its small-molecule regulators, and identifying the metabolic pathways governed by a signal transduction network. *
* The treatment of the existing literature on the research topic by the manuscript's authors is balanced and fair. *
* This well-organized and clearly written manuscript is a must-read for anyone interested in the molecular mechanisms of cellular signaling. *
* My field of expertise involves exploring the molecular dynamics of complex cellular processes using advanced genetic, cell biological and biochemical approaches (including the mass spectrometry-based analyses of the cellular proteomes, lipidomes and water-soluble metabolomes). *
* I recommend accepting this manuscript for publication in any journal affiliated with Review Commons. **
*
We thank reviewer 1 heartily for their comments and for the time they spent reviewing our work.*
** Reviewer #2 (Evidence, reproducibility and clarity (Required)):
In this manuscript, Rechiling et al., have used a unique approach by exploiting a temporal dynamic high-throughput metabolome profiling (using flow-injection time-of-flight mass spectrometry) to measure the metabolome profiles of many mutants in yeast, which allow them to newly annotate 3 genes in the TOR signaling pathway. This work demonstrate elegantly that dynamic perturbation of the cell allows inferring gene function when using a metabolomics-based guilt-by-association scheme. They were able to successfully find genes like CFF1, BCK1 and CLA4 which might act as positive regulators in the TOR pathway. This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. Overall, the manuscript was concisely well written, and the finding has a great potential to improve our understanding of gene function and genetic determinism of metabolism in model organisms.
Major comments: As the cellular response to rapamycin is not restricted to changes at the level of the metabolome, the authors investigated the proteomic response of each mutant to reaffirm their functional relationship to the TOR pathway. In this regard, it is not clear why the author did not consider a time-course analysis of the proteome as they did for the metabolome. The measurable steady-state proteomic signature might also reflect a buffered cellular state which might hide other response.
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A time course analysis of the proteome for all mutants would be extremely interesting. However, due to the time and cost of proteomic analysis we instead focused on one time point for rapamycin treatment in the context of proteomics analysis. The proteome response data from mutants treated with rapamycin for 1 hour was used to identify the altered proteome responses of the mutants presented in figure 3A. This allowed us to explore the proteome responses of the mutants to rapamycin, and overcome the cellular buffering that our reviewer correctly points to.
* **Minor comments: Many typos in the methodology section (e.g., potassium phosphate ...) *
The typos in the methods section have been corrected.
* **Supplementary Fig 1: Not clear what this figure show and how it supports the author's claim that FIA-MS was validated by LC-MS. *
The positive linear relationship between the values for the measured metabolites by FIA-MS and LC-MS indicate that similar results were obtained with both methods. We have altered the text to make this clearer.*
**Reviewer #2 (Significance (Required)):
This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. The finding has a great potential to improve our understanding of gene function and genetic determinism of metabolic adaptation in model organisms. *
We thank reviewer 2 for their helpful comments, and the time they spent reviewing our work.
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, Rechiling et al., have used a unique approach by exploiting a temporal dynamic high-throughput metabolome profiling (using flow-injection time-of-flight mass spectrometry) to measure the metabolome profiles of many mutants in yeast, which allow them to newly annotate 3 genes in the TOR signaling pathway. This work demonstrate elegantly that dynamic perturbation of the cell allows inferring gene function when using a metabolomics-based guilt-by-association scheme. They were able to successfully find genes like CFF1, BCK1 and CLA4 which might act as positive regulators in the TOR pathway.
This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. Overall, the manuscript was concisely well written, and the finding has a great potential to improve our understanding of gene function and genetic determinism of metabolism in model organisms.
Major comments:
As the cellular response to rapamycin is not restricted to changes at the level of the metabolome, the authors investigated the proteomic response of each mutant to reaffirm their functional relationship to the TOR pathway. In this regard, it is not clear why the author did not consider a time-course analysis of the proteome as they did for the metabolome. The measurable steady-state proteomic signature might also reflect a buffered cellular state which might hide other response.
Minor comments:
Many typos in the methodology section (e.g., potassium phosphate ...) Supplementary Fig 1: Not clear what this figure show and how it supports the author's claim that FIA-MS was validated by LC-MS.
Significance
This an interesting study since it provides an alternative approach to annotate gene function and their contribution to known signaling pathways by analyzing the dynamic of the soluble metabolome. The finding has a great potential to improve our understanding of gene function and genetic determinism of metabolic adaptation in model organisms.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Reichling et al. used flow-injection time-of-flight mass spectrometry (FIA-MS), a chromatography-free high-throughput method of untargeted metabolomics, to assess the temporary changes in the concentrations of polar metabolites within budding yeast exposed to the TORC1 (target of rapamycin complex 1) inhibitor rapamycin. The water-soluble metabolomes of the rapamycin-treated wild-type (WT) strain, many mutants in the non-essential genes lacking known protein components of the TORC1-upstream and -downstream signaling and numerous gene-deletion mutants missing the redundant small-molecule protein receptors that might participate in TORC1 signaling were analyzed by FIA-MS. The quality of the quantitative metabolome profiling performed by the FIA-MS method was validated with the help of a time-consuming liquid chromatography/mass spectrometry-based metabolomics of WT cells.
Using the FIA-MS-based metabolome profiling, the authors revealed that rapamycin treatment upregulates or downregulates the polar metabolomes specific to a distinct set of cellular processes. Similar patterns of the temporal dynamics of rapamycin-induced changes in the metabolomes characteristic of these cellular processes were observed in WT cells and mutants deficient in known protein components of TORC1 signaling. Remarkably, the authors found that three mutants (i.e., BCK1, CLA4 and CFF1) impaired in the small-molecule protein receptors that were unknown for their roles in TORC1 signaling exhibit the temporal dynamics of rapamycin-induced metabolome changes comparable to the mutants deficient in the known positive protein regulators of TORC1 signaling. Furthermore, the comparative metabolome-based analyses of relationships between rapamycin-treated cells defective in the many known and several unknown protein regulators of TORC1 signaling allowed authors to conclude that such analyses objectively reflect the functional connectivity of these protein regulators. Moreover, the authors provided evidence that comparing the metabolome profiles in rapamycin-treated WT and TORC1 signaling mutant cells enables the identification of new metabolic reactions and pathways affected by and/or contributing to the TORC1-dependent nutrient signaling network.
In this proof-of-principle study, the authors also employed the liquid chromatography-tandem mass spectrometry for a quantitative proteomic comparison of rapamycin-treated WT cells and mutants impaired in the known and previously unknown protein components of TORC1 signaling. They found that rapamycin induces comparable changes in the proteomes of all these cells. This proteomic analysis confirmed that the small-molecule protein receptors previously unknown as TORC1 signaling components and identified as such components only with the help of dynamic metabolome are integrated into the TORC1 signaling network. The authors further confirmed the essential contribution of the novel protein components of the TORC1 signaling to this type of nutrient signaling in the experiments on measuring growth rate changes following a nitrogen source upshift and assessing metabolome and proteome alterations after a nitrogen source downshift.
Comments:
The manuscript is clearly written and of high technical quality. All claims are convincing, fully supported by the experimental data and appropriately discussed in the context of previous literature. The authors have been fair in their treatment of previous literature. They provided the methodological detail sufficient for others to reproduce the experiments. No additional experiments are needed to support the claims made in the manuscript. The experiments are adequately replicated and statistical analysis is appropriately performed.
Significance
This pioneering study represents a major conceptual advancement in the fields of using condition-specific, dynamic metabolome profiling for the deep understanding of relationships between genes integrated into a signaling pathway, discovering novel genes incorporated into a cellular signaling network and its small-molecule regulators, and identifying the metabolic pathways governed by a signal transduction network.
The treatment of the existing literature on the research topic by the manuscript's authors is balanced and fair.
This well-organized and clearly written manuscript is a must-read for anyone interested in the molecular mechanisms of cellular signaling.
My field of expertise involves exploring the molecular dynamics of complex cellular processes using advanced genetic, cell biological and biochemical approaches (including the mass spectrometry-based analyses of the cellular proteomes, lipidomes and water-soluble metabolomes).
I recommend accepting this manuscript for publication in any journal affiliated with Review Commons.
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Reply to the reviewers
Reviewer #1 CROSS-CONSULTATION COMMENTS
All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).
Response: In our fist submission, our full PDF file did not use the high-resolution figures due to large file size of some images. For re-submission, we uploaded our original, high-resolution figures. We also re-performed several immunostaining experiments and took confocal images to increase the quality of our figures. Please see below for detailed response.
Reviewer #1 (Evidence, reproducibility, and clarity (Required)):
Summary:
In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation.
The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.
Response: ____ To show the co-localization of multiple proteins, immunofluorescence staining is necessary. ____We added and described, in detail, how we performed macrophage quantification in the Methods section.
Reviewer #2 (Evidence, reproducibility, and clarity (Required)):
Summary:
Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.
Major comments:
Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).
Response: ____ As reviewer pointed out, the previous study only reported that Ngn3 expression and YAP1 loss occur in parallel within the same cells, and NGN3 can turn off Yap1 transcription in cell culture setting. However, our study showed that NGN3 alone is not sufficient to turn off Yap1 gene in vivo. Using our genetic model, we revealed that the Hippo pathway controling the nuclear localization of YAP1 is essential at the initiation of endocrine differentiation and allows YAP1 to be turned off at the transcriptional level. ____Our rescue model (NLTY mice) confirmed that the endocrine development defects observed in NL pancreas are caused by YAP1/TAZ, suggesting the necessity of controlling YAP1/TAZ by the Hippo pathway during endocrine development. Failure to sequester YAP1/TAZ outside of nuclei by the Hippo pathway in Ngn3-expressing endocrine progenitors halts the development of these cells.
Reviewer #2:
In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?
Response: ____ Our NL model (deletion of Lats1/2 in Ngn3-expressing cells) showed endocrine development defects, suggesting that Lats1/2 are essential for normal endocrine specification and differentiation. Removal of YAP1/TAZ in Lats1/2 null cells rescued the endocrine defects of NL pancreas. Genetic removal of YAP1/TAZ mimics Hippo pathway-mediated sequestration/degradation of YAP1/TAZ by LATS1/2. Uncontrolled YAP1/TAZ due to genetic removal of Lats1/2 blocked the endocrine specification and differentiation. This further demonstrates the necessity for functional Lats1/2 for inhibition of YAP1/TAZ in Ngn3-expressing cells during endocrine development. Ngn3CreYap1fl/flTazfl/fl mice have no endocrine defects, which is consistent with previous reports that Ngn3 expression and YAP1 loss occur in parallel within the same cells. Thus, we did not present this result.
Reviewer #2:
In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?
Response:____ YFP expression shows Cre activity which can be used as an indicator for Lats1/2 deletion. However, the expression level of YFP does not correlate with the expression level of Lats1/2 because YFP is controlled by the Rosa26 gene promoter. We did observe a higher level of YFP expression in endocrine cells compared with non-endocrine cells in NL pancreas. It is possible that the Rosa26 promoter is more active in endocrine cells. However, this is beyond the scope of our research.
Reviewer #2:
Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.
Response:____ In Figure 6, we intend to show that loss of YAP1/TAZ can rescue NL mice defects at P1 where Ngn3 expression is low. The NLTY pancreas showed much more endocrine cells compared to NL pancreas, suggesting that Lats1&2 functions to control YAP1/TAZ. Genetic ablation of YAP1/TAZ in Ngn3-expressing endocrine cells equals sequestering of YAP1/TAZ outside of nuclei by LATS1/2. Thus, we did not show expression of YFP, NGN3, and YAP1/TAZ in Figure 6A. Instead, we reperformed immunostaining for Figure 5B and took high-magnification confocal images to show expression pattern of NGN3 and YAP1 in endocrine progenitors.
Reviewer #2:
Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.
Response:____ At E12.5, the Hippo pathway plays important roles in pancreas development. See reference 11. Ngn3 peaks at E15.5 and only expresses in endocrine progenitors. We have published a review paper (ref. 7) “Wu Y, Aegerter P, Nipper M, Ramjit L, Liu J, Wang P. Hippo Signaling Pathway in Pancreas Development. Front Cell Dev Biol. 2021;9: 663906. doi:10.3389/fcell.2021.663906” as a detailed background for this manuscript, including pancreas development, up-to-date publication on Hippo pathway in pancreas development, and a model for YAP1 function in endocrine development. Thus, we did not include this background information in this manuscript.
Reviewer #2:
Minor comments: Please describe the affiliation of authors (number 3 and 4).
Response:____ It has been included in the manuscript: 3Department of Molecular Medicine, 4Department of Population Health Sciences.
Reviewer #2:
In table 1, the details of secondary antibodies were not described.
Response:____ We have added tables to show all primary and secondary antibodies used in the paper.
Reviewer #2 (Significance (Required)): Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.
Response:____ The primary aim of our study is to understand if the Hippo pathway plays an important function in endocrine pancreas development.
Reviewer #2:
Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.
Response:____ The defects in our NL (Lats1/2 null) pancreas in endocrine development strongly suggest the essential role of Hippo pathway in endocrine development. The null conditions of both Lats1/2 and Yap1/Taz (NLTY mice) mimic the effects of LATS1/2-mediated control of YAP1/TAZ in endocrine progenitors via genetic ablation rather than through the canonical Hippo kinase cascade. This further demonstrates the importance of proper Hippo signaling during endocrine development to inhibit YAP1/TAZ via the Hippo kinase cascade in Ngn3-expressing endocrine progenitors.
Reviewer #3 (Evidence, reproducibility, and clarity (Required)):
Summary: In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.
Major comments:
Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.
Response:____ We did not upload high resolution images for Bioxriv publication. We now uploaded the high resolution images. We also repeated a few immunostaining experiments and have taken confocal images to increase clarity.
Reviewer #3:
The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.
Response:____ As reviewer pointed out that there are technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells. We can only quantify gene expression level through RT-qPCR. We have discussed the high level of Krt19 and unchanged level of Sox9 in Figure 3B and 3C when we performed immunostaining. The results from the two experiments are consistent. We observed KRT19 positive staining but no SOX9 staining in Lats1/2 null Ngn3-expressing cells. This result is consistent with RT-qPCR result. We have postulated in the Discussion section that Krt19 may be directly controlled by YAP1.
Furthermore, Lats1/2 controls the protein level and localization of YAP1/TAZ, thus immunostaining can show the cellular localization.
In addition, we added the n’s used to perform RT-qPCR experiments to our manuscript and figures. We also added details to provide clarity on calculations made for relative mRNA expression from RT-qPCR experiments in the Methods section.
Reviewer #3:
The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.
Response:____ The increase of YAP1 staining is restricted to endocrine progenitor cells and blocks the endocrine differentiation. The endocrine cells in NL pancreas are escapers of Lats1/2 deletion and have no YAP1 expression, which is consistent with George et al. findings. The model we proposed is that in Ngn3-expressing cells, Lats1/2 are required to keep YAP1/TAZ out of nuclei so that Ngn3 can repress YAP1 expression. Loss of Lats1/2 led to high nuclear YAP1 which may block Ngn3’s ability to suppress YAP1. ChIP-seq or ChIP-PCR on YAP1 promoter will answer the question whether YAP1 auto-regulates on the transcriptional level. However, the small number of cells in mouse pancreas limits us to perform this experiment.
Reviewer #3:
The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.
Response:____ We added n’s to all figures and in our manuscript.
Reviewer #3:
The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.
Response:____ Reviewer is correct that the dataset of TEAD1 ChIP-seq from Cebola et al. was generated from human embryonic pancreatic progenitor cells. These data are in line with our mouse experimental results where cells with high YAP1, due to loss of Lats1&2, continue to have high YAP1, CDH1 and KRT19. We do not have evidence to point out the potential differences between mouse and human.
Reviewer #3:
Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context.
Response:____ We did not claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in NL pancreas. ISL1 and NKX2.2 are markers for endocrine differentiation. The lack of ISL1 and NKX2.2 expression indicates that endocrine differentiation has been blocked.
Reviewer #3:
P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.
__Response:____ No Sox9 expression was observed in Ngn3-expressing cells in both Control and NL pancreases (Figure 3B and 3C) while we observed YAP1 nuclei staining in NGN3-positive cells (Figure 5B), suggesting that YAP1 does not control Sox9 expression.
__
Reviewer #3:
Minor comments:
It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?
Response:____ We showed that there was no positive staining for ISL1 and NKX2.2 in progeny of Lats1&2 null Ngn3-expressing cells, suggesting the block of endocrine differentiation including all endocrine cells. The INS+ and GCG+ cells in NL pancreas are the escapers in which Lats1&2 were not deleted. Other endocrine cells should be affected too. We observed smaller sized NL mice with low blood glucose levels. We have postulated that brain expression of Ngn3Cre may contribute to these phenotypes.
Reviewer #3:
The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.
Response:____ We have added mRNA in the appropriate places where we discuss results from RT-qPCR experiments. All other places are protein expression results by immunostaining. We followed the general guideline for formatting gene and protein name throughout the manuscript: mouse gene symbols are italicized, with only the first letter in upper-case; protein symbols are not italicized, and all letters are in upper-case.
Reviewer #3:
Figure S1D is mis-referred to as S1E in the text. Figure 7G is missing. Table S1 is mis-referred to as S Table 2 in the text. Typo on page 19: "Controlcontrol" Figure S6B is mis-referred to as Figure S6A in the text.
Response:____ We have made all appropriate changes to the manuscript to correct these mistakes.
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Referee #3
Evidence, reproducibility and clarity
Summary
In this study, Wu et al. use murine Cre-lox model systems to demonstrate that the core Hippo pathway components, Lats1/2, promote pancreatic endocrine specification and differentiation through Yap1/Taz. The roles of the Hippo pathway in mammals are complicated and context-dependent. Prior studies have implicated the core kinase cascade of the Hippo pathway, containing MST1/2, LATS1/2 and YAP1/TAZ, in pancreatic cell lineage differentiation and morphogenesis. However, the necessity of the Hippo pathway in the development of the endocrine pancreas in vivo remains unsettled. This study by Wu et al. demonstrates that the Hippo pathway is essential for endocrine progenitor specification and differentiation but not for pancreatic beta cell function in mice. Their results are in line with prior studies, but some major issues ensue largely due to the lack of data quantification to substantiate the authors' claims, the use of ambiguous/imprecise terminologies, and making unsubstantiated claims.
Major comments:
- Immunofluorescence was used to evaluate the expression, co-localization, and subcellular localization of proteins of interest (Figures 2~7). However, except for the estimation of macrophage densities (4D and 6C), none of the other immunofluorescence experiments are accompanied by quantifications and statistical analyses to substantiate the authors' claims. In addition, the red channel in several figure panels (eg. 2A and 4A) was suboptimal making interpretations very difficult. Quantification of immunofluorescence data can be done by manual counting or automated counting using software such as ImageJ or QuPath, followed by statistical analyses to provide objective evidence.
- The endocrine component constitutes only a portion of the pancreas. The authors use whole pancreases to compare the expression levels of cell type-specific or Yap1 target genes between the knockout and control mice through qPCR. While there may be technical feasibility reasons limiting the direct assessments of gene expression in the endocrine progenitor cells, the caveats of the experiments (eg. inferring cell type-specific gene expression changes from whole organ) should be highlighted along with any inconsistent results. For example, among the three ductal genes tested in Figure 1C, only Krt19 has significantly higher mRNA expression in NL vs control, but the possible reasons for such discrepancy between ductal markers were not discussed. The authors used immunofluorescence, which is only semi-quantitative, to determine that Yap1 protein abundance is increased in Lats1&2 knockout cells. qPCR should also be performed for Yap1 in addition to Yap1 target genes to augment their claim of Yap1 expression being increased. More details on how the relative mRNA expression was computed is also necessary for readers to accurately interpret the results.
- The increased immunofluorescent detection of Yap1 in the NL pancreases at E16.5 is interesting but warrants further investigation. Is the increase restricted to endocrine progenitor cells or all endocrine compartments? George et al. (Mol Endocrinol. 2015) used RNA in situ hybridization and found that Yap1 mRNA expression is undetectable in the endocrine pancreas at E16.5, but here the authors observe increased Yap1 protein detected by immunofluorescence in the pancreases of animals with Lats1&2 knockout at E16.5. Although the authors speculate on a possible mechanism that may explain the discrepancies, it is important to evaluate whether the knockout really results in reactivation of Yap1 transcription and whether Yap1 auto-regulates on the transcriptional level.
- The numbers of animals used are unclear except for those depicted by the barplots. There is also no mention of the number of cells or fields analyzed for immunofluorescence experiments, which is essential for any quantitative comparisons and claims. The n's should be added throughout.
- The authors repeatedly refer to the dataset from Cebola et al. (Nat Cell Biol 2015), which was generated from human embryonic pancreatic progenitor cells, to speculate that TEAD1 binds to the promoter regions of YAP1, CDH1 and KRT19 and therefore may promote YAP1 autoregulation or expression of CDH1 and KRT19, explaining some of their immunofluorescence observations. However, they fail to acknowledge potential differences that may ensue due to the different species examined. Co-staining of Yap1 and Cdh1/Krt19 would indicate whether co-expression of Yap1 and Cdh1/Krt19 is indeed evident in the context of their study and provide further evidence to support their speculations.
- Several observations are over-interpreted or over-stated, and should be qualified as preliminary or speculative with proper wording. For example, a. P14: "differentiation...was blocked by lack of expression of ISL1 and NKX2.2". Although the authors observe low Isl1 and Nkx2.2 staining in NL vs control pancreases, no experiments were done to substantiate the claim that the reduction in ISL1 and NKX2.2 directly block the differentiation in this context. b. P15: "...KRT19 expression is not controlled by SOX9, but instead by YAP1". The authors observe that Krt19 proteins are increased in Lats1&2-null Ngn3+ cells, whereas Sox9 proteins were unchanged. However, they do not provide evidence that Yap1 controls Sox9 expression.
Minor comments:
- It is mentioned that deletion of Lats1&2 in Ngn3+ cells results in fewer acinar cells and smaller islets, evident by reduction in Ins+ or Gcg+ cells, whereas such genetic ablation in pancreatic beta cells does not result in any phenotype. Did the deletion of Lats1&2 in Ngn3+ cells similarly lead to reduction in other endocrine cell types?
- The authors use the word "expression" without specification to refer to both mRNA expression and marker fluorescence levels throughout the text. This is inaccurate and potentially confusing. More specific terminologies should instead be used to avoid ambiguity.
- Figure S1D is mis-referred to as S1E in the text.
- Figure 7G is missing.
- Table S1 is mis-referred to as S Table 2 in the text.
- Typo on page 19: "Controlcontrol"
- Figure S6B is mis-referred to as Figure S6A in the text.
Significance
The exact roles of the Hippo pathway in pancreas organogenesis are far from being delineated. The current study proposes a new model wherein the Hippo pathway kinase cascade acts to sequester and inhibit Yap1 in the cytosol for Ngn3 to drive endocrine specification, including inhibition of Yap1 expression. This work is potentially valuable to further our understanding of the physiological roles of the Hippo pathway in the context of endocrine pancreas development.
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Referee #2
Evidence, reproducibility and clarity
Summary:
Wu et al. examined the roles of Hippo pathway mediated YAP1/TAZ inhibition in the development stages of endocrine specification and differentiation in vitro and in vivo. This study concluded Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for endocrine specification and differentiation. The present study is conducted by solid experiment in some parts, however, there are several major concerns as follows.
Major comments:
- Authors concluded that proper Hippo activity was required for the Ngn3 driven differentiation program, further expanding our fundamental understanding of Hippo pathway participation in pancreatic endocrine development. This sentence is too vague. Previous study has already reported that Ngn3 expression and Yap loss occur in parallel within the same cell during development of the endocrine pancreas (Mol Endocrinol Baltim Md. 2015;29: 1594-607. doi:10.1210/me.2014-1375).
- In the present study, removal of YAP1/TAZ rescued the defect in endocrine specification and differentiation in LATS1/2-null pancreas. These results indicate that both of LATS1/2 and YAP/TAZ are not essential for the normal endocrine specification and differentiation. Furthermore, these results suggest that Hippo pathway-mediated YAP1/TAZ inhibition is also unnecessary for proper pancreatic endocrine specification and differentiation. Additionally, these results suggest that just a deletion of YAP/TAZ is sufficient for endocrine specification and differentiation. How is the endocrine specification and differentiation in Ngn3creyap1fl/flTazfl/fl mice?
- In Figure 2B and 3B, YFP expression (an indicator for LATS1/2 deletion) is more detectable in control compared to LATS1/2 null mice, suggesting that the LATS1/2 expression is more decreased in control mice. Is this true?
- Please show the expression of YFP, NGN3, and YAP/TAZ in figure 6A to confirm their expression status.
- Please present the sequential changes of LATS1/2, YAP/TAZ, NGN3 expressions in the control mice and LATS1/2 null mice at least E12.5, E16.5, and P1, to make ii easy to understand them for general readers.
Minor comments:
Please describe the affiliation of authors (number 3 and 4).
In table 1, the details of secondary antibodies were not described.
Significance
Previous studies have already reported that Ngn3 expression and Yap loss occur in parallel manner during development of the endocrine pancreas. The primary aim of the present study is whether the YAP loss is mediated by LATS1/2 in Ngn3 positive cells.
Furthermore, although authors concluded that Hippo pathway (LATS1/2)-mediated YAP1/TAZ inhibition is essential for proper pancreatic endocrine specification and differentiation, the null condition both of LATS1/2 and YAP/TAZ in Ngn3 positive cells provided the normal endocrine specification and differentiation (Figure 6). These findings do not support the conclusion.
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Referee #1
Evidence, reproducibility and clarity
In this study, Wu et al. confirmed the prominent cross talk of the Hippo pathway in beta-cell development, namely of Ngn3-YAP and beta-cell maturation. Deletion of the Hippo kinases Lats1 and 2 in Ngn3-expressing endocrine progenitor cells activated YAP1/TAZ transcriptional activity and reduced islets size and morphology and induced pancreas inflammation. This is in line with the previously established role of YAP/TAZ as regulator of viral response pathways In contrast, deletion of Lats1&2 later in development had no effect on beta-cell function and morphology. The authors conclude that Hippo pathway-mediated YAP1/TAZ inhibition in endocrine progenitors is a prerequisite for beta-cell maturation.
While the effect of Lats1 and 2 in beta-cell development has never been investigated, the outcome of the study is largely confirmative; namely YAP's necessity to switch off whenever an endocrine cell is formed while it also balances inflammation. The study depends on cell-specific Lats1 and 2-KO mouse models and in-vitro assessments of mechanisms how loss of LATS leads to beta-cell derangement at the time of ngn3 expression and how the inflammatory pathway is activated are missing. Observations are mostly based on stainings of quite low quality and it is unclear how authors performed quantitative evaluations and how many cells from how many mice were counted. Figures show high background, e.g. CD45 in Fig.3 which would make a robust evaluation impossible. Also YAP staining, usually well-expressed in ductal cells, shows low quality and high background. Usually the antibody is well-know for its weak performance in fluorescence and should only be used with chromogenic labels.
Referees cross-commenting
All reviewers have similar doubts on quality and quantification of the IHC analyses, so I would agree to give a chance to the authors for a revision, and it is probably doable in between 3 and 6 months (I wrote >6 before).
Significance
Largely confirmative. Single model approach with relatively low quality of the IHC analyses.
My expertise: inflammation, beta-cell, islets, Hippo pathway
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Reply to the reviewers
Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary: The authors compared the various multinucleated cells, osteoclasts, LCG and FBGC. Overall, the manuscript shows rigor in the analyses, and also very interesting approaches for retrieving mononuclear cells, for instance using DC-STAMP siRNA. This work adds very much to understanding the biological differences, as summarized in figure 6h. A lot of work in osteoclast field with for instance qPCR is hampered because, inevitably, a mix of mononuclear and multinucleated cells is always measured. Here, a solid attempt to separate those mixes with cell sorting and subsequent analysis on the mononuclear and multinucleated isolates, really adds. Choice of figures is good, also the extra info of the supplementary figures is relevant and makes it easy to read.
Major and minor concerns:
- For osteoclasts, various markers exist for their biological characterization, for instance the ability to resorb bone. What, apart from the arrangement and number of nuclei, were the biological parameters that confirmed that the cells made by addition of IFN or IL-4 were LCG and FBGC? [Authors’ reply]. In order to address this point, we focused on gene sets that characterize LCGs and FBGCs. By doing so, we aimed to identify (i) lineage dependent factors and (ii) markers of LGCs and FBGCs. (See new Supplementary Figure 1B and C, New Supplementary Table 1 and highlighted text in Results). As expected, and in line with the lineage-determining factors, the transcriptomics comparison between mononucleated/multinucleated IFN-γ and IL-4-differentiated macrophages showed predominance of IFN-γ and IL-4-related pathways, respectively (Supplementary Figure 1B and C and Supplementary Table 1). Among known LGC and FBGC markers, we confirmed up-regulation of CCL7 [1] and CD86 [2], respectively.* As per the biological parameters, we indeed confirm that FBGCs show enhanced phagocytosis properties (Figure 5C) while LGCs can form granuloma-like clusters in vitro (Figure 4D and E). Altogether, we characterize LGCs and FBGCs with (i) polykaryon-specific nuclear arrangement, (ii) polykaryon-specific gene expression markers, (iii) previously shown and new phenotypic characteristics such as LGCs’ unique ability to form in vitro clusters containing CD3+ cells. *
In fig 2c: did the authors perform stainings with isotype control antibodies? In my experience, quite often, antibodies stain mononuclear cells much intenser, since the cytoplasm is much more condense, less spread over a large area.
[Authors’ reply]. According to the reviewer’s suggestion, we provide isotype control staining for MRC1 in IFN-g-stimulated mononucleated/multinucleated cells by ImageStream (left panel) and immunofluorescence in LGCs, FBGCs and osteoclasts (right panel). There was negligible staining with the isotype control antibody for MRC1 in both settings (Figure provided to the journal).
*We did not observe a potential artefact of staining in multinucleated cells when compared to mononuclear cells. In fact, some markers of multinucleation such as B7-H3 is augmented in LGCs (Figure 4E). *
Resorption assay in 6 is not clear. It is weird that osteoclasts apparently display so limited resorption? Also the traces are not typical for osteoclasts. Please explain.
[Authors’ reply]. Human osteoclasts are cultured for 2 days on hydroxyapatite-coated plates and the amount of resorption is dependent on the healthy donor the peripheral blood is derived from. In addition to genetic variability, the support (hydroxyapatite) is different from dentine, which is also widely used for measuring osteoclast resorptive activity. The visualization of the human osteoclast resorption is made by transparency (area not coated by hydroxyapatite due to its resorption) on image J.
Provide a better image Supplementary 2A, even at 250% the lettering is vague. What do the colours in 2A mean?
[Authors’ reply]. *According to the reviewer’s suggestion, we now provide the Supplementary Figure 2A with better resolution. In STRING protein-protein interaction analysis, there is no particular meaning of the node color itself. *
CROSS-CONSULTATION COMMENTS
I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology!
[Authors’ reply]. We appreciate that the reviewer provides cross-consultation comments which we address in our revised manuscript. As such, we discuss future avenues regarding the translatability of these results to human pathology involving MGCs.
Reviewer #1 (Significance (Required)):
This manuscript is particularly interesting to those who are interested in the BIOLOGY of MNCs. In essence, three types of MNCs were cultured and compared, with each of them a specific function.
I am an osteoclast expert (76 publications), and have two publications on FBGCs
[Authors’ reply]. *We sincerely thank the reviewer for his/her pertinent comments, enthusiasm for our findings and for providing us an overall summary of our findings in view of all other reviewer comments. *
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary:
In this manuscript, the authors performed a comparative transcriptome analysis of mononuclear and multinuclear human osteoclasts, LGCs and FBGCs. They found that multinucleation triggers a significant downregulation of macrophage identity in all three types of MGCs. Furthermore, RNA-seq data and in-vitro functional analysis of multinucleated cells showed that macrophage cell-cell fusion and multinucleation enhance phagocytosis and contribute to lysosome-dependent intracellular iron homeostasis. Furthermore, multinucleation of osteoclasts promoted mitochondrial activity and oxidative phosphorylation, resulting in maximal respiration. This unique and interesting study addresses the fundamental question of how cell-cell fusion and multinucleation contribute to cellular activity and biological homeostasis.
Major comments
1 The authors generated mature multinucleated cells by stimulating human PBMC-derived macrophages with either IFN-g, IL-4, or RANKL. However, no quantitative data have been presented to determine how effectively IL-4, IFN-g, and RANKL can induce multinucleated giant cells from mononuclear macrophages. Quantitative data showing induction efficiency would provide a more detailed picture of the overall experiment.
[Authors’ reply]. According to the reviewer’s suggestion, quantitative data showing the efficiency of these cytokines to induce multinucleation (i.e. fusion index) is now provided as part of the revised Supplementary Figure 1A (right panel).
2 The authors mentioned, "The distinct morphological appearance of these three types of MGCs (Figure 1B) suggested cell type-specific functional properties and shared mechanisms underlying macrophage multinucleation". However, there is no discussion or data showing how the nuclear arrangement and intracellular location affect the biological function of multinucleated cells.
[Authors’ reply]. This is good point and is now discussed in the revised manuscript (see highlighted text in revised manuscript and below).
Whether MGC-specific nuclear arrangements and/or numbers are indicative of specialized function is currently unclear. Intracellular nuclei arrangement is likely to be important for the sealing zone formation in a polarized bone-resorbing osteoclast. Furthermore, whether distinct transcriptional activities are assigned to different nuclei of the MGC also remain to be tested. Recent elegant work performed in multinucleated skeletal myofibers suggest transcriptional heterogeneity among the different nuclei of the polykaryon [3].
3 Based on the results of DC-stamp knockdown experiments, the authors concluded that cell-cell fusion and multinucleation suppress the mononuclear phagocytic gene signature. However, to strengthen this hypothesis, it would be necessary to provide at least data showing that DC-stamp knockdown reduces the number of multinucleated cells.
[Authors’ reply]. According to the reviewer’s suggestion, we provide data showing that DCSTAMP knockdown reduces multinucleation in LGCs and FBGCs (see below and new Supplementary Figure 2F). For human osteoclasts, the data was included in our previously published paper ([4] and figure provided to the journal).
4 In Figure4, the authors showed that transcripts in LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.
[Authors’ reply]. We agree with the reviewer that our data on a potential role of LGCs’ on T cell activation is based on increased surface expression of B7-H3 and the unique CD3+ cluster forming ability of LGCs. In order to check for further markers of antigen presentation, we have performed MHC-1 and MHC-2 quantification by ImageStream in 3 types of MGCs (figure provided to the journal).
Although there was no difference in MHC-I/MHC-2 between the mononucleated and multinucleated macrophages, the mean fluorescent intensity (MFI) range was the highest in IFN-g-stimulated macrophages, suggesting that LGCs may be better equipped for antigen presentation than the other 2 types of MGCs. A more comprehensive analysis of antigen presentation requires enzymatic digestion and isolation and phenotyping of LGCs from clusters in vitro and human tissues in vivo. This is a program of research that we have initiated as part of a separate study, which will focus on the in vivo relevance of the current findings such as the unique Ag presentation ability of LGCs in a non-sterile tissue environment.
5 Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway?
[Authors’ reply]. This is a point worth expending (see also discussion; highlighted text). Osteoclast multinucleation is characterized by increased mitochondrial gene expression which also translates into increased spare respiratory capacity (SRC or maximal respiration). This metabolic rewiring does not modify glycolysis and basal respiration rate. As the reviewer correctly states, increased SRC may be a way to supply more ATP to the energy-demanding polykaryon.
As per the production of NAD(P)H as an electron source for ETC, it could indeed be through glutamine rather than glucose usage in multinucleated osteoclasts. Furthermore, as iron is an essential cofactor for ETC activity through activity of iron-sulfur clusters, the mitochondrial concentration of iron is likely to be critical for the mitochondrial activity of multinucleated osteoclasts (see also discussion).
Minor comments:
6 Supplementary tables 1-6 were not provided.
[Authors’ reply]. We apologize for this. The revised versions of supplementary tables are provided as part of the revised manuscript.
7 Figure 2D right panel, difficult to see DAPI+ nuclei.
[Authors’ reply]. Thanks for pointing this out. We have now replaced Figure 2D with a more pronounced DAPI+ nuclei.
Reviewer #2 (Significance (Required)):
Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.
[Authors’ reply]. We thank the reviewer for finding our study unique and biologically meaningful. We also thank the reviewer for all the suggestions that improved significantly the overall message of the manuscript.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variants - osteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below.
Minor comments:
- The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts. [Authors’ reply]. We thank the reviewer for this point and we agree it is important to highlight markers for each polykaryon early in the manuscript. In accordance with this reviewers’ comment (and also with Reviewer 1’s point), we first verified existence of lineage-dependent factors and markers of LGCs and FBGCs as these cells are relatively less well-defined compared to osteoclasts. (New Supplementary Figure 1B and C and New Supplementary Table 1). As expected, and in line with the lineage-determining effects, the transcriptomics comparison between mononucleated/multinucleated IFN-γ and IL-4-differentiated macrophages showed predominance of IFN-γ and IL-4-related pathways, respectively (New Supplementary Figure 1B and C and New Supplementary Table 1). Among known LGC and FBGC markers, we confirmed up-regulation of CCL7 [1] and CD86 [2], respectively (New Supplementary Table 1). We have added this information in the revised manuscript (see highlighted text). Osteoclast phenotyping is provided by TRAP staining and resorption assay (Figure 6C) and we also confirm that CTSK is indeed significantly up-regulated upon multinucleation (LogFc=1.69; P=9.2 x 10E-6; highlighted in the revised manuscript).
The overall decrease in phagocytic identity of all the MGCs, and the specific upregulation of phagocytic pathways in the FBGCs are conflicting. Are there subsets of phagocytic pathways that were down and upregulated during the formation of FBGCs?
[Authors’ reply]. This is a very good point. As the reviewer indicates, the results suggest that subsets of phagocytic pathways are changed upon multinucleation. All three types of MGCs show a downregulation of transcripts that belong to Fc receptors and complement C1Q family. However only FBGCs show an up-regulation of S. Aureus bioparticle-mediated phagocytosis. Hence the exact surface receptors responsible for this pathogen clearance remain to be identified. FBGC phagocytosis is a complex process including non-canonical phagocytosis pathways and participation of increased membrane area and endoplasmic reticulum [5, 6]*. Whether these pathways are specifically induced in human FBGCs remain to be identified. We now discuss this point in the revised manuscript (see highlighted text in Discussion). *
What are the identities of the mononuclear cells in each of the MGC experiment? They appeared to be quite heterogeneous based on the DEGs identified, beyond the common phagocyte signature. Can the authors comment on the difference between the mononuclear cells and whether this will affect the DEG analysis?
[Authors’ reply]. This is also a very relevant point that we now address in the revised manuscript (New Supplementary Figure 1B and C; New Supplementary Table 1 and highlighted revised text in Results). The reviewer is correct that MGC-specific pathways are in line with the known function of each polykaryon (Figure 4A, 5A and 6A). To what extent lineage-dependent effects (e.g. IFN-g and IL-4) are conserved between the mononucleated and multinucleated state is yet to be determined. In order to address this point, we compared DEG in IFN-g and IL-4-differentiated mononucleated macrophages to the ones obtained in multinucleated macrophages (New Supplementary Figure 1B and C; New Supplementary Table 1). The results showed that the multinucleated cell state preserves the majority of the lineage-dependent pathways which are very significantly represented at the mononucleated cell state (e.g. IFN-g and IL-4-related pathways). Interestingly, although less significant, this analysis also showed pathways that were specific to the mononucleated or multinucleated state in IFN-γ-differentiated macrophages when compared to IL-4-differentiated ones and vice versa. (Supplementary Figure 1B and C). For instance, TRAF3-dependent IRF activation pathway is specific to mononucleated IFN-g-differentiated macrophages (Supplementary Figure 1B).
The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study.
[Authors’ reply]. We thank the reviewer for this point and we now discuss our results from a clinical/diagnostic perspective (see highlighted text in the Discussion and below).
From a clinical perspective, since lysosome-regulated intracellular iron homeostasis appears to be a general condition for macrophage multinucleation across different tissues, its blockade may hold therapeutic potential. However, it is still unclear whether granulomatous disease can benefit from targeting LGC fusion. For non-granulomatous inflammatory diseases, inhibiting MGC formation by targeting lysosomes may be a therapeutic avenue. This approach would avoid FBGC-related adverse effects during foreign body reaction or inhibit the formation of MGCs of white adipose tissue during obesity. v-ATPase inhibitors have been previously proposed to inhibit osteoclast activity and bone resorption [7]* so their selective targeting in the lysosomal compartment may be generalized to other MGCs such as FBGCs. In addition to potential clinical translation, the results presented in this study require confirmation in tissues originating from human pathology involving MGCs. *
Major comments:
- As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues. [Authors’ reply]. This is an important point that we would like to explore in a comprehensive way. We have initiated a 2-year program to undertake a Multiplexed Immunohistochemistry (mIHC) using MILAN (Multiple Iterative Labeling by Antibody Neodeposition) https://www.lpcm.be/multiplex-ihc-milan approach in human biopsies using >100 antibodies. The current study is pivotal in selecting the gene targets (i.e. common and MGC-specific markers) for prioritization. We foresee to gain critical pathophysiological information about the tissue characteristics of MGCs. The reviewer would acknowledge that these high-throughput and biopsy-based initiatives are lengthy and not the primary scope of our current findings which set the foundation of major cellular events governing multinucleation in macrophages.
Reviewer #3 (Significance (Required)):
Significance:
- The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However, the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored. [Authors’ reply]. *We are grateful to Reviewer 3 for raising relevant and constructive points regarding the main findings. His/her review significantly improved the clarity of the overall manuscript. *
We recognize our study lacks human clinical association, but we highlight the prospective translatability of our findings and the usage of donor-based human macrophages throughout the manuscript. As also recommended by Reviewer 1 in his/her cross-consultation, we discuss the potential clinical impact of our findings in the Discussion of our revised manuscript.
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My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg LGCs and FBGCs is limited. References
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Chen Y, Jiang H, Xiong J, Shang J, Chen Z, Wu A, Wang H: Insight into the Molecular Characteristics of Langhans Giant Cell by Combination of Laser Capture Microdissection and RNA Sequencing. J Inflamm Res 2022, 15:621-634.
- McNally AK, Anderson JM: Foreign body-type multinucleated giant cells induced by interleukin-4 express select lymphocyte co-stimulatory molecules and are phenotypically distinct from osteoclasts and dendritic cells. Exp Mol Pathol 2011, 91(3):673-681.
- Petrany MJ, Swoboda CO, Sun C, Chetal K, Chen X, Weirauch MT, Salomonis N, Millay DP: Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers. Nat Commun 2020, 11(1):6374.
- Pereira M, Ko JH, Logan J, Protheroe H, Kim KB, Tan ALM, Croucher PI, Park KS, Rotival M, Petretto E et al: A trans-eQTL network regulates osteoclast multinucleation and bone mass. Elife 2020, 9.
- McNally AK, Anderson JM: Multinucleated giant cell formation exhibits features of phagocytosis with participation of the endoplasmic reticulum. Exp Mol Pathol 2005, 79(2):126-135.
- Milde R, Ritter J, Tennent GA, Loesch A, Martinez FO, Gordon S, Pepys MB, Verschoor A, Helming L: Multinucleated Giant Cells Are Specialized for Complement-Mediated Phagocytosis and Large Target Destruction. Cell Rep 2015, 13(9):1937-1948.
- Qin A, Cheng TS, Pavlos NJ, Lin Z, Dai KR, Zheng MH: V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption. Int J Biochem Cell Biol 2012, 44(9):1422-1435.
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Referee #3
Evidence, reproducibility and clarity
Summary:
The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variants - osteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below.
Minor comments:
• The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts.
• The overall decrease in phagocytic identity of all the MGCs, and the specific upregulation of phagocytic pathways in the FBGCs are conflicting. Are there subsets of phagocytic pathways that were down and upregulated during the formation of FBGCs?
• What are the identities of the mononuclear cells in each of the MGC experiment? They appeared to be quite heterogeneous based on the DEGs identified, beyond the common phagocyte signature. Can the authors comment on the difference between the mononuclear cells and whether this will affect the DEG analysis?
• The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study
Major comments:
• As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues.
Significance
Significance:
• The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored.
• My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg LGCs and FBGCs is limited.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors performed a comparative transcriptome analysis of mononuclear and multinuclear human osteoclasts, LGCs and FBGCs. They found that multinucleation triggers a significant downregulation of macrophage identity in all three types of MGCs. Furthermore, RNA-seq data and in-vitro functional analysis of multinucleated cells showed that macrophage cell-cell fusion and multinucleation enhance phagocytosis and contribute to lysosome-dependent intracellular iron homeostasis. Furthermore, multinucleation of osteoclasts promoted mitochondrial activity and oxidative phosphorylation, resulting in maximal respiration. This unique and interesting study addresses the fundamental question of how cell-cell fusion and multinucleation contribute to cellular activity and biological homeostasis.
Major comments:
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The authors generated mature multinucleated cells by stimulating human PBMC-derived macrophages with either IFN-g, IL-4, or RANKL. However, no quantitative data have been presented to determine how effectively IL-4, IFN-g, and RANKL can induce multinucleated giant cells from mononuclear macrophages. Quantitative data showing induction efficiency would provide a more detailed picture of the overall experiment.
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The authors mentioned, "The distinct morphological appearance of these three types of MGCs (Figure 1B) suggested cell type-specific functional properties and shared mechanisms underlying macrophage multinucleation". However, there is no discussion or data showing how the nuclear arrangement and intracellular location affect the biological function of multinucleated cells.
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Based on the results of DC-stamp knockdown experiments, the authors concluded that cell-cell fusion and multinucleation suppress the mononuclear phagocytic gene signature. However, to strengthen this hypothesis, it would be necessary to provide at least data showing that DC-stamp knockdown reduces the number of multinucleated cells.
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In Figure4, the authors showed that transcripts in LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.
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Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway?
Minor comments:
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Supplementary tables 1-6 were not provided.
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Figure 2D right panel, difficult to see DAPI+ nuclei.
Significance
Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.
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Referee #1
Evidence, reproducibility and clarity
Summary:
The authors compared the various multinucleated cells, osteoclasts, LCG and FBGC. Overall, the manuscript shows rigor in the analyses, and also very interesting approaches for retrieving mononuclear cells, for instance using DC-STAMP siRNA. This work adds very much to understanding the biological differences, as summarized in figure 6h. A lot of work in osteoclast field with for instance qPCR is hampered because, inevitably, a mix of mononuclear and multinucleated cells is always measured. Here, a solid attempt to separate those mixes with cell sorting and subsequent analysis on the mononuclear and multinucleated isolates, really adds. Choice of figures is good, also the extra info of the supplementary figures is relevant and makes it easy to read.
Major and minor concerns:
For osteoclasts, various markers exist for their biological characterization, for instance the ability to resorb bone. What, apart from the arrangement and number of nuclei, were the biological parameters that confirmed that the cells made by addition of IFN or IL-4 were LCG and FBGC? In fig 2c: did the authors perform stainings with isotype control antibodies? In my experience, quite often, antibodies stain mononuclear cells much intenser, since the cytoplasm is much more condense, less spread over a large area. Resorption assay in 6 is not clear. It is weird that osteoclasts apparently display so limited resorption? Also the traces are not typical for osteoclasts. Please explain. Provide a better image Supplementary 2A, even at 250% the lettering is vague. What do the colours in 2A mean?
CROSS-CONSULTATION COMMENTS
I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology!
Significance
This manuscript is particularly interesting to those who are interested in the BIOLOGY of MNCs. In essence, three types of MNCs were cultured and compared, with each of them a specific function.
I am an osteoclast expert (76 publications), and have two publications on FBGCs
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www.biorxiv.org www.biorxiv.org
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Reply to the reviewers
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.
Major comments: 1. Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications.
Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4.
Different regions should be included, at least as supplementary figures, to reinforce their conclusions.
__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states. We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.
Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically, heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol?
__Response: __The reviewer is correct that chromatin structure may alter accessibility which may bias absolute measurements but since the accessibility biases based to chromatin-structure are identical for both the histone PTM-specific antibody and the isotype control and cancel out in the enrichment score.
How biased is the cut&run sample compare to the ChIP-seq sample?
__Response: __We have included this in Supplementary Figure 1. The H3K4me3 and H3K9me3 enrichment scores are strongly correlated both between CUT&RUN replicates and between CUT&RUN and previously published ChIP-seq results.
In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions.
__Response: __We agree that this would be of interest to the reader. For this reason, the full genome-wide enrichment tracks were made available for all datasets in this study. We have added language to draw further attention to this availability.
Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.
__Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states, including 2 var genes (PF3D7_0808600 and PF3D7_0808700) and two rifin genes (PF3D7_0808800 and PF3D7_0808900). We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7, which also include subtelomeric and non-subtelomeric heterochromatin loci containing these genes. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.
- For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol.
__Response: __Since such biases are sequence dependent, they would impact raw coverage but cancel out in the enrichment plots since the sequence-based biases are identical in both samples. While PCR-free amplification may be desirable for some applications, we feel this is outside the scope of this study to implement these changes.
It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.
Response: As indicated by the title, this protocol was optimized specifically for profiling of histone modifications. CUT&RUN has been used in other systems to profile genome-wide binding of other proteins but this was not our aim and outside the scope of this study.
- The step by step protocol is very detailed, however there are some parts that need to be better explained:
In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL".
According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point?
Response: For this study we enriched for trophozoites using a percoll/sorbitol density gradient, which we have now clarified in step 8. However, whether and which enrichment strategy is employed will vary based on the desired parasite stages and experimental design.
In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization.
The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.
Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.
- With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.
Minor comments: - Abstract. A closing bracket is missing.
Response: Corrected - Step 11: Split each sample into 1mL aliquots at ?
Response: Corrected
- The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work
Response: Corrected
- Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77.
Response: The vendor and catalog number for the low-bind tubes are specified in the Reagents, Materials & Equipment list.
Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.
Response: Thank you for catching that error. The target was 10M read pairs per library but since CUT&RUN is so specific the isotype controls release less DNA and the resulting libraries produces fewer clusters than aimed for leading to slight over sequencing of the remaining samples.
- Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. Response: After excluding the telomere repeats 100 bp (50+50) are sufficient for uniquely mapping 98.3% of the nuclear. Paired-end sequencing was chosen over single-end because it provides the actual size of each fragment.
In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.
Response: Corrected
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Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why? Response: It is inherent in biological replicates that one would have the greatest amount of noise but we unfortunately have no further insight into why Sample 2 had a higher elevated background that Samples 1 and 3. Furthermore, even with this slightly higher background the relative enrichment of signal to noise ratio enrichment peaks are readily identifiable.
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Below some suggestions that may help the authors improve the presentation of their data and conclusions:
The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end.
Response: A “Limitations” section was added.
Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs).
Response: As mentioned above, we have not applied to CUT&RUN to profile chromatin other than Histone PTMs, as this was not the aim of our study. Since chromatin-bound histones always occur within a nucleosomal context, we are hesitant to make claims to the utility of this specific protocol for profiling DNA-binding proteins with smaller DNA-binding footprints. That said, CUT&RUN has been used to great success in other systems to profile a wide range of chromatin-bound proteins. We have included mention of this at the end of the introduction.
Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis.
Response: For the enrichment scores, sequence and accessibility biases cancel out since they are the same for both the PTM-specific antibody and the isotype controls.
The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures.
Response: Gene Expression Omnibus under accession number GSE210062 as indicated in the Quantification & Statistical Analysis and data availability sections.
Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here.
Response: As stated in the note following step 10, we did not optimize the minimum number of parasites required in this study since at the 1e7 iRBC required for each sample correspond as little as 1mL of bloodstage culture 2% parasitemia and 5% hematocrit. The down-sampling analysis in figure 3 suggests that the number of input cells can likely be reduced at least 10-fold.
Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.
Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.
Significance (Required) The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.
This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.
Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.
Reviewer #3 (Evidence, reproducibility and clarity (Required)): ____ In general the paper is very clear and convincing and I have only minor comments for the authors to address.
Introduction The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement? Response: Additional references (Baranello et al, O’Neill et al) were added.
In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.
Response: The reference was added.
Figure 2 A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.
Response: Thank you for the suggestion. We have made these changes.
Figure 3 An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.
Response: Correct, no HP1 CUT&RUN was performed, we are just trying to show that H3K9me3 CUT&RUN recapitulates ChIP-seq of both H3K9me3 and HP1, which binds to H3K9me3.
Figure 4 The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.
Response: The reason we included the down-sampling of CUT&RUN sequence reads was to explore whether were over-sequencing our CUT&RUN libraries not to provide a comparison to ChIP-seq. For simplicity we have therefore kept the figure as is.
DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?
Response: RNAse A is already present since it was included in the STOP buffer at step 35.
The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.
Response: TE buffer recipe was added.
Clean-up of PCR amplified library Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure. Response: Corrected
Typos and writing It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there. Response: It is defined in the 3rd paragraph of the introduction
I've mostly seen ChIP-seq with a dash between the IP and the seq.
Response: Corrected
Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.' Response: Corrected
Figure 2 legend. “(purple) in of three biological replicates” should be “(purple) in three biological replicates” Response: Corrected
Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'. Response: Corrected
Post-ligation Clean-up “Wash twice with 200µl of 80% Ethanol freshly prepared” should be “Wash twice with 200µl of freshly prepared 80% Ethanol” Response: Corrected
Library PCR amplification “Fragments are PCR amplified using Kapa polymerase, which it is more efficient” should be “Fragments are PCR amplified using Kapa polymerase, which is more efficient” Response: Corrected
Figure 7 legend “Indicated in the tope left panel” Should be “Indicated in the top left panel” Response: Corrected
Expected outcomes “to dismiss any sort of contamination” should be “To dismiss contamination” Response: Corrected
Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate. Response: Corrected
CROSS-CONSULTATION COMMENTS Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.
Reviewer #3 (Significance (Required)):
This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.
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Referee #3
Evidence, reproducibility and clarity
In general the paper is very clear and convincing and I have only minor comments for the authors to address.
Introduction
The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement?
In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.
Figure 2
A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.
Figure 3
An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.
Figure 4
The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.
DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?
The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.
Clean-up of PCR amplified library
Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure.
Typos and writing
It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there.
I've mostly seen ChIP-seq with a dash between the IP and the seq.
Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.'
Figure 2 legend.
(purple) in of three biological replicates
Should be
(purple) in three biological replicates
Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'.
Post-ligation Clean-up Wash twice with 200µl of 80% Ethanol freshly prepared
Should be
Wash twice with 200µl of freshly prepared 80% Ethanol
Library PCR amplification Fragments are PCR amplified using Kapa polymerase, which it is more efficient
Should be
Fragments are PCR amplified using Kapa polymerase, which is more efficient
Figure 7 legend Indicated in the tope left panel
Should be
Indicated in the top left panel
Expected outcomes to dismiss any sort of contamination
Should be
To dismiss contamination
Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate.
CROSS-CONSULTATION COMMENTS
Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.
Significance
This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.
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Referee #2
Evidence, reproducibility and clarity
This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.
Major comments:
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Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications. Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4. Different regions should be included, at least as supplementary figures, to reinforce their conclusions. Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol? How biased is the cut&run sample compare to the ChIP-seq sample? In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions. Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.
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For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol. It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.
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The step by step protocol is very detailed, however there are some parts that need to be better explained: In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL". According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point? In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization. The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.
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With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.
Minor comments:
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Abstract. A closing bracket is missing.
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Step 11: Split each sample into 1mL aliquots at ?
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The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work
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Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77. Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.
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Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.
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Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why?
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Below some suggestions that may help the authors improve the presentation of their data and conclusions: The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end. Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs). Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis. The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures. Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here. Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.
Significance
The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.
This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.
Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.
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Referee #1
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)): *In the manuscript " Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans" the authors show that a hormetic heat shock requires the peroxidase catalase ctl-2 for longevity and thermotolerance. Furthermore the authors characterize the hormetic stress response in ctl-2 mutants and show that ctl-2 is required for the proper formation of HSF-1 stress granules, and that ctl-2 mutants have a changed transcriptional response to heat shock and a changed induction of heat-stress induced oxidative stress. The authors go on to characterize peroxisomes, and peroxidase-regulated fatty acids, as well as mitochondria in both ctl-2 mutants and upon heat shock. The authors conclude that functional peroxisomes play an important role in the hormetic heat stress response.
This is an interesting study, that demonstrates that the loss of the peroxidase catalase ctl-2 plays an important role in the heat stress response. The authors need to provide more details on experimental repeats and experiments strengthening the conclusion that peroxisomes are generally important for the hormetic stress response would also improve this manuscript.
Furthermore, some textual changes would clarify some specifics in this manuscript.*
**Major issues:** *1. Throughout the manuscript it is unclear how many times the experiments were conducted (e.g. Lifespan, thermotolerance) or how many biological replicates were used (qRT-PCR, peroxisome characterization etc). This critical information should be included. A table with the individual lifespan experiments and thermotolerance experiments is expected. *
We are grateful to the Reviewer for drawing our attention to this and we have now included this information throughout the manuscript.
- The authors are characterizing the ctl-2 mutant upon heat stress and find some compelling differences to wild-type animals. The authors state "We measured the expression levels of two peroxisomal transport proteins, PRX-5 and PRX-11, and found no differences in expression between WT and Δctl-2 strains, suggesting that morphogenesis should not be impaired and that proteins needed for their proper function should be present, with the exception of ctl-2 in the Δctl-2 strain (Figure 3A)." The authors however conclude that functional peroxisomes are required for the benefits of a hormetic HS. What the authors are in fact demonstrating is that ctl-2 specifically is required for the hormetic HS response. To demonstrate that peroxisomes in general are required they should disrupt peroxisome function by additional means. What the authors also demonstrate is that heat shock has an effect on peroxisomes. These differences need to be clarified. It is also not fully clear whether peroxisomes are functional in ctl-2 mutants? Further peroxisome-relevant enzymes should be tested for levels and functionality in ctl-2 mutants and upon HS. *
This is correct and we have now addressed the above-mentioned issues throughout the manuscript. As the Reviewer noted, the peroxisomes are functional if monitored as the transcript levels of two peroxisomal transport proteins, implying that they have the potential to import their components. On the other hand, they are lacking the catalase, so they do not have a complete set of their components. Our phrasing in the original manuscript indeed was not optimal and we have now modified the conclusions, stating the peroxisomal catalase is important for the proper heat shock response.
Moreover, it is partially correct to say that a thorough explanation is missing as to what heat shock does to peroxisomes. We would like to emphasize that we measured the transcript levels of three enzymes from the very long chain fatty acid oxidation: straight-chain acyl-CoA oxidase, ACOX-1, MAO-C-like dehydratase domain protein, MAOC-1, and propanoyl-CoA C-acyltransferase, DAF-22. We have now measured the transcript levels of several other peroxisomal enzymes in the WT and the ctl-2 mutant under optimal conditions and heat shock and reported about it in the manuscript.
- The TORC1 experiments are too indirect. The authors should perform WesternBlots with a S6Kinase -Phospho antibody to determine whether TORC1 is inhibited or not. Alternatively, the authors may choose to remove this experiment from the manuscript without disrupting the main message of the manuscript, since no link between TORC1 and peroxisome function nor with HS has been established.
*
We are aware that the classical experiment to measure the activity of TORC1 is the Western Blot based assay to observe the phosphorylation of S6 kinase. However, we find the available antibodies not good enough to perform this experiment. We, therefore, conducted a different experiment to estimate the TORC1 activity, and it is the one described in our manuscript. We suggest that, as the phosphorylation of S6 is a downstream readout of TORC1 activity, so is the transcript level of the group of genes we measured, as reported previously (Das, Melo et al. 2017; Kenyon 2010; Kenyon 2011; Robida-Stubbs, Glover-Cutter et al. 2012; from the manuscript). While we are aware of the shortcomings of the assay we used, we find it valuable to report that the heat shock has an inhibitory effect on the TORC1 activity in WT C. elegans but not in the peroxisomal mutant. We would like to ask the Reviewer to reconsider this comment.
**Textual changes:** *1. The authors should adhere to C. elegans nomenclature convention (at least once), by referring to the ctl-2 deletion with the specific allele name [ctl-2(xx)] (unlike in yeast, ∆ is not commonly used). Furthermore a more thorough description of the specific allele (complete gene deletion? Point mutation? Truncation?) should be included. *
We have now taken care of this issue.
- Throughout the manuscript the authors should refer back to their specific hormetic heat shock paradigm, since several studies (also cited here) have shown differences in the specific physiological changes (e.g. HS on day 1 of adulthood has profound effects on broodsize, whereas the authors show here that a HS at L4 does not, other differences in terms of activation of other stress reporters has also been reported). *
Since our data regarding brood size were not put into context of the present study and were not studied in-depth, we have decided to remove these results from the present manuscript.
- The abbreviation OGT (original growth temperature??) should be defined *
OGT stands for optimal growth temperature and we have now defined it in the manuscript.
- The authors should change the sentence "The binding of HSF-1 to the HSEs can be visualized by a HSF-1::GFP fusion protein". This is not technically true. While HS has been shown to induce HSF-1 stress granules and these stress granules have been indirectly shown to correlate with transcription, it has however not been demonstrated that HSF-1 is bound to HSEs or whether it is bound to other DNA stretches or what is in fact transcribed by HSF-1 when it localizes to these foci. *
We are grateful to the Reviewer for this clarification, which we have included into the manuscript.
- The authors describe the morphological changes of peroxisomes upon HS, however they missed the opportunity to fully interpret these results in the discussion. Again, their conclusion that peroxisomes may be impaired in ctl-2 mutants is very vague. *
We agree with this comment and we have now taken care of this issue by introducing additional clarifications.
Reviewer #1 (Significance (Required)):
*Hormesis and the role of peroxisomes in stress responses is an important and interesting topic. *
The role of peroxisomes in stress responses has not been addressed.
*Researchers with interest in stress responses will be interested in this work.
My expertise lies in C.elegans stress and longevity with a specific focus on hormetic mechanisms.*
**Referee Cross-commenting**
While I agree with most comments from the other referees, I don't believe it is feasible to ask the authors to generate c. Elegans cell cultures for any follow up experiments. I would be satisfied with a more thorough comparison of the HS response between WT and Ctl-2 mutants, I.e: compare preconditiöning: 1h and 4h HS at L4 and d1 and then do thermotolerance experiments
We thank the Reviewer for this comment. We agree that to generate C. elegans cell cultures is not feasible. We have, however, performed the HS treatments in mammalian cells in culture.
HEK cells were transfected with Lipofectamine 3000 for 48h with either negative siRNA control (siCont), siPEX5 or siCAT at 100 nM, according to the manufacturer’s protocol. For HSR induction, cells were moved for 1h to a humidified incubator containing 5% CO2 at 42,5ºC, or maintained at 37ºC. After, RNA was immediately isolated with NucleoSpin RNA Columns for RNA purification (Macherey-Nagel), and 1000 ng of total RNA was converted to cDNA with iScriptTM cDNA Synthesis Kit (Biorad). Relative gene expression of PEX5, catalase (CAT), HSP90AA1, HSP70 (Hspa1b) and small HSPs (Hspb1, Hsph1 and Hspe1) were determined by qPCR, which were performed in a CFX Opus 384 Real-Time PCR System (Biorad) in a final volume of 5 µL using 2× SYBR Green PCR master mix and 0.3 μM of each primer pair. Relative gene expressions were calculated according to the Pfaffl method, with the results being normalized to the three most stable housekeeping genes in this experimental setting (HPRT, B2M and RPL7), as determined with NormFinder. The Ct averages of the four siCont samples at 37ºC were used as the calibrators to determine ΔCt for each gene. Data comparisons were conducted with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests and data is presented as mean ± SEM (Figures 1 and 2).
Although the siRNA-mediated silencing of CAT and PEX5 was successful (93% and 60% respectively), as determined by qPCR (Figure 1), the expression of several HSPs (HSP70, HSP90 and the small HSPs Hspb1, Hsph1 and Hspe1) remained similar following HS between either CAT- or PEX5- silenced cells and siCont-transfected cells (Figure 2). Preliminary data from SH-SY5Y cells treated as described above yielded similar results (data not shown). These observations may suggest that catalase and/or functional peroxisomes are not necessary for HSR induction and hormesis, but these experiments were limited to two cell lines and one form of HS (1h at 42,5ºC). Therefore, it is possible that different protocols may yet unravel a still undescribed link between peroxisomes, HSR, and life extension, but we to pursue this will take a long time and would be far outside the scope of this manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Summary:** In the manuscript entitled "Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans", Musa et al. explored the role of functional peroxisomes in the heat shock (HS)-induced hormesis in C. elegans using peroxisomal catalase ctl-2 deletion mutants. They showed that ctl-2 deletion abolished HS-induced longevity and suppressed the HS-induced upregulation of small heat shock proteins and HS-induced HSF-1 nuclear accumulation. Furthermore, the authors analyzed the differences in HS-induced phenotypes between wild type animals and ctl-2 mutants: the activation of the antioxidant response, the pentose phosphate pathway, and increased triglyceride content, which are the most prominent changes observed during heat shock in wild-type animals. However, there is only weak evidence supporting their claim that functional peroxisomes are required for HS-induced hormesis. * **Major comments:** 1. The authors conclude that the functional peroxisomes are essential for the HS-induced hormesis based only on one observation that the transient heat shock treatment did not increase the lifespan in the short-lived ctl-2 mutants. *
This is only partially correct. In addition to the absence of lifespan extension in the ctl-2 mutants, our conclusion was based also on the lower extent of the heat shock protein expression during HS in ctl-2 mutant. In the revision, we have reinforced this conclusion by adding the measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.
*Because the ctl-2 mutants are short-lived, the author should have carefully examined the relevant role of functional peroxisomes in hormesis response by conducting lifespan measurements using ctl-2 RNAi-treated animals or mutants deficient in other genes essential for peroxisome function. *
- *
We entirely agree with this comment. Since we are unable to perform such an elaborate study in this moment, and due to other similar comments from other Reviewers, we have decided to modify the conclusion, pointing out that it is the lack of the peroxisomal catalase that is associated to the lower extent of the heat shock response activation.
- The authors examined the difference between wild-type animals and ctl-2 mutants in many aspects; however, there is no rational explanation or examination that their observations have a role in HS-induced hormesis. *
We acknowledge that we are lacking the mechanistic insight into the reported phenomenon. This will be the focus of future studies, while in our present study, we have modified the conclusions to match the presented results.
- There is a severe shortage in the explanation of their experiments, especially for the methods of the experiments and statistical procedures. The authors should clarify the details of the experiments with proper statistical analyses. Follows are the points to be clarified for Figure 1 as insufficiency examples in the explanation in their study.
*
We have now taken care of this issue throughout the manuscript.
*Fig. 1A, B: Is this the representative survival curve of several experiments? The authors should clarify this point. The authors should include a table with the statistical numbers: the number of animals they used, the number of animals they measure in each experiment, and the p-value. *
- *
We have now clarified this issue in the manuscript.
*Fig. 1C: Is this a representative brood size of one animal, the mean brood size of some animals of one representative experiment, or the mean brood sizes from some experiments? The authors should clarify this point. Also, the authors should perform statistical analysis. *
We have removed the results related to the brood size from the present version of the manuscript.
*Fig. 1D-F: The authors should state the number of experiments they conducted. The author should perform multiple comparison statistical analyses instead of t-test without any correction. *
Done.
*Fig. 1H: Is this a representative plot of a representative experiment or the plot of the mean values from some experiments? The authors should clarify this point. The author should also clarify the tissue they analyzed and the exact number of the experiment (the number of nuclei in each animal they analyze, the number of animals they analyze in each experiment). The author should also clarify the detailed statistical procedure: one-way ANOVA or two-way ANOVA; which multiple comparison method they used in their analyses. *
We have now clarified all these issues.
**Minor comments:** *The author should explain the details for the experiments in either the Methods section or the figure legends. The authors should integrate the figures 6B-J (6B-D -> one figure, 6E-G -> one figure, and 6H-J -> one figure) into three figures because there are redundancies in these figures. In addition, the authors should perform the statistical analyses with multiple comparison procedures instead of a simple t-test. *
We have now done as suggested by the Reviewer.
*The authors should perform statistical analyses on Figs. 6N and 6O. *
Done.
*The authors would better cite relevant articles when referring to representative target genes of UPRER, UPRmt, and TORC1. *
Done.
Reviewer #2 (Significance (Required)): *Understanding the mechanism underlying the HS-induced hormesis in a multicellular organism is essential in the research field. Their finding that functional peroxisomes play a pivotal role in the HS-induced hormesis, if properly demonstrated, would provide us with the significant progress in this field; however, much more proper experiments are required to support their conclusion. Nevertheless, their finding can stimulate the attention of researchers who study the aging process, stress response, and especially peroxisome function.
I am an expertise in the study of aging in C. elegans. *
Reviewer #3 (Evidence, reproducibility and clarity (Required)): In this MS, the authors aim at the understanding of the role of peroxisome related-anti-oxidant capacity for the effect of a heat conditioning treatment on the HSR and associated longevity. Despite the finding that ctl-2 mutants they used show reduced resistance upon heat pre-conditioning, neither the mechanism (Ctl-2 is required for HSR) nor the claim that functional peroxisomes are required for heat shock-induced hormesis are, in my view, fully proven by the data in this MS. What the data basically show is that, related to a fragile status of the ctl-2 mutants, pre-conditioning was either to severe (toxic) or/and lead to development defects such that it was no longer effective in priming organismal resistance, likely to HSR-independent features. * **Major comments:** Figure 1: The level of pre-conditioning induced resistance (as opposed to intrinsic sensitivity) that can be induced in a given genetic background is dependent on a number of things, one of which is the severity of the priming dose. A more severe heat shock (that initially causes more damage) leads to a slower rate of tolerance development but the level of tolerance (of the surviving cells) is much higher. However, if too toxic, the priming treatment will result is loss of cells, which - at the organismal level- not or less reveal the resistance of the surviving primed and thus tolerant cells. *
Agreed.
Furthermore, such intrinsic sensitivity (un-primed) is determined by many more factors that only the capacity to induce the HSR. It is thus important to better evaluate the relevance of differences in intrinsic sensitivity of wildtype and ctl-2 mutants to the priming heat shock (figure 1F). Albeit interesting that Ctl-2 strains are hypersensitive to heat, this data also could imply that the real mechanism of being able to build up induced-resistance and longevity is not mechanistically due to an altered regulation of the HSR, but merely a reflection of that intrinsic difference in the sensitivity to the damage inflicted by the priming heat treatment. *
Also, as the treatment was given during the L4 stage, many of the effects may be blurred by differences in the sensitivity to heat treatment on developmental processes, conditions under which also many HSP are differently regulated (also HSF-1 independently). Whilst still interesting, this is even more complex to interpret mechanistically.
*
We chose the late L4 stage as opposed to adults since worms are mostly developed at L4, and have no embryos, which is a big advantage. The presence of embryos may confound the measurements, especially in the qPCR experiments since they may express HSPs due to their role in developmental processes as opposed to heat stress. It was also reported elsewhere that L4 stage is the most sensitive to HS in terms of HSR, while exhibiting higher survival rate than younger or older worms following HS. We have included this explanation in the manuscript.
*In fact, to conclude on the mechanistic involvement on peroxisome redox status for resistance inducting by heat priming, one of would require to e.g. derive cell lines from wildtype and ctl-2 mutant worms and perform an induced-thermotolerance / survival experiments (a iso-toxic and iso-dose heat priming treatments) to see whether ctl-2 truly have an impaired HSR due to cell autonomous features.
*
We are not able to establish cell lines from C. elegans, however, we have performed the suggested experiment in the mammalian cells in culture.
HEK cells were transfected with Lipofectamine 3000 for 48h with either negative siRNA control (siCont), siPEX5 or siCAT at 100 nM, according to the manufacturer’s protocol. For HSR induction, cells were moved for 1h to a humidified incubator containing 5% CO2 at 42,5ºC, or maintained at 37ºC. After, RNA was immediately isolated with NucleoSpin RNA Columns for RNA purification (Macherey-Nagel), and 1000 ng of total RNA was converted to cDNA with iScriptTM cDNA Synthesis Kit (Biorad). Relative gene expression of PEX5, catalase (CAT), HSP90AA1, HSP70 (Hspa1b) and small HSPs (Hspb1, Hsph1 and Hspe1) were determined by qPCR, which were performed in a CFX Opus 384 Real-Time PCR System (Biorad) in a final volume of 5 µL using 2× SYBR Green PCR master mix and 0.3 μM of each primer pair. Relative gene expressions were calculated according to the Pfaffl method, with the results being normalized to the three most stable housekeeping genes in this experimental setting (HPRT, B2M and RPL7), as determined with NormFinder. The Ct averages of the four siCont samples at 37ºC were used as the calibrators to determine ΔCt for each gene. Data comparisons were conducted with two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests and data is presented as mean ± SEM (Figures 1 and 2).
Although the siRNA-mediated silencing of CAT and PEX5 was successful (93% and 60% respectively), as determined by qPCR (Figure 1), the expression of several HSPs (HSP70, HSP90 and the small HSPs Hspb1, Hsph1 and Hspe1) remained similar following HS between either CAT- or PEX5- silenced cells and siCont-transfected cells (Figure 2). Preliminary data from SH-SY5Y cells treated as described above yielded similar results (data not shown). These observations may suggest that catalase and/or functional peroxisomes are not necessary for HSR induction and hormesis, but these experiments were limited to two cell lines and one form of HS (1h at 42,5ºC). Therefore, it is possible that different protocols may yet unravel a still undescribed link between peroxisomes, HSR, and life extension, but we to pursue this will take a long time and would be far outside the scope of this manuscript.
*Related to the actual data in figure 1A-c, also relative effects need to be taken into account as the Ctl-2 mutants are short lived. E.g., if one looks at maximum life span, the differences between the strains seem minimal (20/17 = 1,17 fold for wildtype and 15/13 = 1,15 fold for Ctl-2), so to conclude on no effect of heat pre-conditioning in ctl-2 strains seems an overstatement. *
This is not entirely correct. Usually, in lifespan measurements in C. elegans, we compare the median lifespan. For WT worms, we measured increased median and maximum lifespan; median lifespan was increased from 11 to 14 days post HS (≈20% increase), and maximum lifespan from 17 to 20 days (15% increase) (Figure 1A). In contrast, while maximum lifespan of the ctl-2(ua90)II strain after HS was increased from 13 to 15 days (≈13% increase), the median lifespan was unchanged and was 10 days for both HS and OGT ctl-2(ua90)II worms (Figure 1B). Overall, mild HS did not significantly affect ctl-2(ua90)II strain lifespan.
*Regarding to the brood size, it is not only true that these are smaller for clt-2 worms, but also that there was no effect of the pre-conditioning treatment in wildtype whereas a reduction was caused by the pre-conditioning of the ctl-2 worms. What does this imply?
*
Since the results of brood size were not sufficiently understood and out of context of the present study, we have removed them from the current version of the manuscript.
*Regarding the HSF/HSP data. First of all, a better visual insight in the quantitative differences in basal transcription levels between the strains should be provided. It looks as if they could be significantly lower for at least HSP16.1 in clt-2 strains. *
We have now included description of these results in the manuscript.
*Next, it would then be essential to evaluate the responses relative to these basal levels in the ctl-2 lines themselves (and not relative to that in wildtype animals). Second, looking at Hsp70, the HSP being most dependent of HSF1 upon a heat shock, the data imply that there is nothing wrong with the heat shock activated HFS-1 response in ctl-2 as such. As stated above, magnitude differences might also be a matter of kinetics, so measuring this at a single time point (4h after HS) may e.g. too early for being at its peak in ctl-2 cells. *
- *
We agree with this comment. Therefore, we have performed the experiment where we evaluated the kinetics of the heat shock response in the WT and the ctl-2 mutant worm. The measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant reveal that there are no differences in the kinetics between the strains, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.
*Third, it must be emphasized that HSF-1 foci/granules formation is a well-known feature of the response of HSF-1 to heat shock, but these granules are not the site of Hsp transcription, i.e they are not functionally related to HSP expression and not necessarily correlate quantitatively to HSR activation. So, the conclusion that HSF-1 activation/the HSR is truly attenuated in ctl-2 strains is -in my view- not fully proven. In fact, there is an intricate related between small HSP and oxidative stress and the lower (if correct) Hsp16.1 and Hsp16.2 expression could rather be related to such features.
*
We thank the Reviewer for drawing our attention to this issue, which we have now addressed in the manuscript by modifying the conclusion of a specific set of results related to the HSF-1 foci formation during heat shock. We have also discussed more extensively the connection with the oxidative stress.
*Figure 2: As for the HSR, kinetics may be different for wildtype and ctl-2 strains for all these endpoints, reflecting the higher intrinsic, non-primed heat sensitivity of the ctl-2 strain. Again, whilst interesting phenotypically and maybe relevant physiologically (i.e. being able to be primed as weak animal to show organismal resistance), this means that the data are elusive in terms of mechanisms.
*
We acknowledged from the beginning that we are missing mechanistic details of the presented result, and we therefore agree with this comment. As for the possible differences in the kinetics of the heat shock response activation in the WT and the ctl-2 worms: the measurements of the kinetics of the heat shock response activation in the WT and the ctl-2 mutant reveal that there are no differences in the kinetics between the strains, whereby the possible differences in the kinetics between the strains have been excluded as a possible interpretation. The results are now reported in the manuscript.
*In panel 2E, the lack of elevation in CellROX fluorescence by heat shock in wildtype cells in explained as due to the result in activation of the antioxidant defenses. Whereas this may sound OK, it is contradictory the reasoning given above that heat stress induced oxidative stress and hence cause G6PD upregulation (data panel 2D). In addition, whist the authors suggest they are the same, to me the CellROX data for ctl-2 strains appear to be lower (rather than, if anything, higher) for unstressed ctl-2 strains than wildtype strains. Is this not surprising given they are used as model for an impaired oxidation status? And does this not (also) indicate that the knockout lines have developed compensating strategies? Anyway: I got confused here.
*
First, there is no significant difference between the CellROX signal of the unstressed WT and the ctl-2 mutants. Why exactly the WT and ctl-2 mutant worms have the same CellROX signal is hard to say; the absence of the peroxisomal catalase could have triggered the activation of the cytosolic antioxidant defenses in optimal conditions already, or other compensatory strategies. It is also possible that the absence of the peroxisomal catalase has its consequences only in stressful conditions. In addition, our results during heat shock suggest that the WT strain is able to neutralize the ROS produced during heat shock, unlike the ctl-2 mutant. The increase in the G6PD expression may have helped with that, since it fed the activation of the pentose phosphate pathway. Our reasoning is that the ctl-2 mutant was for some reason not able to respond in the same way as the WT, and not that there was no need. The anti-ROS protection seems to have been insufficient during heat shock in the ctl-2 strain.
*Figure 3: First of all, it seems dangerous to conclude anything on peroxisome NUMBER here as what is measured is the presence of an imported FGP-tagged protein into peroxisomes and hence difference may be (also) due to import related effects. In fact, EM data would be required to make firmer conclusions on peroxisomal morphology, size and numbers. *
- *
In principle we agree with this comment, however, we demonstrate in Figure 3 that Prx-5 and Prx-11 expression levels do not display any differences in the WT and ctl-2 worms. Therefore, we remain confident about the analysis of the peroxisome number. We hesitate to perform an EM analysis due to the lack of specific markers; anything even slightly resembling a peroxisome could falsely be counted as one. In other words, every methodological approach comes with its own imperfections.
*In panel C, it is unclear what is significantly different from what; is the signal in clt-2 strains truly lower in ctl-2 strains that in wildtype strains? *
We thank the Reviewer for drawing our attention to this. In Figure 3C, for the significance analysis, everything was compared to the WT in optimal growth conditions. We have now clarified this in the text. The difference between the ctl-2 mutant and the WT is debatable: while the medians of the data sets are significantly different, this difference may not have any biological significance. Still, we reported the result of the statistical analysis.
*Figure 4 - 6: I sympathize with the comprehensive analysis presented in these figures. It is clear to me that different things are either (not) up or down in unprimed ctl-2 strains and that heat shock does or does not cause similar effects on these endpoints in wildtype and ctl-2 strains. Whilst this indeed shows that they do respond differently, I do not understand from these that what it all means and, in particular if and how it related causally or consequentially to the impaired priming effects (if true) of the heat shock in ctl-2 strains. *
The results presented in Figure 4-6 have not been put into a mechanistic context in the present study. In the mentioned figures, we do not report any causal or consequential relationships, but we do wish to report on the differential phenotypes of lipid metabolism and storage, as well as mitochondrial morphology in the WT and ctl-2 mutant worms. However, the presented analyses were performed well and we believe that these results may benefit to other researchers working in related topics.
**Minor additional comments (textual only)**
*Whereas this paper discusses the possibility of pre-heat conditioning to induce (long term) resistance, the generality of this as being a hormesis response (or general stress responses) related to other challenges is not warranted and all text concerning that should be deleted. Stresses damaging primarily DNA (ionizing radiation) or proteins & lipid (heat shock) are fundamentally different and each of them requires entirely different and largely independent systems to respond to. *
Done.
*Moreover, whilst the induced HSR is clearly an established hermetic response, this is still far less clear for e.g DNA damage responses. Therefore, it is also relevant define the types of stress to which is referred to e.g. when mentioning the envorinmental stimuli to which ctl-2 mutants are apparently hypersensitive. *
Done.
Also, the text related to cell non-autonomous response is irrelevant to this study and should be deleted. *
*
Done.
*Page 3 lines 1-4: It is incorrect to write that the role HSP has been put forward as most relevant to heat-induced hormesis. This is how they were discovered, but it is now clear that they play a role in many other pre-conditioning induced resistant phenotypes as well as in the cell-intrinsic sensitivity to proteotoxic stresses in general. Please also be aware that for unprimed, intrinsic resistance to e.g. heat shock, pre-existing levels of HSP are more relevant than the ability to activate the HSR. Activating the HSR is more relevant to resistance to more chronic temperature elevations and acquired resistance via the priming (hormesis).
*We thank the Reviewer for drawing our attention to this. We agree about the relevance of these points and have modified the manuscript accordingly.
Reviewer #3 (Significance (Required)): * Regulation of the cell intrinsic heat shock response is an important item to understand how cels may be primed to become resilient to (certain) other stresses. Besides the main studied regulator (HSF-1), many other levels of regulation likely exist and intra-organellar communication and proteostasis might be an important aspect for controlling such regulation.
As such, peroxisome proteostasis (that, unlike other organelles, are not (also) controlling a organellar unfolded protein response) is an interesting organelle that could co-control the cytosolic heat shock response. So, the aim of this study per se is quite interesting, However, although for evaluation of physiological relevance c. elegance is a good model of choice, for the mechanistic studies, cellular experiments would have been better suited. *
-
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Referee #3
Evidence, reproducibility and clarity
In this MS, the authors aim at the understanding of the role of peroxisome related-anti-oxidant capacity for the effect of a heat conditioning treatment on the HSR and associated longevity. Despite the finding that ctl-2 mutants they used show reduced resistance upon heat pre-conditioning, neither the mechanism (Ctl-2 is required for HSR) nor the claim that functional peroxisomes are required for heat shock-induced hormesis are, in my view, fully proven by the data in this MS. What the data basically show is that, related to a fragile status of the ctl-2 mutants, pre-conditioning was either to severe (toxic) or/and lead to development defects such that it was no longer effective in priming organismal resistance, likely to HSR-independent features.
Major comments:
Figure 1: The level of pre-conditioning induced resistance (as opposed to intrinsic sensitivity) that can be induced in a given genetic background is dependent on a number of things, one of which is the severity of the priming dose. A more severe heat shock (that initially causes more damage) leads to a slower rate of tolerance development but the level of tolerance (of the surviving cells) is much higher. However, if too toxic, the priming treatment will result is loss of cells, which - at the organismal level- not or less reveal the resistance of the surviving primed and thus tolerant cells.
Furthermore, such intrinsic sensitivity (un-primed) is determined by many more factors that only the capacity to induce the HSR. It is thus important to better evaluate the relevance of differences in intrinsic sensitivity of wildtype and ctl-2 mutants to the priming heat shock (figure 1F). Albeit interesting that Ctl-2 strains are hypersensitive to heat, this data also could imply that the real mechanism of being able to build up induced-resistance and longevity is not mechanistically due to an altered regulation of the HSR, but merely a reflection of that intrinsic difference in the sensitivity to the damage inflicted by the priming heat treatment.
Also, as the treatment was given during the L4 stage, many of the effects may be blurred by differences in the sensitivity to heat treatment on developmental processes, conditions under which also many HSP are differently regulated (also HSF-1 independently). Whilst still interesting, this is even more complex to interpret mechanistically.
In fact, to conclude on the mechanistic involvement on peroxisome redox status for resistance inducting by heat priming, one of would require to e.g. derive cell lines from wildtype and ctl-2 mutant worms and perform an induced-thermotolerance / survival experiments (a iso-toxic and iso-dose heat priming treatments) to see whether ctl-2 truly have an impaired HSR due to cell autonomous features.
Related to the actual data in figure 1A-c, also relative effects need to be taken into account as the Ctl-2 mutants are short lived. E.g., if one looks at maximum life span, the differences between the strains seem minimal (20/17 = 1,17 fold for wildtype and 15/13 = 1,15 fold for Ctl-2), so to conclude on no effect of heat pre-conditioning in ctl-2 strains seems an overstatement. Regarding to the brood size, it is not only true that these are smaller for clt-2 worms, but also that there was no effect of the pre-conditioning treatment in wildtype whereas a reduction was caused by the pre-conditioning of the ctl-2 worms. What does this imply?
Regarding the HSF/HSP data. First of all, a better visual insight in the quantitative differences in basal transcription levels between the strains should be provided. It looks as if they could be significantly lower for at least HSP16.1 in clt-2 strains. Next, it would then be essential to evaluate the responses relative to these basal levels in the ctl-2 lines themselves (and not relative to that in wildtype animals). Second, looking at Hsp70, the HSP being most dependent of HSF1 upon a heat shock, the data imply that there is nothing wrong with the heat shock activated HFS-1 response in stl-2 as such. As stated above, magnitude differences might also be a matter of kinetics, so measuring this at a single time point (4h after HS) may e.g. too early for being at its peak in ctl-2 cells. Third, it must be emphasized that HSF-1 foci/granules formation is a well-known feature of the response of HSF-1 to heat shock, but these granules are not the site of Hsp transcription, i.e they are not functionally related to HSP expression and not necessarily correlate quantitatively to HSR activation. So, the conclusion that HSF-1 activation/the HSR is truly attenuated in ctl-2 strains is -in my view- not fully proven. In fact, there is an intricate related between small HSP and oxidative stress and the lower (if correct) Hsp16.1 and Hsp16.2 expression could rather be related to such features.
Figure 2: As for the HSR, kinetics may be different for wildtype and ctl-2 strains for all these endpoints, reflecting the higher intrinsic, non-primed heat sensitivity of the ctl-2 strain. Again, whilst interesting phenotypically and maybe relevant physiologically (i.e. being able to be primed as weak animal to show organismal resistance), this means that the data are elusive in terms of mechanisms.
In panel 2E, the lack of elevation in CellROX fluorescence by heat shock in wildtype cells in explained as due to the result in activation of the antioxidant defenses. Whereas this may sound OK, it is contradictory the reasoning given above that heat stress induced oxidative stress and hence cause G6PD upregulation (data panel 2D). In addition, whist the authors suggest they are the same, to me the CellROX data for ctl-2 strains appear to be lower (rather than, if anything, higher) for unstressed ctl-2 strains than wildtype strains. Is this not surprising given the are used as model for an impaired oxidation status? And does this not (also) indicate that the knockout lines have developed compensating strategies? Anyway: I got confused here.
Figure 3: First of all, it seems dangerous to conclude anything on peroxisome NUMBER here as what is measured is the presence of an imported FGP-tagged protein into peroxisomes and hence difference may be (also) due to import related effects. In fact, EM data would be required to make firmer conclusions on peroxisomal morphology, size and numbers.
In panel C, it is unclear what is significantly different from what; is the signal in clt-2 strains truly lower in ctl-2 strains that in wildtype strains?
Figure 4 - 6: I sympathize with the comprehensive analysis presented in these figures. It is clear to me that different things are either (not) up or down in unprimed ctl-2 strains and that heat shock does or does not cause similar effects on these endpoints in wildtype and ctl-2 strains. Whilst this indeed shows that they do respond differently, I do not understand from these that what it all means and, in particular if and how it related causally or consequentially to the impaired priming effects (if true) of the heat shock in ctl-2 strains.
Minor additional comments (textual only)
Whereas this paper discusses the possibility of pre-heat conditioning to induce (long term) resistance, the generality of this as being a hormesis response (or general stress responses) related to other challenges is not warranted and all text concerning that should be deleted. Stresses damaging primarily DNA (ionizing radiation) or proteins & lipid (heat shock) are fundamentally different and each of them requires entirely different and largely independent systems to respond to.
Moreover, whilst the induced HSR is clearly an established hermetic response, this is still far less clear for e.g DNA damage responses. Therefore, it is also relevant define the types of stress to which is referred to e.g. when mentioning the envorinmental stimuli to which ctl-2 mutants are apparently hypersensitive.
Also, the text related to cell non-autonomous response is irrelevant to this study and should be deleted.
Page 3 lines 1-4: It is incorrect to write that the role HSP has been put forward as most relevant to heat-induced hormesis. This is how they were discovered, but it is now clear that they play a role in many other pre-conditioning induced resistant phenotypes as well as in the cell-intrinsic sensitivity to proteotoxic stresses in general. Please also be aware that for unprimed, intrinsic resistance to e.g. heat shock, pre-existing levels of HSP are more relevant than the ability to activate the HSR. Activating the HSR is more relevant to resistance to more chronic temperature elevations and acquired resistance via the priming (hormesis).
Significance
Regulation of the cell intrinsic heat shock response is an important item to understand how cels may be primed to become resilient to (certain) other stresses. Besides the main studied regulator (HSF-1), many other levels of regulation likely exist and intra-organellar communication and proteostasis might be an important aspect for controlling such regulation.
As such, peroxisome proteostasis (that, unlike other organelles, are not (also) controlling a organellar unfolded protein response) is an interesting organelle that could co-control the cytosolic heat shock response. So, the aim of this study per se is quite interesting, However, although for evaluation of physiological relevance c. elegance is a good model of choice, for the mechanistic studies, cellular experiments would have been better suited.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In the manuscript entitled "Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans", Musa et al. explored the role of functional peroxisomes in the heat shock (HS)-induced hormesis in C. elegans using peroxisomal catalase ctl-2 deletion mutants. They showed that ctl-2 deletion abolished HS-induced longevity and suppressed the HS-induced upregulation of small heat shock proteins and HS-induced HSF-1 nuclear accumulation. Furthermore, the authors analyzed the differences in HS-induced phenotypes between wild type animals and ctl-2 mutants: the activation of the antioxidant response, the pentose phosphate pathway, and increased triglyceride content, which are the most prominent changes observed during heat shock in wild-type animals. However, there is only weak evidence supporting their claim that functional peroxisomes are required for HS-induced hormesis.
Major comments:
- The authors conclude that the functional peroxisomes are essential for the HS-induced hormesis based only on one observation that the transient heat shock treatment did not increase the lifespan in the short-lived ctl-2 mutants. Because the ctl-2 mutants are short-lived, the author should have carefully examined the relevant role of functional peroxisomes in hormesis response by conducting lifespan measurements using ctl-2 RNAi-treated animals or mutants deficient in other genes essential for peroxisome function.
- The authors examined the difference between wild-type animals and ctl-2 mutants in many aspects; however, there is no rational explanation or examination that their observations have a role in HS-induced hormesis.
- There is a severe shortage in the explanation of their experiments, especially for the methods of the experiments and statistical procedures. The authors should clarify the details of the experiments with proper statistical analyses. Follows are the points to be clarified for Figure 1 as insufficiency examples in the explanation in their study.
<Points for Figure 1: they should also carefully consider the clarification on other figures> Fig. 1A, B: Is this the representative survival curve of several experiments? The authors should clarify this point. The authors should include a table with the statistical numbers: the number of animals they used, the number of animals they measure in each experiment, and the p-value. Fig. 1C: Is this a representative brood size of one animal, the mean brood size of some animals of one representative experiment, or the mean brood sizes from some experiments? The authors should clarify this point. Also, the authors should perform statistical analysis. Fig. 1D-F: The authors should state the number of experiments they conducted. The author should perform multiple comparison statistical analyses instead of t-test without any correction. Fig. 1H: Is this a representative plot of a representative experiment or the plot of the mean values from some experiments? The authors should clarify this point. The author should also clarify the tissue they analyzed and the exact number of the experiment (the number of nuclei in each animal they analyze, the number of animals they analyze in each experiment). The author should also clarify the detailed statistical procedure: one-way ANOVA or two-way ANOVA; which multiple comparison method they used in their analyses.
Minor comments:
The author should explain the details for the experiments in either the Methods section or the figure legends. The authors should integrate the figures 6B-J (6B-D -> one figure, 6E-G -> one figure, and 6H-J -> one figure) into three figures because there are redundancies in these figures. In addition, the authors should perform the statistical analyses with multiple comparison procedures instead of a simple t-test. The authors should perform statistical analyses on Figs. 6N and 6O. The authors would better cite relevant articles when referring to representative target genes of UPRER, UPRmt, and TORC1.
Significance
Understanding the mechanism underlying the HS-induced hormesis in a multicellular organism is essential in the research field. Their finding that functional peroxisomes play a pivotal role in the HS-induced hormesis, if properly demonstrated, would provide us with the significant progress in this field; however, much more proper experiments are required to support their conclusion. Nevertheless, their finding can stimulate the attention of researchers who study the aging process, stress response, and especially peroxisome function.
I am an expertise in the study of aging in C. elegans.
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Referee #1
Evidence, reproducibility and clarity
In the manuscript " Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans" the authors show that a hormetic heat shock requires the peroxidase catalase ctl-2 for longevity and thermotolerance. Furthermore the authors characterize the hormetic stress response in ctl-2 mutants and show that ctl-2 is required for the proper formation of HSF-1 stress granules, and that ctl-2 mutants have a changed transcriptional response to heat shock and a changed induction of heat-stress induced oxidative stress. The authors go on to characterize peroxisomes, and peroxidase-regulated fatty acids, as well as mitochondria in both ctl-2 mutants and upon heat shock. The authors conclude that functional peroxisomes play an important role in the hormetic heat stress response.
This is an interesting study, that demonstrates that the loss of the peroxidase catalase ctl-2 plays an important role in the heat stress response. The authors need to provide more details on experimental repeats and experiments strengthening the conclusion that peroxisomes are generally important for the hormetic stress response would also improve this manuscript.
Furthermore, some textual changes would clarify some specifics in this manuscript.
Major issues:
- Throughout the manuscript it is unclear how many times the experiments were conducted (e.g. Lifespan, thermotolerance) or how many biological replicates were used (qRT-PCR, peroxisome characterization etc). This critical information should be included. A table with the individual lifespan experiments and thermotolerance experiments is expected.
- The authors are characterizing the ctl-2 mutant upon heat stress and find some compelling differences to wild-type animals. The authors state "We measured the expression levels of two peroxisomal transport proteins, PRX-5 and PRX-11, and found no differences in expression between WT and Δctl-2 strains, suggesting that morphogenesis should not be impaired and that proteins needed for their proper function should be present, with the exception of ctl-2 in the Δctl-2 strain (Figure 3A)." The authors however conclude that functional peroxisomes are required for the benefits of a hormetic HS. What the authors are in fact demonstrating is that ctl-2 specifically is required for the hormetic HS response. To demonstrate that peroxisomes in general are required they should disrupt peroxisome function by additional means. What the authors also demonstrate is that heat shock has an effect on peroxisomes. These differences need to be clarified. It is also not fully clear whether peroxisomes are functional in ctl-2 mutants? Further peroxisome-relevant enzymes should be tested for levels and functionality in ctl-2 mutants and upon HS.
- The TORC1 experiments are too indirect. The authors should perform WesternBlots with a S6Kinase -Phospho antibody to determine whether TORC1 is inhibited or not. Alternatively, the authors may choose to remove this experiment from the manuscript without disrupting the main message of the manuscript, since no link between TORC1 and peroxisome function nor with HS has been established.
Textual changes:
- The authors should adhere to C. elegans nomenclature convention (at least once), by referring to the ctl-2 deletion with the specific allele name [ctl-2(xx)] (unlike in yeast, ∆ is not commonly used). Furthermore a more thorough description of the specific allele (complete gene deletion? Point mutation? Truncation?) should be included.
- Throughout the manuscript the authors should refer back to their specific hormetic heat shock paradigm, since several studies (also cited here) have shown differences in the specific physiological changes (e.g. HS on day 1 of adulthood has profound effects on broodsize, whereas the authors show here that a HS at L4 does not, other differences in terms of activation of other stress reporters has also been reported).
- The abbreviation OGT (original growth temperature??) should be defined
- The authors should change the sentence "The binding of HSF-1 to the HSEs can be visualized by a HSF-1::GFP fusion protein". This is not technically true. While HS has been shown to induce HSF-1 stress granules and these stress granules have been indirectly shown to correlate with transcription, it has however not been demonstrated that HSF-1 is bound to HSEs or whether it is bound to other DNA stretches or what is in fact transcribed by HSF-1 when it localizes to these foci.
- The authors describe the morphological changes of peroxisomes upon HS, however they missed the opportunity to fully interpret these results in the discussion. Again, their conclusion that peroxisomes may be impaired in ctl-2 mutants is very vague.
Significance
Hormesis and the role of peroxisomes in stress responses is an important and interesting topic.
The role of peroxisomes in stress responses has not been addressed.
Researchers with interest in stress responses will be interested in this work.
My expertise lies in C.elegans stress and longevity with a specific focus on hormetic mechanisms.
Referee Cross-commenting
While I agree with most comments from the other referees, I don't believe it is feasible to ask the authors to generate c. Elegans cell cultures for any follow up experiments. I would be satisfied with a more thorough comparison of the HS response between WT and Ctl-2 mutants, I.e: compare preconditiöning: 1h and 4h HS at L4 and d1 and then do thermotolerance experiments
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Reply to the reviewers
The authors do not wish to provide a response at this time. We aim to provide a revised version of the manuscript within two month.
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Referee #3
Evidence, reproducibility and clarity
In recent years, significant progress has been made in defining the molecular details of many structural features of the nuclear pore complex (NPC). However, one area that remains ill-defined is the interface between the core structures of the NPC and the pore membrane domain. This is an especially intriguing area when one considers that the NPC contains several integral proteins and numerous peripheral membrane proteins contain amphipathic helices whose functions and interactions with the membrane, as well as with one another, remain largely undefined.
In this manuscript by Amm et al., the authors have examined the functional role of the integral membrane Nup Ndc1 and its interactions with various peripheral membrane Nups, including members of the Nup84 complex (termed the Y-complex) and the linker Nups Nup53 and Nup59. The authors show that Ndc1 interacts with specific members of the Nup84 complex, namely Nup120 and Nup133, supporting the idea that Ndc1 functions, in part, to anchor this NPC substructure to the pore membrane. In addition, they identified an amphipathic helix (AH) within the C-terminal half of Ndc1, and they showed that it can directly bind to membranes. Importantly, they have used genetic assays to show that the Ndc1-AH functionally interacts with AHs present at the C-terminus of Nup53 and Nup59. Strikingly, they show that the lethal phenotype detected in strains lacking Ndc1 can be suppressed by the deletion of NUP53, but not NUP59, and, more specifically, only the loss of the C-terminal AH Nup53 was required to suppress the lethal phenotype of the ndc1 null mutation. Further ultrastructural analysis of these mutants revealed that, while these mutants were viable, they exhibited extensive NE expansion phenotypes.
Overall, the data presented in this manuscript are of high quality, and the experiments are well controlled. My specific comments are relatively minor and listed below.
Minor points
1) The authors state "Serial ultrathin sections of fixed yeast cells overexpressing ProtA-CtNdc1 revealed that these unusual extranuclear membrane proliferations exhibited pore-like structures with diameters similar to the diameter of NPCs within the nuclear membrane (Fig. 2C)." This is not entirely clear from the data. I suggest the authors provide direct measurements that support their statement.
2) The authors examined the total cellular lipid content following overexpression of Ndc1-AH-containing constructs, as well as ProtA-ScHmg1. There is little discussion of the significance of these results, which would provide a clear justification for including these data in the manuscript.
3) There are numerous typographical and grammatical errors throughout the manuscript that need to be addressed.
Significance
The results presented in this manuscript provide further insight into the molecular interactions between Nups and the pore membrane. They suggest that AHs present in a subset of Nups perform linked functions and contribute, in part, to nuclear membrane biogenesis. As such, these results are an important advance in our knowledge of NPC structure and function. They will be of general interest to those studying the function of NPCs and, more generally, NE and organelle biogenesis.
Reviewer expertise: NPC structure and function, NE biogenesis, yeast model system.
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Referee #2
Evidence, reproducibility and clarity
Amm et al report on the role of new motifs and interactions between the essential and conserved integral nuclear pore membrane protein Ndc1 and other key components of the yeast nuclear pore complex. They show that members of the Y-subcomplex that coats the pore membrane bind directly to Ndc1 and identify an amphipathic helix at the C-terminus of Ndc1 that displays genetic interactions with other nucleoporins carrying analogous amphipathic helices. The authors find that cells can survive without Ndc1 when these related amphipathic helices from other nups are coincidentally deleted.
Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.
Specific experimental issues and clarifications:
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A major part of the manuscript describes a detailed structure-function analysis of Ndc1. The link between the two domains of ScNdc1 studied and their effects on membrane proliferation could be better defined: specifically, can the authors exclude that the N-domain of Ndc1 that includes its transmembrane domain, is not also involved in the membrane proliferation phenotype shown in Fig2A and C? It also seems as if GAL-ProtA-ScNdc1 (1-260) also causes growth inhibition (Fig. 2F). How do cells with GAL-ProtA-ScNdc1 (1-260) look like? Finally, although the authors convincingly show that overexpression of 261-655 inhibits growth, from the EM it seems as its effects on membrane proliferation is not the same as that of the overexpression of full-length Ndc1 (compare Fig. 3D vs Fig. 2D).
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Figure 1A: Do the CtNups shown under "Input" represent 100% of what used in the binding reaction? If so, please indicate at the figure.
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CtNup120 and CtPom133 would migrate close to CtPom152, which could make visualization by Coomassie stain a bit tricky - if the authors could provide SDS PAGE gels with lower %, that would be helpful. Along similar lines, how do the authors know that CtNup120beta does not bind the CtNdc1 if these two appear to migrate at the same size (Fig. 1D)?
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Figure 1B, GUVs: Why do the authors use CtNup85 for the GUV experiment instead of CtNup84 that was used in Fig. 1A?
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Moreover, CtNup120 and CtNup133 ...BC08/SCL1 (Fig. 1C)" Don't see this in Fig. 1C
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The imaging of ProtA-AHNdc1-eGFP (Fig. 3C) is not great and the localization of the AH does not look very clear - can the authors provide better micrographs? Perhaps co-expression of a red ER reporter or similar reporter would also help.
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The ndc1 nup53 double mutant appears to display a striking cold-sensitive growth defect (Supplemental Figure 6A, compare 23 vs 30C). Can the authors comment on this?
Significance
Despite significant recent advances in our structural understanding of the nuclear pore complex, how the NPC associates with the curved nuclear membrane remains poorly understood. Previous studies in yeast have uncovered significant redundancy in this association but again the basis for this remains unclear. Therefore, I find this study on the amphipathic helix of Ndc1 and its interaction with other membrane binding components of the NPC an important and timely contribution to the field. Technically, the paper is solid and I find that most of the authors' conclusions are well supported by the evidence they provide (but see below for few experimental issues). Overall, the paper is well written, and despite the use of several mutants and methodologies, it is easy to read. I think the paper's significance would improve if the authors could present some "larger picture" view on how the Ndc1 helix and/or domains they describe interact with the Nup84 complex and the pore membrane or other elements of the NPC. For example, the authors make the remarkable finding that removal of Nup53 makes ndc1 nulls able to survive. Would it be possible to use existing models of the yeast NPC and provide some structural explanation of why that is? However, I would like to emphasize that this is not required to support the main claims of the paper and should only be considered if the authors wish to provide a more "molecular" view of their findings.
Audience: Mostly the following - Nuclear pore complex, nuclear envelope, and possibly some membrane biologists.
My field of expertise: Cell biology, Nuclear envelope.
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Referee #1
Evidence, reproducibility and clarity
Ndc1 is a transmembrane nucleoporin, essential for insertion of the nuclear pore complex (NPC) and spindle pole body (SPB) into the nuclear envelope (NE). How NE-associated proteins contribute to the bending and fusion of membranes during NPC insertion has not been fully elucidated. Here, the authors report a number of loosely connected, interesting observations related to Ndc1 function. Their main findings are the following: (i) The N-terminal transmembrane domain of Ndc1 mediates the membrane recruitment of two Y-complex nucleoporins. Therefore, these interactions are likely to contribute to NPC biogenesis. (ii) Over-expression of a novel amphipathic helix (AH) in the non-essential C-terminus of Ndc1, and of a similar AH in the non-essential nucleoporin Nup53, alters the lipid composition and nuclear morphology of yeast cells, although the underlying mechanisms remain unknown. (iii) The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53, or by deleting the transmembrane nucleoporin POM34. Surviving strains have altered nuclear morphology (NE expansions), and are sensitive to membrane-fluidizing drugs, suggesting that NPC assembly is somehow linked to lipid homeostasis.
Overall, the experiments are of high technical quality, are presented in a clear way, and the conclusions are well-supported by the data. I have some minor suggestions for clarifications, which can be addressed by textual changes or by additional experiments.
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When overexpressed in budding yeast, the C-terminal domain of Ndc1 is toxic and induces membrane expansion with NPC-like openings, which the authors describe as enlarged ER membranes (Figure 2). Could these be NE expansions instead? ER and NE membranes are continuous but perhaps this issue could be addressed by examining the distribution of fluorescent markers specific for each compartment.
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The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53 or by deleting POM34. These experiments are done using a plasmid shuffle strategy, in which Ndc1 is temporarily expressed from a low copy plasmid. I wonder if surviving strains are stable, or whether they survive for a limited time only due to stabilisation of the Ndc1 protein in the absence of Nup53 or Pom34. Could the authors discard this possibility, for example by checking whether viable double mutants are recovered after backcrossing of the survivor strains?
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Cells over-expressing Ndc1, and surviving ndc1-delta strains display ER and/or NE expansions. It would be interesting to discuss these observations in the context of nuclear morphology studies by the Cohen-Fix and Liakopoulos labs, among others, showing NE expansion is partially dependent on the coordination between lipid synthesis, cell growth rate, and cell cycle progression (doi: 10.1091/mbc.E18-04-0204, 10.1091/mbc.e05-09-0839, 10.1016/j.cub.2012.04.022).
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Related to the previous point: nuclear membrane expansions caused by metaphase arrest usually overlap with the nucleolus, and appear DAPI-negative. Did the authors examine nucleolar distribution relative to NE expansion in cells shown in figure 4C? Along the same lines, what is the cell cycle distribution of cells with ER/NE expansion? If they are delayed in mitosis, nuclear morphology defects may be a secondary consequence of cell cycle progression defects, themselves due to NPC and/or SPB insertion problems.
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I suggest to rephrase the last sentence of the abstract: "nuclear membrane biogenesis dependent on a balanced ratio between amphipathic motifs in diverse nucleoporins is essential for interphase NPC biogenesis". This study does not directly assess NPC biogenesis and therefore, the interesting link between lipids and NPC biogenesis remains correlative.
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It would be useful to include some information on the number of cells observed in the EM figures.
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Results, first page: "Moreover, CtNup120 and CtNup133 did not associate with GUVs containing the unrelated inner nuclear membrane protein BC08/SCL1 (Fig. 1C)" should be Figure S1C.
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P. 19: "Prompted by the finding that Ndc1 and Nup53/Nup59 amphipathic motifs may (modify?) the nuclear ... "
I am an expert in yeast genetics and cell cycle progression.
Significance
Significance: This report describes novel functional motifs in the Ndc1 protein that may be important for NPC assembly, and intriguing genetic interactions between NPC assembly and lipid homeostasis pathways. Although the mechanisms linking Ndc1 motifs with NE expansion and lipid composition remain unclear, these observations will be interesting for researchers working on NPC biogenesis and nuclear morphology.
Reviewer Expertise: yeast genetics, cell cycle progression and NPCs.
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Reviewer #1
Major #1
This study primarily uses the genetic mouse model in which LSD1 gene is inactivated after tamoxifen injection in 8 weeks old mice, as shown in supplemental figure 1 B and C. 8 weeks after birth postnatal growth of muscle is not complete and the contribution of satellite cells to muscle growth is still significant. Therefore the timing of tamoxifen injection used cannot discriminate if the observed phenotype involves the function of LSD1 during the post-natal growth of the muscle or in the muscle fibers or both. One way to demonstrate the real contribution of LSD1 in the maintenance of muscle fibers plasticity under environmental stress would be to inject Tamoxifen later (around 10-12 weeks of age), in order to remove a possible bias caused by the contribution of satellite cells during the post-natal growth. At least key findings should be confirmed at this later stage.
In this study, we used ACTA1-CreERT mice to conditionally knockout LSD1 in the skeletal muscle. The ACTA1 promoter is derived from human muscle actin gene, which is not expressed in the satellite cells, and has been widely used for the transgene expression in myofibers (Stantzou et al. Development 2017). Thus, the inactivation of LSD1 occurs in the existing myofibers, and alterations in satellite cell function, if any, would be indirect effects of the loss of LSD1 in mature myocytes or differentiating myoblasts.
To test whether postnatal muscle growth was affected in our LSD1-mKO mice, we administrated tamoxifen (4OHT) to pre-weaning mice (11 days old). LSD1 depletion did not affect the expression of muscle fiber genes, when muscle tissues were isolated from mice 11 days after the start of 4OHT (Additional Data).
These evidences exclude the contribution of satellite cells in the phenotypes observed in the LSD1-mKO mice. __Additional Data __will be added in the revised manuscript.
Major #2
LSD1 m-KO muscles seem to have more type I and IIA fibers than WT, even without DEX treatment. Is it possible to quantify the results in supplemental figure 4C?
As suggested, we quantitatively analyzed the fiber type compositions in Supplemental Fig. 4C using the data from WT (n=4) and LSD1-mKO (n=5) mice (Additional Data). We did not find a significant difference between these mice, confirming our finding that the loss of LSD1 accelerates the Dex-driven phenotypic changes. __Additional Data__will be added in the revised manuscript.
Major #3
The effect on fiber type is convincing, while variations in gene expression are of quite low amplitude. However, the atrophy should be induced by other means to ensure that the effects are specific to GC/nuclear receptors pathways; Denervation? Starvation? Not all the experiments need to be repeated, just key results such as, for example, exacerbation of atrophy in LSD1 m-KO, Foxk1 increase.
We agree that testing alternative atrophy models is important for generalizing our findings. For this, we employed a model for diabetes-related muscle atrophy. A pro-diabetic agent streptozotocin (STZ) disturbs the function of pancreatic islet leading to fast-fiber atrophy (O’Neill et al. Diabetes 2019). LSD1-mKO did not affect the muscle weight in STZ-treated mice (Additional Data). Consistently, there were no major difference in the expression of atrophy genes in STZ-treated WT and LSD1-mKO mice (Additional Data). These results suggest that the LSD1 function depends on the source of atrophy-inducing stress, and that the loss of LSD1 sensitized the muscle to GC-mediate signaling. Additional Data will be added in the revised manuscript.
Major #4
Autophagy data: the effect on the LC3I/LC3II ratio are modest. The autophagy part should be removed or completed with additional data to convincingly show that autophagy is affected. Links between LSD1 and mTOR have been published, so the mTOR pathway could be investigated in the model (S6k, S6 and 4EBP1 phosphorylation). Given AKT levels and phosphorylation are affected by the absence of LSD1 + DEX, it can be predicted that mTOR activity will change.
We have analyzed the expression of additional autophagy markers p62 and phosphorylated 4EBP1. Consistent with the upregulated expression of atrophy genes and increased LC3I/II ratio, LSD1-mKO mice had elevated levels of p62 and phosphorylated 4EBP1 (Additional Data). Altogether, the data suggest that Dex-induced muscle atrophy was exacerbated by the loss of LSD1. Additional Data will be added in the revised manuscript.
Major #5 The ability of LSD1 to retain FOXK1 in the nucleus is an important information that should be better supported experimentally. In the absence of such information, no mechanism can be proposed for the effect of LSD1 of FOXK1. The immunofluorescence images provided are not convincing and moreover they could be interpreted by a reduction in the level of FOXK1 protein (degradation?) rather than by a nuclear exclusion in the presence of DEX. This point should be addressed, authors could include western blot of nuclear and cytoplasmic fractions to better quantify the nuclear level of FOXK1 in absence of LSD1.
We agree that performing the suggested experiment would further enhance the quality of our study.
Major #6 The absence of centralized nuclei indicates that there is no fiber regeneration but it does not exclude the possibility that satellite cells were recruited to existing fibers and thus participated to hypertrophy. To eliminate this possibility, the average nuclei/cytoplasm volume should decrease if hypertrophy results from increased protein synthesis and not myonuclei accretion. This should be checked.
We histologically analyzed the sections of Gas muscles after Dex treatment and found that there is no evidence of central nuclei in either WT or KO mice (Supplemental Fig. 4D).
As mentioned above (Major #1), it is unlikely that the satellite cell function was responsible for the enhanced atrophic phenotype.
Major #7 The upregulation of ERR____g in the absence of LSD1 is convincing in the VWR conditions. ERR____g level should be evaluated in the sedentary LSD1 KO mice.
We have analyzed the expression of ERRg in sedentary mice, and found no significant difference between WT and KO mice (Additional Data). This suggests that the loss of LSD1 in combination with VWR training led to the increased expression of ERRg. Additional Data will be added in the revised manuscript.
Minor #1
There is a clear difference in the number of mouse replicates between treated (Dex or VWR) and non-treated mice, regardless the genotype. Experiments with non-treated mice lack adequate numbers to make a definitive conclusion. For example, there is a huge spread in the data in Figure 1 B and 4 D. If the number of animals would have been increased, would the absence of difference hold up?
We increased the number of non-treated animals in Figures 1B and 4B as suggested. Nonetheless, we did not find any significant differences in the muscle weight (Additional Data). These changes will be reflected on original Figures 1B and 4B.
Minor #2 The authors claim that: "Consistent with the results of the augmented endurance capacity, the Sol muscle in the KO mice showed enhanced succinate dehydrogenase (SDH) staining, indicating that the number of oxidative fibers increased (Figure 4F and Supplemental Figure 8F)". However, supplemental figure 8 D indicates that the number of type I fibers does not change compared to WT. Authors should clarify this statement.
Indeed, we found that the area of type I fiber but not the number was increased in the LSD1-KO Sol (Fig. 4D and Supplemental Fig. 8D). Because SDH staining reflects the OXPHOS capacity in all fiber types, it is possible that the OXPHOS capacity in the fibers other than type I had been augmented by LSD1-KO. Thus, for clarification, we will change the statement as follows: OXPHOS capacity of Sol was enhanced by the loss of LSD1.
Reviewer #2
__Methods
1__
The authors used the Cre-lox system with tamoxifen to generate skeletal muscle-specific LSD1 KO mice. It is clear that both the mRNA and protein levels of LSD1 in various muscles were dramatically reduced, but there is still some LSD1 expressed in skeletal muscle, especially in Sol muscle (Supplemental Figure 1C). The author needs to think about whether it is appropriate to use the term "LSD1 knockout" or "LSD1 deficiency".
We thank the reviewer for this comment. In this study, we crossed LSD1-floxed mice with ACTA1-creERT mice. This enables the deletion of critical exons of LSD1 in mature myocytes and myogenic precursors that have initiated the differentiation program. LSD1 is a ubiquitously expressed gene, and it is known that immature myogenic cells (e.g., satellite cells, Tosic et al. Nat Commun. 2018) and other non-myogenic cells such as hematopoietic and vascular cells abundantly express LSD1 (Kerenyi et al. Elife 2013, Yuan et al. Biochem Pharmacol. 2022). Thus, it is likely that LSD1 expression by these cell types were detected in our whole muscle western blots. We will add these statements in the text for clarification.
__Results
2__
To identify the transcriptional regulators that mediate the regulation of atrophy-associated genes by LSD1, the authors performed motif analyses on the promotor regions of upregulated genes in LSD1-mKO Gas. Based on the results and other reports, they focused on Foxk1 and proved LSD1 and Foxk1 cooperatively regulate the atrophy transcriptome in the presence of Dex. However, Figure 3C showed that a transcription factor Nfatc1 is also reduced in Sol muscle similar to Foxk1. Also, other studies demonstrated that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. More specifically, NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300 (Cell Rep. 2014 Sep 25; 8(6): 1639-1648). Furthermore, it has been reported that LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription (Cell Rep. 2017 Feb 21;18(8):1996-2006. doi: 10.1016/j.celrep.2017.01.078). It is unclear how the authors exclude Nfatc1 for the LSD1-mediated effects in different muscle fibers. Further experiments may be necessary to exclude Nfatc1.
We thank the reviewer for an insightful comment. In addition to Foxk1, we tested the involvement of NFATc1 in the gene regulation under LSD1-depleted state. We treated C2C12 with an LSD1 inhibitor S2101 in combination with a calcium ionophore that promotes the transcriptional function of NFATc1 by inducing its nuclear localization (Meissner et al. J Cell Physiol. 2007). While LSD1 inhibition promoted the expression of Pgc1a and Myh7, ionophore treatment had no additive effects (Additional Data). Because we found a physical association of Foxk1 with LSD1, we focused on the functional involvement of Foxk1 in LSD1-mediated repression of atrophy genes. We recently performed an ATAC-seq analysis in Dex-treated muscle, and found that the Foxk1 motif but not the NFATc1 motif was enriched in the LSD1-KO-specific open chromatin regions. This data further suggests the significant contribution of Foxk1 in the transcriptional regulation under LSD1 depletion.
#3
In figure 3D, only merged images were colored. It would be better to show colored images for Foxk1 and DAPI.
We will replace the images with the colored ones.
#4
Immunofluorescence analysis in C2C12 myotubes showed that Dex exposure reduced the nuclear retention of Foxk1, which was further promoted by the addition of T-3775440, an LSD1 inhibitor (Figure 3D). The author also used Foxk1-KO C2C12 myotubes to prove LSD1 and Foxk1 cooperation to regulate the expression of type I /IIA fiber and atrophy genes in Foxk1-KO cells. Are the effects of LSD1 dependent on Foxk1 or synergistically acting with Foxk1? The treatment of LSD1 inhibitor in Foxk1-KO C2C12 may be helpful to answer this question.
As suggested, we will examine the combination effect of LSD1 inhibition and Foxk1-KO. In addition, we will analyze chromatin association of LSD1 in Foxk1-KO cells by ChIP experiments, to test whether the function of LSD1 depends on Foxk1.
#5
In supplementary figure 2, body weight in the mKO+Dex group was reduced in comparison to that of WT+Dex. How about the body weight of mKO mice without Dex injection compared to that of WT? This data will be helpful to understand the effect of muscle-specific LSD1 deficiency on whole-body energy balance.
We measured the body weight of untreated mice, and found that there is no genotype effect (Additional Data). Thus, we think that LSD1-mKO alone does not influence the whole-body energy balance. We will include this data in the revised version.
#6
The authors analyzed the size distribution of myofibers and mentioned that large type I and type IIA fibers preferentially increased in the LSD1-mKO muscle, whereas large type IIB + IIX fibers decreased (Supplemental Figure 4, B, E, and F). It is better to show the results of statistics. If no significance were found, it should be mentioned in the result section.
We have performed statistical analyses on Supplemental Fig. 4E and F, and found that a fraction of large type I fibers was significantly larger in KO mice. This result will be added in the next version.
#7
Page 11, To reveal the genes regulated by LSD1 under the VWR condition, the authors performed additional RNA-seq analysis using Sol muscle. The non-hierarchical clustering analysis was informative and showed signaling pathways related to ‘mitochondrion’, ‘mitochondrion organization’, and ‘oxidative phosphorylation’ were altered in the Sol muscle deficient in LSD1 under the VWR condition (Figure 5B). However, it is unclear why they focus on Err-gamma to explain LSD1-KO phenotypes in Sol muscle. Is this gene also derived from RNA seq? It is better to show whether Err-gamma expression is also significantly altered based on RNA seq data.
Indeed, ERRg was upregulated by LSD1-KO+VWR and was included in the Cluster 6 genes together with the OXPHOS and mitochondria-related genes (Additional Data and Fig. 5A). These data prompted us to focus on ERRg as a potential factor that explains the LSD1-KO phenotype. Additional Data will be included in the revised version.
#8
The authors claim that LSD1 serves as an "epigenetic barrier" that optimizes fiber type-specific responses and muscle mass under stress conditions. This claim is derived from the loss of function studies. To generalize the functions of LSD1, the gain of function studies will be also necessary. Adding the characteristics of LSD1 overexpression in C2C12 cells will further improve the quality of the manuscript.
We agree that the gain of function studies will further strengthen the quality of our manuscript. As suggested, we will perform an LSD1 overexpression experiment using C2C12 cells and analyze the expression of atrophy and fast fiber related genes. Because Esrrg is completely silenced in C2C12 cells, it is difficult to monitor ERRg-mediated gene regulation in these cells. To overcome this, we will use a cardiomyocyte cell line, in which ERRg is functionally involved in differentiation (Sakamoto et al. Nat Commun 2022). We will overexpress LSD1 in these cells and examine whether the expression of ERRg and its downstream targets are altered.
__Discussion
9__
The authors mentioned supplementary figure 10 only at the end of the manuscript of the discussion section (page 15) without a specific explanation of the figures in the result section. The data are important in that LSD1 expression in human muscles declined with age and showed a negative correlation with the expression of the atrophy gene. It should be presented in the result section with a more detailed description.
We agree that these data are important and need further explanations. We will describe the details in the Results section and move the entire figure to the main figure.
#10
There are other studies to examine LSD1 and muscle regeneration or functions (e.g. Nat Commun 9, 366 (2018). ____https://doi.org/10.1038/s41467-017-02740-5____). More discussion to compare the current study and other studies will be necessary.
We thank the reviewer for this comment. We will add the discussion accordingly.
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Referee #2
Evidence, reproducibility and clarity
The authors investigated the functions of LSD1 in skeletal muscle in response to different stimulations. The study is quite interesting in that it revealed the dual roles of an epigenetic regulator LSD1 in the types of muscle fibers using a muscle-specific LSD1 KO mouse model and related cell lines with big data analysis. The LSD1 deficiency sensitized the skeletal muscle to Dex-induced atrophy and VWR-induced hypertrophy. LSD1 appears to suppress the catabolic pathways and the formation of type I fibers. Also, LSD1 may exert its effects together with Foxk1 to suppress genes related to protein catabolism, while repressing ERRg and its downstream oxidative genes, depending on the stimulus and muscle location. These findings will shed light on related fields in that LSD1 may be an epigenetic molecular switch that induces fiber-type-specific responses. Nevertheless, some questions need to be further answered to make up for the shortcomings of the study. The specific comments are as follows;
Methods
- The authors used the Cre-lox system with tamoxifen to generate skeletal muscle-specific LSD1 KO mice. It is clear that both the mRNA and protein levels of LSD1 in various muscles were dramatically reduced, but there is still some LSD1 expressed in skeletal muscle, especially in Sol muscle (Supplemental Figure 1C). The author needs to think about whether it is appropriate to use the term "LSD1 knockout" or "LSD1 deficiency".
Results
- To identify the transcriptional regulators that mediate the regulation of atrophy-associated genes by LSD1, the authors performed motif analyses on the promotor regions of upregulated genes in LSD1-mKO Gas. Based on the results and other reports, they focused on Foxk1 and proved LSD1 and Foxk1 cooperatively regulate the atrophy transcriptome in the presence of Dex. However, Figure 3C showed that a transcription factor Nfatc1 is also reduced in Sol muscle similar to Foxk1. Also, other studies demonstrated that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. More specifically, NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300 (Cell Rep. 2014 Sep 25; 8(6): 1639-1648). Furthermore, it has been reported that LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription (Cell Rep. 2017 Feb 21;18(8):1996-2006. doi: 10.1016/j.celrep.2017.01.078). It is unclear how the authors exclude Nfatc1 for the LSD1-mediated effects in different muscle fibers. Further experiments may be necessary to exclude Nfatc1.
- In figure 3D, only merged images were colored. It would be better to show colored images for Foxk1 and DAPI.
- Immunofluorescence analysis in C2C12 myotubes showed that Dex exposure reduced the nuclear retention of Foxk1, which was further promoted by the addition of T-3775440, an LSD1 inhibitor (Figure 3D). The author also used Foxk1-KO C2C12 myotubes to prove LSD1 and Foxk1 cooperation to regulate the expression of type I /IIA fiber and atrophy genes in Foxk1-KO cells. Are the effects of LSD1 dependent on Foxk1 or synergistically acting with Foxk1? The treatment of LSD1 inhibitor in Foxk1-KO C2C12 may be helpful to answer this question.
- In supplementary figure 2, body weight in the mKO+Dex group was reduced in comparison to that of WT+Dex. How about the body weight of mKO mice without Dex injection compared to that of WT? This data will be helpful to understand the effect of muscle-specific LSD1 deficiency on whole-body energy balance.
- The authors analyzed the size distribution of myofibers and mentioned that large type I and type IIA fibers preferentially increased in the LSD1-mKO muscle, whereas large type IIB + IIX fibers decreased (Supplemental Figure 4, B, E, and F). It is better to show the results of statistics. If no significance were found, it should be mentioned in the result section.
- Page 11, To reveal the genes regulated by LSD1 under the VWR condition, the authors performed additional RNA-seq analysis using Sol muscle. The non-hierarchical clustering analysis was informative and showed signaling pathways related to 'mitochondrion', 'mitochondrion organization', and 'oxidative phosphorylation' were altered in the Sol muscle deficient in LSD1 under the VWR condition (Figure 5B). However, it is unclear why they focus on Err-gamma to explain LSD1-KO phenotypes in Sol muscle. Is this gene also derived from RNA seq? It is better to show whether Err-gamma expression is also significantly altered based on RNA seq data.
- The authors claim that LSD1 serves as an "epigenetic barrier" that optimizes fiber type-specific responses and muscle mass under stress conditions. This claim is derived from the loss of function studies. To generalize the functions of LSD1, the gain of function studies will be also necessary. Adding the characteristics of LSD1 overexpression in C2C12 cells will further improve the quality of the manuscript.
Discussion
- The authors mentioned supplementary figure 10 only at the end of the manuscript of the discussion section (page 15) without a specific explanation of the figures in the result section. The data are important in that LSD1 expression in human muscles declined with age and showed a negative correlation with the expression of the atrophy gene. It should be presented in the result section with a more detailed description.
- There are other studies to examine LSD1 and muscle regeneration or functions (e.g. Nat Commun 9, 366 (2018). https://doi.org/10.1038/s41467-017-02740-5). More discussion to compare the current study and other studies will be necessary.
Significance
The study is quite interesting in that it revealed the dual roles of an epigenetic regulator LSD1 in the types of muscle fibers using a muscle-specific LSD1 KO mouse model and related cell lines with big data analysis.
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Referee #1
Evidence, reproducibility and clarity
Summary:
In this manuscript, Araki et. al show that LSD1 is required for muscle fiber maintenance upon chemical and environmental stress. By using a LSD1 conditional KO mouse model, along with C2C12 cell culture, the authors demonstrate that loss of LSD1 exacerbates glucocorticoid-induced muscle atrophy in fast fiber-dominant muscle. On the other hand, loss of LSD1 together with endurance exercise induces a hypertrophy in slow fiber-dominant muscle. Thus, these data suggest that LSD1 acts as a modulator of the muscle response to chemical and environmental stress, might be a target for therapeutic strategies against stress-induced myopathies. However, some concerns should be addressed in a revised version.
Major comments:
This study primarily uses the genetic mouse model in which LSD1 gene is inactivated after tamoxifen injection in 8 weeks old mice, as shown in supplemental figure 1 B and C. 8 weeks after birth post natal growth of muscle is not complete and the contribution of satellite cells to muscle growth is still significant. Therefore the timing of tamoxifen injection used cannot discriminate if the observed phenotype involves the function of LSD1 during the post-natal growth of the muscle or in the muscle fibers or both. One way to demonstrate the real contribution of LSD1 in the maintenance of muscle fibers plasticity under environmental stress would be to inject Tamoxifen later (around 10-12 weeks of age), in order to remove a possible bias caused by the contribution of satellite cells during the post-natal growth. At least key findings should be confirmed at this later stage.
- LSD1 m-KO muscles seem to have more type I and IIA fibers than WT, even without DEX treatment. Is it possible to quantify the results in supplemental figure 4C?
- The effect on fiber type is convincing, while variations in gene expression are of quite low amplitude. However, the atrophy should be induced by other means to ensure that the effects are specific to GC/nuclear receptors pathways; Denervation? Starvation? Not all the experiments need to be repeated, just key results such as, for example, exacerbation of atrophy in LSD1 m-KO, Foxk1 increase.
- Autophagy data: the effect on the LC3I/LC3II ratio are modest. The autophagy part should be removed or completed with additional data to convincingly show that autophagy is affected. Links between LSD1 and mTOR have been published, so the mTOR pathway could be investigated in the model (S6k, S6 and 4EBP1 phosphorylation). Given AKT levels and phosphorylation are affected by the absence of LSD1 + DEX, it can be predicted that mTOR activity will change.
- The ability of LSD1 to retain FOXK1 in the nucleus is an important information that should be better supported experimentally. In the absence of such information, no mechanism can be proposed for the effect of LSD1 of FOXK1. The immunofluorescence images provided are not convincing and moreover they could be interpreted by a reduction in the level of FOXK1 protein (degradation?) rather than by a nuclear exclusion in the presence of DEX. This point should be addressed, authors could include western blot of nuclear and cytoplasmic fractions to better quantify the nuclear level of FOXK1 in absence of LSD1.
- The absence of centralized nuclei indicates that there is no fiber regeneration but it does not exclude the possibility that satellite cells were recruited to existing fibers and thus participated to hypertrophy. To eliminate this possibility, the average nuclei/cytoplasm volume should decrease if hypertrophy results from increased protein synthesis and not myonuclei accretion. This should be checked
- The upregulation of ERR in the absence of LSD1 is convincing in the VWR conditions. ERR level should be evaluated in the sedentary LSD1 KO mice.
Minor comments:
- There is a clear difference in the number of mouse replicates between treated (Dex or VWR) and non-treated mice, regardless the genotype. Experiments with non-treated mice lack adequate numbers to make a definitive conclusion. For example, there is a huge spread in the data in Figure 1 B and 4 D. If the number of animals would have been increased, would the absence of difference hold up?
- The authors claim that: "Consistent with the results of the augmented endurance capacity, the Sol muscle in the KO mice showed enhanced succinate dehydrogenase (SDH) staining, indicating that the number of oxidative fibers increased (Figure 4F and Supplemental Figure 8F)". However, supplemental figure 8 D indicates that the number of type I fibers does not change compared to WT. Authors should clarify this statement.
Significance
Conceptually, Araki and colleagues have described the potential contribution of LSD1 in the maintenance of muscle fiber typing upon environmental stress. However, the technical approach used only partially allows to demonstrate the concept.
Field of expertise: Muscle atrophy and regeneration, Muscle stem cells, Epigenetic regulation in muscle, LSD1.
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Reply to the reviewers
Thank you for giving us the opportunity to submit a revised draft of the manuscript “Tup1 is Required for Transcriptional Repression Necessary in Quiescence in S. cerevisiae” to Review Commons. We appreciate the time and effort that you and the other reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We believe that the experiments suggested in these comments would bring clarity to the manuscript, and wish that we had the ability to perform them all. Unfortunately our lab is closing, and the only remaining lab member is the PI, so we are only able to perform limited experiments to address some of the concerns raised during review. We have incorporated several changes in response to comments from the reviewers. Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript Word file with tracked changes.
Of particular note is the discussion of cellular morphology of the tup1∆ and sds3∆ strains. We realize that our findings are purely descriptive, and are not surprised that all three reviewers had comments on this data. This was the source of much discussion among the authors and consultation with other labs; we debated even including these observations in the manuscript, since we were unable to figure out the underlying mechanism. Ultimately we decided that it was worth reporting in case other labs may benefit from the knowledge, and we have altered the language in the manuscript (page 6) to better reflect this. However, if the reviewers feel that this observation would be better left out of the manuscript, we would be willing to remove Figure 6 and any discussion of these images.
Reviewer #1 Comments:1. The authors chose to examine 3-day SP cells to interrogate quiescence because tup1∆ cells are highly flocculant, interfering with the isolation of purified quiescent cells. These cells are a mixture of both nonquiescent and quiescent cells, so it is not correct to state that they represent a quiescent cell population. The addition of EDTA to the gradients used to isolate quiescent cells could eliminate flocculation and permit the isolation of quiescent cells. EDTA is also often added to media in low amounts to reduce flocculation. The authors need to indicate the proportion of quiescent cells in their SP cultures by applying these tools.
We appreciate the suggestion, but the phenotype of this strain is not typical flocculation (see photo below, also added to the paper as Supplementary Figure 1). We did add EDTA (pH 8.0) to a final concentration of 10 mM to two separate tup1∆ and it did not visibly affect the clumping of cells. Furthermore, changes to the cell wall are a distinct feature of quiescent S. cerevisiae and contribute to the ability to separate different cell types by density-gradient centrifugation, so it is difficult to anticipate how EDTA would affect our ability to isolate Q cells. We have provided more explanation in the manuscript to better explain this (page 3).
- The authors reported that while Xpb1 and Tup1 share many overlapping binding sites, but that Xbp1 does not regulate Tup1's binding. What other factors might be responsible for their shared binding? Could histone deacetylation play a role? This could be addressed by a Tup1 ChIP in an sds3∆ mutant.
This is a good thought; histone acetylation levels may have a role in regulating Tup1 localization and we would have liked to address this if we had more time. Unfortunately, we had some difficulty performing ChIP of Tup1, because initially we used a FLAG tag which caused a phenotype similar to deletion of Tup1, and had to switch to making myc-tagged strains. This delay meant we did not have time to pursue creating myc-tagged Tup1 in an sds3∆ strain, and now we do not have the ability to follow up on this for revisions.
- Has PolII occupancy been examined in Log vs SP cells of tup1∆ to determine if Tup1 inhibits PolII association with its genes that are repressed ?
We did not look at PolII occupancy in our Tup1 deletion, and could not find any existing datasets with this information. It is our hope that another lab is able to carry out this experiment, because it could be very enlightening, but it is beyond the scope of this work.
- The observation that tup1∆ cells have several nuclear puncta is intriguing, although the cytological images need to be improved.
The nuclear puncta we see in the tup1 deletion are definitely a puzzle. We had limited time to investigate this phenomena, and in discussing the matter with some other labs it seemed doubtful that more advanced imaging would yield anything of use to us. We realized that we accidentally omitted important details for this figure and have updated the manuscript to add them. We imaged 2 biological replicates for each strain and imaged many yeast samples for each strain (which has been added to the caption for Figure 6) and found that our findings were statistically significant (p
Reviewer #2 Comments1. The authors acknowledge that it would be better to work with purified quiescent cells but couldn't isolate pure populations. As a result, a mixture of quiescent and nonquiescent cells are analyzed in stationary phase. They say this is because Tup1 deletion strains are flocculent. But they performed ChIP-Seq on Myc-tagged Tup1 strain. Don't these cells express Tup1? If not, could this be performed in wild-type yeast with Myc-tagged Tup1? It seems important to separate quiescent from nonquiescent yeast for the authors' conclusions.
It is true that we could have done ChIP-seq for Tup1 in purified Q cells. We considered it, but decided to look at the mixed population so that we could directly compare our RNA-seq results from the tup1∆ strain. It’s a balance between having some results that are specific to quiescence, versus being able to directly compare the effects of deletion of Tup1 at the sites where it binds. We are now unable to perform this experiment, but we have updated the language in the manuscript (page 3) to better reflect this choice.
- The Chipseq data in Fig 1B do not have a y axis and it is consequently not clear whether these data are normalized and shown with the same axis.
Thank you for pointing this out - these data are normalized to RPKM during processing, and we have updated the caption for figure 1 and the methods on page 10 to reflect this information. Normalizing the data in IGB itself, however, causes an adjustment in the y-axis that makes the tracks appear to be inconsistent. In any case, we are not making claims about the relative amount of signal, and as it is common in the field to not include y-axes on IGB tracks, we have opted to keep the y-axis for Figure 1B as-is.
- In Fig 2, it seems important to determine how many genes are different between WT and Tup1 deletion strains in log phase. Are just as many genes different? Or is Tup1 more important in diauxic shift and stationary phase than log phase?
We did intend to focus only on diauxic shift and stationary phase data for this paper, since there has already been so much work on the role of Tup1 in log phase. As mentioned above, comparisons of RNA between log and DS/Q is difficult. We attempted to find a publicly available dataset to perform some analysis for revisions, but unfortunately most previous work on the effect of Tup1 on transcription was performed via tiling arrays, which is not comparable.
- Are the genes that are regulated by Tup1 normally regulated during diauxic shift or stationary phase compared with log growth?
Because there is a massive global decrease in the level of total RNA in diauxic shift and quiescence (McKnight, Boerma, et al., 2015) it is impossible to directly compare transcript levels between these states in our experiments. If there was time, we could have attempted to repeat these experiments with an external spike-in control; this is potentially something another lab could do to follow up on our findings.
- What fraction of the genes that are differentially expressed in Tup1 knockout yeast have Tup1 binding at the promoter? Enhancer? What fraction can be explained by Tup1, Hap1, Nrg1, Mig1 individually and together?
We have added the number of genes that are differentially expressed in Tup1 knockout yeast during DS to the manuscript (page 3). Regarding enhancers, the genome of S. cerevisiae is very compact, and there is not evidence of long-distance activation of genes as seen in metazoans (Dujon, 1996; Dobi & Winston, 2007; Spiegel and Arnone, 2021). Upstream activating sequences (UASs) are generally considered the closest equivalent to enhancers in cerevisiae, and they tend to function within a few hundred base pairs of the promoter. Our analysis only identifies the nearest gene; it would be difficult to parse out locations in the promoter versus a UAS without a more advanced analysis that is beyond our capabilities now.
As for the effect of Hap1, Nrg1, and Mig1, we were able to look for their motifs in the genes that are differentially expressed in the Tup1 knockout but we do not have binding data for these factors in quiescence or stationary phase so it is impossible to conclusively state what role those TFs play. This would be a very interesting followup to our work, but is outside the scope of this manuscript.
References:
Dujon, B. 1996. The Yeast Genome Project: What did we learn? Trends Genet. 12, 263-270.
Dobi, K.C.; Winston, F. 2007. Analysis of Transcriptional Activation at a Distance in Saccharomyces cerevisiae. Mol Cell Biol. 27(15), 5575-5586. https://doi.org/10.1128/MCB.00459-007
Spiegel, JA; Arnone, J.T. 2021.Transcription at a Distance in the Budding Yeast Saccharomyces cerevisiae. Appl. Microbiol. 1(1), 142-149. https://doi.org/10.3390/applmicrobiol1010011
- The methodology used to generate the gene ontology enrichments should be described in the methods.
Thank you for noticing this omission; we have added the relevant information to the manuscript (page 10) and have also added the related citation (page 11).
- The authors should provide genomewide data to support the statement that Tup1 and Rpd3 ChIP datasets have substantial overlap. They should also provide genomewide data to support the statement that there is substantial overlap between Rpd3 and Tup1. How much overlap is observed and how much is expected by chance?
We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B), along with a p-value.
- For Sds3, similar to Tup1 inactivation, it would be helpful to know how many genes change in with Sds3 inactivation in log phase in addition to diauxic shift and stationary phase.
As with our response to comment #3, we focused only on diauxic shift and stationary phase data for this paper, and analysis of this data would be difficult without a spike-in control. While there are some existing datasets for RNA-seq of Rpd3 knockouts, this would include both Rpd3L and Rpd3S activity, rather than just Rpd3L, which is our focus with the Sds3 deletion strains. As such, we did not perform RNA-seq of sds3∆ in log phase.
- If the argument is that Sds3 and Xbp1 cooperate with Tup1 to affect gene expression, testing the gene expression changes that are associated with Tup1 in Sds3 or Xbp1 knockout strains would help the authors make this point.
We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.
- The final phenotype of extra DAPI positive blobs in the nucleus is not very specific or clear.
We agree, please see our comments at the top of this letter.
Reviewer #3 (Major comments):Did tup1∆/sds3∆ double mutant show the same phenotype with tup1∆ (or sds3∆) single mutant in G0? If Tup1 actually plays role in tandem with Sds3 in the gene regulation during G0, the epistatic relationship might be estimated.
We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.
The histone acetylation was not synergistically augmented in the above double mutant?
Please see the response above.
The authors showed that tup1∆ but not sds3∆ cells contain multiple DAPI signals but sds3∆ cells show abnormal cell shape in G0 phase. These phenotypic abnormalities in these mutants suggest a potential mitotic defect. Both mutants showed very similar abnormalities in H3K23 acetylation and gene expressions in quiescent state. Why these showed distinctly different abnormality in cell morphology during G0?
Unfortunately we were unable to investigate this further.
Did iswi2∆ cells also show abnormality in G0 phase?
No, they did not; thank you for asking, this is a good question. We have added this information to the manuscript (page 6).
(Minor comments)Supplementary figure1. This data seems to be very important. I recommend to use this data in the main figure with statistical analysis (p-values) to show the significant overlap of Tup1 and Rdp3 distribution.
We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B) . We do feel that this data belong in the supplement, however, because the data is not exactly equivalent to our studies: quiescent cells and 3-day cultures are not the same, and knockout of Rpd3 eliminates function of both Rpd3L and Rpd3S complexes, while knocking out Sds3 targets only the Rpd3L complex.
Figure 4. Histone acetylation level data in Figure 4A and the data for gene repressions by Tup1 and Sds3 in Figure 4C seem to be very important. However, statistical analysis data (p-values) was not presented. Please show the statistical analysis data (p-values) as in figure 3 to show that the Tup1 and Sds3 contribute similarly in histone deacetylation and repression. The author did not find the significant changes of histone deacetylation in xbp1∆ cells but said that when filtered in Xbp1 binding motif Xbp1 depletion has similar effect on the acetylation level. Please show this data.
We have added language in the manuscript comparing genes with altered acetylation levels to those that are differentially expressed in our RNA-seq datasets, along with a p-value, to page 5.
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Referee #3
Evidence, reproducibility and clarity
In this study, the authors found that Tup1 corepressor coordinates with Rpd3L HDAC complex in the deacetylation of histone H3K23 during quiescence entry. They also found that Tup1 coordinates with ISWI2 to regulate +1 nucleosome at HXT family genes for the gene repression during G0. Finally, they showed that loss of Tup1 results in abnormal shape of nuclei during G0. These data suggest a critical role of Tup1 corepressor in the proper gene regulations in tandem with Rpd3 HDAC and ISWI2 remodeler upon quiescence entry. This study seems to be a critical progress from their previous study on the roles of Rpd3 complex in the quiescence entry (McKnight et al. 2015 Mol. Cell). My comments were listed bellow.
Major comments
Did tup1∆/sds3∆ double mutant show the same phenotype with tup1∆ (or sds3∆) single mutant in G0? If Tup1 actually plays role in tandem with Sds3 in the gene regulation during G0, the epistatic relationship might be estimated.
The histone acetylation was not synergistically augmented in the above double mutant?
The authors showed that tup1∆ but not sds3∆ cells contain multiple DAPI signals but sds3∆ cells show abnormal cell shape in G0 phase. These phenotypic abnormalities in these mutants suggest a potential mitotic defect. Both mutants showed very similar abnormalities in H3K23 acetylation and gene expressions in quiescent state. Why these showed distinctly different abnormality in cell morphology during G0?
Did iswi2∆ cells also show abnormality in G0 phase?
Minor comments
Supplementary figure1. This data seems to be very important. I recommend to use this data in the main figure with statistical analysis (p-values) to show the significant overlap of Tup1 and Rdp3 distribution.
Figure 4. Histone acetylation level data in Figure 4A and the data for gene repressions by Tup1 and Sds3 in Figure 4C seem to be very important. However, statistical analysis data (p-values) was not presented. Please show the statistical analysis data (p-values) as in figure 3 to show that the Tup1 and Sds3 contribute similarly in histone deacetylation and repression. The author did not find the significant changes of histone deacetylation in xbp1∆ cells but said that when filtered in Xbp1 binding motif Xbp1 depletion has similar effect on the acetylation level. Please show this data.
Significance
The current work by the authors significantly progressed their work (McKnight et al. 2015 Mol. Cell) showing important role of Rpd3 deacetylase complex in the quiescence entry. This study will significantly contribute to understanding the gene regulation mechanisms in the G0 entry.
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Referee #2
Evidence, reproducibility and clarity
In this paper, the authors investigate the role of the Tup1 transcriptional co-repressor in transcriptional repression after glucose depletion in quiescent S. cerevisiae. Tup1 N terminal helix facilitates oligomerization and interacts with histone tails and a beta propeller domain interacts with histone deacetylases and DNA binding factors. Tup1 interacts with nucleosomes by binding histone H3 and H4 tails, binds to hypoacetylated tails and stabilizes +1 nucleosomes repositioned by Isw2. Tup1 had been identified as a gene essential for viability of cells in G0 and implicated in glucose repression in yeast. The authors found that Tup1-Ssn6 binds new targets when S cerevisiae are glucose deprived and as they enter G0. Tup1 was found to repress the expression of genes involved in glucose metabolism and glucose transport. Tup1 was discovered to coordinate with the Rpd3L complex to deacetylate H3K23. Tup1 was also found to coordinate with Isw2 to affect nucleosome positions at hexose transporter family genes. Some cells with Tup1 deletion had multiple DAPI puncta in stationary phase, suggesting a possible role for Tup1 in mitosis.
Overall Comments
In Fig 1, the shift in Tup1 binding with diauxic shift and stationary phase is clear. The data clearly show an effect of Tup1 on histone acetylation and nucleosome positioning. However, whether Tup1 has an important functional role in quiescence is not clear.
Comments
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The authors acknowledge that it would be better to work with purified quiescent cells but couldn't isolate pure populations. As a result, a mixture of quiescent and nonquiescent cells are analyzed in stationary phase. They say this is because Tup1 deletion strains are flocculent. But they performed ChIP-Seq on Myc-tagged Tup1 strain. Don't these cells express Tup1? If not, could this be performed in wild-type yeast with Myc-tagged Tup1? It seems important to separate quiescent from nonquiescent yeast for the authors' conclusions.
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The Chipseq data in Fig 1B do not have a y axis and it is consequently not clear whether these data are normalized and shown with the same axis.
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In Fig 2, it seems important to determine how many genes are different between WT and Tup1 deletion strains in log phase. Are just as many genes different? Or is Tup1 more important in diauxic shift and stationary phase than log phase?
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Are the genes that are regulated by Tup1 normally regulated during diauxic shift or stationary phase compared with log growth?
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What fraction of the genes that are differentially expressed in Tup1 knockout yeast have Tup1 binding at the promoter? Enhancer? What fraction can be explained by Tup1, Hap1, Nrg1, Mig1 individually and together?
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The methodology used to generate the gene ontology enrichments should be described in the methods.
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The authors should provide genomewide data to support the statement that Tup1 and Rpd3 ChIP datasets have substantial overlap. They should also provide genomewide data to support the statement that there is substantial overlap between Rpd3 and Tup1. How much overlap is observed and how much is expected by chance?
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For Sds3, similar to Tup1 inactivation, it would be helpful to know how many genes change in with Sds3 inactivation in log phase in addition to diauxic shift and stationary phase.
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If the argument is that Sds3 and Xbp1 cooperate with Tup1 to affect gene expression, testing the gene expression changes that are associated with Tup1 in Sds3 or Xbp1 knockout strains would help the authors make this point.
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The final phenotype of extra DAPI positive blobs in the nucleus is not very specific or clear.
Significance
Discovering transcription factors that are critical for the entry into and maintenance of quiescence is an important area of discovery as relatively little is known about gene regulation during quiescence and understanding this process is fundamentally important for our understanding of cell biology. Previous studies had implicated Tup1 in stationary phase in yeast. These studies provide additional insight into the mechanism.
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Referee #1
Evidence, reproducibility and clarity
In this manuscript, the authors have investigated the role of the Tup1 corepressor in transcriptional repression that occurs during the establishment of quiescence in S. cerevisiae. Using ChIP-seq, they found that Tup1 is present at many sites during log phase growth, and that it localizes to new targets upon glucose exhaustion at a point called diauxie; post-diuaxie, Tup 1 remains associated with these sites in stationary phase (SP), which they define as growth of yeast cells for 3 days after inoculation in glucose-rich medium. To understand the significance of Tup1 relocalization during quiescence establishment, they performed differential expression (DE) analysis following RNA-seq in WT and tup1∆ cells at the diauxic shift and 3-day SP. The large number of DE genes in both cell states were consistent with a role for Tup1 in the regulation of key targets associated with the initiation of quiescence, although there was not a strict correlation between Tup1 occupancy at these DE genes. Next, they investigated the relationship of Tup1 to Xbp1 and Rpd3, factors that play a role in transcriptional repression during quiescence. They noted significant overlap between the binding sites for the three factors in either isolated quiescent cells or 3-day SP cells using published and newly acquired ChIP-seq data. However, the absence of Xbp1, a transcription factor that recruits the Rpd3 histone deacetylase in quiescent cells, did not alter Tup1 binding to its targets in these cells. RNA-seq analysis of xbp1∆ and tup1∆ SP cells found a significant overlap between the genes that were repressed in the absence of the two transcription factors. Moreover, Tup1, Xbp1, and Rpd3 were noted to be required for H3K23 deacetylation at repressed genes in SP. Finally, the authors asked if the Tup1 affects the position of nucleosomes at TSSs in SP cells by performing MNase-seq. They found that Tup1 and the chromatin remodeler Isw2, which interacts with Tup1, are required to position nucleosomes at the promoters of a family of glucose transporter genes, targets of Tup1 during the initiation of quiescence.
Comments:
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The authors chose to examine 3-day SP cells to interrogate quiescence because tup1∆ cells are highly flocculant, interfering with the isolation of purified quiescent cells. These cells are a mixture of both nonquiescent and quiescent cells, so it is not correct to state that they represent a quiescent cell population. The addition of EDTA to the gradients used to isolate quiescent cells could eliminate flocculation and permit the isolation of quiescent cells. EDTA is also often added to media in low amounts to reduce flocculation. The authors need to indicate the proportion of quiescent cells in their SP cultures by applying these tools.
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The authors reported that while Xpb1 and Tup1 share many overlapping binding sites, but that Xbp1 does not regulate Tup1's binding. What other factors might be responsible for their shared binding? Could histone deacetylation play a role? This could be addressed by a Tup1 ChIP in an sds3∆ mutant.
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Has PolII occupancy been examined in Log vs SP cells of tup1∆ to determine if Tup1 inhibits PolII association with its genes that are repressed ?
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The observation that tup1∆ cells have several nuclear puncta is intriguing, although the cytological images need to be improved.
Significance
This manuscript presents some new information that links Tup1, a multifacteted transcriptional co-repressor, to the repression of transcription that occurs during the establishment of yeast quiescence. The McKnight lab has previously shown that the Rpd3 HDAC plays an important role in this process, in part through its recruitment by the TF Xbp1. Tup1 also binds to Rpd3, and the overlap between the sites where Tup1 and Xpb1 bind and the targets that they repress, suggests a shared function in establishing transcriptional reprogramming. Whether this shared function is based only on the recruitment of the Rpd3 histone deacetylation is unclear, and, more importantly, there is also some question if deacetylation is in fact the main factor driving transcriptional repression during the initiation of quiescence. The finding that Tup1 is responsible for repositioning nucleosomes at a class of glucose transporters to repress the transcription of these genes suggests a specific function for Tup1 during the establishment of quiescence; however, this is apparently not a broad function. Thus, we are left with a lot of nice descriptive information as a result of well-done experiments that has not revealed much about the mechanism by which Tup1 regulates the global transcriptional reprogramming that occurs during quiescence.
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Reply to the reviewers
Manuscript number: RC-2022-01536
Corresponding author(s): Michael Glotzer
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- General Statements We thank the reviewers for their thoughtful and helpful comments. In general, the reviews were highly positive, although their reviews indicated parts of the manuscript that needed further clarification. We have made extensive changes that improve the clarity and rigor of this submission. We have performed several additional experiments which have extended our analysis in several ways detailed below. None of the conclusions have changed.
The following is a list of eight major changes implemented during the revisions. Point-by-point responses to the reviewers comments follow on subsequent pages.
- The reviews made clear that we needed to more explicitly discuss the AIR-1 depletion phenotype. This phenotype is complex, it does not result in a complete loss of asymmetry, unlike, for example, depletion of the centrosome component SPD-5. This is because, in AIR-1 depleted embryos, a PAR-2 and cortical flow-dependent pathway induces PAR-2 accumulation at both anterior and posterior poles that induces flows from each pole to the lateral region (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019; PMIDs 31155349, 31636075, 30861375, 30801250). These flows also modulate ECT-2 localization. To clarify this point which came up in multiple reviews, we now include an explanation of the complexity of the AIR-1 phenotype and we present an analysis of ECT-2 localization in embryos depleted of both AIR-1 and PAR-2.
In addition to the 95% confidence intervals that were present on our graphs, we now include indications of the results of statistical tests of significance to the results of different treatments.
We have revised the analysis ECT-2 accumulation in two ways. First, in the previous draft, we assessed the anterior accumulation over the anterior 40% and the posterior 15% of the embryo. We have revised this analysis comparing the anterior and posterior 20% of the cortex, respectively. This is simpler and more logical in contexts where embryos are symmetric. In addition, we altered the measurements of the length of the posterior boundary. Previously we used a common threshold value, below which we counted pixels to assess boundary length. During the revisions, we noticed that this value was not appropriate for our mutant transgenes which accumulated to higher levels. Therefore, we revised our analysis pipeline such that, for each embryo, we measure the average intensity of the cortex in the anterior 60% of the embryo. We set a threshold of 0.85* this average anterior intensity value. As before, cortical positions below this threshold contribute to the boundary length. This is a more robust and simpler means of evaluating the size of the posterior domain. Neither of these changes affect any of our conclusions, but they are simpler and more rigorous.
Most of our figures include quantification of the degree of ECT-2 asymmetry as well as the average anterior and posterior accumulation of ECT-2 as a function of time. While the images show the intensity profiles across the embryo, previously, we did not explicitly show a quantification of the average intensity of ECT-2 as a function of position along the embryo. A new graph, Figure 2Bv, shows this for control embryos and embryos in which tubulin is depleted and depolymerized. This shows that the MT depolymerization results in lower accumulation at the posterior of the embryo and higher accumulation at the anterior.
We provide documentary and quantitative evidence that ZYG-9 depletion induces potent cortical flows (Figure 3c and Figure 3, supplement 3), further bolstering the central role of cortical flows in inducing ECT-2 asymmetry.
As requested by reviewer 2 (R2b), we have included the analysis of ECT-2 distribution in Gα depleted embryos. As expected due to the lack of spindle elongation, the displacement of ECT-2 from the posterior cortex is greatly attenuated.
As requested by reviewer 2 (R2d), we now show that ECT-2C fragments accumulate on the cortex in embryos depleted of ECT-2.
One other important point raised by several reviewers concerns the behavior of the ECT-2 T634E allele. This allele, due to the substitution of a phosphomimetic residue, accumulates on the cortex at about 50% the level of the wild-type version. To investigate the possibility that this quantitative difference was the cause of the phenotype, we depleted both the wild-type and mutant ECT-2 constructs by RNAi (these are the sole sources of ECT-2 in the animals). First, we find that wild-type ECT-2 can be depleted to 20% of wild type levels with only a 13% rate of cytokinesis failure (when T634E is depleted to 20%, embryos fail more than 50% of the time). Thus the two-fold reduction in cortical ECT-2 seen in T634E not likely highly significant (ECT-2 is not haploinsufficient). In addition, embryos with ECT-2 T634E initiate ingression in a timely manner, but the furrows ingress more slowly than wild-type. In contrast, depletion of ECT-2 to 20% results in a delay in furrow initiation, but once these furrows form, they ingress at rates similar rates to wild-type. Thus, the T634E variant exhibits a behavior that is quite distinct from that resulting from a (strong) reduction in the levels of wild-type ECT-2.
Point-by-point description of the revisions
This section is mandatory. Please insert a point-by-point reply describing the revisions that were already carried out and included* in the transferred manuscript. *
(Reviewer comments: italicized 9 pt font, author response: plain text 10 pt font. Numbers have been added to the reviewer comments e.g. R2c=Reviewer 2, third comment)
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary
R1a* In this study the authors addressed how Ect2 localization is controlled during polarization and cytokinesis in the one-cell C. elegans embryo. Ect2 is a central regulator of cortical contractility and its spatial and temporal regulation is of uttermost importance. After fertilization, the centrosome induces removal of Ect2 from the posterior plasma membrane. During cytokinesis Ect2 activity is expected to be high at the cell equator and low at the cell poles. Similarly to polarization, the centrosome provides an inhibitory signal during cytokinesis that clears contractile ring components from the cell poles. Whether and how the centrosomes regulate Ect2 localization is not know and investigated in the study. *
This is an accurate summary of the goals of this study.
R1b *The authors start by filming endogenously-tagged Ect2 and find that Ect2 localizes asymmetrically, with high anterior and low posterior membrane levels during polarization and cytokinesis. They reveal that the centrosome together with myosin-dependent flows results in asymmetric Ect2 localization. Previous studies had suggested that Air1, clears Ect2 from the posterior during polarization and the authors expand those finding by showing that Air1 function is also required to displace Ect2 from the posterior membrane during cytokinesis. *
*To elucidate if Ect2 displacement is induced by phosphorylation of Ect2 by Air1, the authors investigate the localization of a C-terminal Ect2 fragment containing the membrane binding PH domain. When the predicted Air1 phosphorylation sites are mutated to alanine, the Ect2 fragment still localizes asymmetrically but exhibits increased membrane accumulation. *
*Finally, they investigate the functional role of Air-1 during furrow ingression. They demonstrate that embryos deficient of Air1 and NOP1 have impaired furrow ingression. Lastly, the authors sought to confirm that there is a direct effect of Air1 on Ect2 function by generating a phosphomimetic point mutation of Ect2 using Crispr. They find that the membrane localization of phosphomimetic Ect2 is reduced and consequently furrow ingression is impaired. *
This is an accurate summary of our results.
Major comments
R1c *It is not convincing that the six putative phosphorylation sites are targeted by the Air1. If Air1 phosphorylation displaces Ect2 from the membrane, a reduction in Ant/Post Ect2 ratio is expected in the phosphodeficient mutants, like after air1 RNAi. However this is not observed for cytokinesis or polarization (Fig. 5D(i); E). This suggests that phosphorylation of those sites is not essential for the asymmetric Ect2 localization. *
In otherwise wild-type embryos, phosphorylation of these sites is not required for asymmetric ECT-2 localization. Non-phosphorylatable ECT-2 variants exhibit asymmetric localization because these proteins relocalize due to myosin-directed flows. To test the role of phosphorylation, we examine the distribution of ECT-2 and ECT-2C fragments in myosin-depleted embryos in which the flows are blocked, under these conditions, transient local depletion is observed with the phosphorylatable variants, Fig 5E.
While AIR-1 promotes normal polarity establishment, as shown in several recent papers, cortical changes nevertheless occur in the absence of AIR-1. Specifically, a parallel PAR-2 dependent pathway induces weaker flows from both poles toward the equator. To further substantiate the effect of PAR-2 accumulation on ECT-2 accumulation in AIR-1 depleted embryos, we assayed ECT-2 accumulation in air-1(RNAi); par-2(RNAi) embryos (Figure 4, supplement 2). These results show that ECT-2 is nearly symmetric in these double depleted embryos. In addition we have edited the text to describe the unusual bi-polar PAR-2 accumulation that occurs in AIR-1 depleted embryos.
R1d *The authors aim to demonstrate that phosphorylation of the identified sites is important for cytokinesis. For this they investigate contractile ring ingression in the phosphomimetic point mutation. Since ring ingression is slower and fails in nop1 mutant they authors conclude that this demonstrates a functional importance of this site. I am not surprised that embryos ingress slower in this mutant since Ect2 localization to the membrane is reduced. This however does not show that this phosphorylation site is the target of the centrosome signal. Importantly, authors would need to demonstrate that Rho signaling and thus Ect2 activity, is increased at the poles, when phosphodeficient Ect2 is the only Ect2 in the embryo. *
The fact that a phosphomimetic residue at this site leads to reduced membrane localization is highly relevant, as we suggest that phosphorylation of this site contributes to the mechanism by which AIR-1 generates asymmetric ECT-2. Given the role of AIR-1 in regulating polarity, a version of ECT-2 that can not be phosphorylated would be predicted to be dominant lethal, necessitating a conditional expression strategy which does not currently exist in the early C. elegans embryo system (indeed we were unable to recover a T-> A allele at this site, despite extensive efforts). To avoid this issue, we used a viable, fertile, hypomorphic allele that is predicted to be less responsive to AIR-1 activity. The goal of this experiment was to evaluate whether the putative AIR-1 sites affect not only the NOP-1 pathway for furrow ingression, but also impact furrowing that is centralspindlin-dependent.
To complement this finding have performed experiments in which ECT-2 was partially depleted We used RNAi to partially deplete ECT-2 and ECT-2 T634E and measured the total embryo fluorescence of each ECT-2 variant and the kinetics of furrow ingression. Partial depletion of wt ECT-2, to ~ 20% of control levels leads to delay in furrow formation and all but 2/18 (11%) of embryos complete cell division. In contrast, a similar depletion of ECT-2T634E depletion results in a failure of furrow ingression in ~52 % of embryos. Furthermore, while ECT-2T634E embryos initiate furrowing with normal kinetics, they exhibit a slower rate of furrow ingression, in contrast, partial depletion of WT ECT-2 results in a delay in furrow initiation, but once initiated, the rate of furrow ingression is not significantly affected. These results demonstrate that ECT-2T634E behavior can not simply be explained by a modest reduction in membrane binding.
R1e *The authors use the Aurora A inhibitor MLN8237: It was shown prior (De Groot et al., 2015) that this inhibitor is not highly specific for Aurora A, and that it also inhibits Aurora B. Thus experiments need to be repeated with MK5108 or MK8745. They should also be conducted during polarization. Why does Aurora A inhibition not abolish asymmetry? That would be expected? *
The role of AIR-1 in symmetry breaking during polarization is previously published, including with chemical inhibitors (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019, PMID 31155349, 31636075, 30861375, 30801250). ECT-2 localization depends on both the spatial regulation of AIR-1 activity and the distribution of cortical factors that contribute to ECT-2 cortical association, as a result of cortical flows. During acute, chemical perturbation of AIR-1 it is likely that these factors, which were polarized prior to drug treatment, remain polarized, allowing the residual cortical ECT-2 to remain asymmetric. The reviewer is correct about the specificity of MLN8237 and we do not rely on it alone to demonstrate the role of AIR-1. Rather this experiment is a complement to our AIR-1 depletion studies, which are sufficient to establish specificity. We present this experiment merely to show that AIR-1 acutely regulates ECT-2 during cytokinesis in embryos that were entirely unperturbed during polarization.
R1f *There is no statistical analysis of the results in the entire study. For all claims stating a change in Ant/Post Ect2 ratio or Ect2 membrane localization selected time points should be statistically compared: for example the main point of Fig.1 is that Ect2 becomes more asymmetric during anaphase. Thus a statistical analysis of the Ect2 ratio at anaphase onset (t=0s) and eg. t=90 s after anaphase onset should be performed; or Fig. 3A nop-1 mutant Ant/Post Ect2 ratio during polarization: again statistical analysis of control and nop-1 mutant embryos is needed at a particular time point. *
All of the graphs were presented with the mean of ~10 embryos per condition and included the 95% confidence intervals. In the revised manuscript, we have included tests of statistical significance, at each time point. While non-overlapping confidence intervals generally suggest statistical significance, we include these analyses on the graphs as it can be difficult to assess statistical significance when the confidence intervals overlap.
R1g *The aim of Fig. 2B is to demonstrate that Ect2 localization is independent of microtubules, however they still observe some microtubules with the Cherry-tubulin marker and those are even very close to the membrane and therefore could very well influence Ect2 on the membrane. Therefore I am not convinced that this experiment rules out that microtubules have no role in regulating Ect2 localization. *
We do not exclude that microtubules play a contributing role in ECT-2 phosphoregulation, but rather we conclude that the primary cue is the centrosome. Indeed, microtubules can play an important role in controlling spindle positioning which affects the proximity of the centrosome to the cortex.
The manuscript states, “Despite significant depletion of tubulin and near complete depolymerization of microtubules (Figure 2B, insets), we observed strong displacement of ECT-2 from a broad region of the posterior cortex during anaphase (Figure 2B).” Thus, despite dramatic reductions in microtubules, not only does ECT-2 become polarized, it becomes hyperpolarized. In contrast, were microtubules directly involved in ECT-2 displacement, one would expect a reduction in polarization as a result microtubule depolymerization. Conversely, though SPD-5 depleted embryos contain far more microtubules than embryos in which microtubule assembly is suppressed, ECT-2 is not polarized in SPD-5 depleted embryos. Thus in the manuscript, we conclude, “Collectively, these studies suggest that ECT-2 asymmetry during anaphase is centrosome-directed.” This conclusion is well supported by the results shown.
R1h *Throughout the paper the authors should tone down their statement that Air1 breaks symmetry by phosphorylating Ect2, since phosphorylation of Ect2 by Air2 is not shown. *
We agree with this comment and will make the necessary edits to the text. Indeed, this is the reason why we had included the final section in our original draft, “Limitations of this study” which makes this point explicitly.
R1i *I understand that the establishment of Ect2 asymmetry is important for polarization. However, how does asymmetric Ect2 localization result in more active Ect2 at the cell equator, which is required for the formation of the active RhoA zone? Would we not expect an accumulation of Ect2 at the cell equator, or if that is not the case more active Ect2 at the equator versus the poles? *
The pseudocleavage furrow forms as a result of the anterior enrichment of active RHO-1 and its downstream effectors. There is no evidence for a local accumulation of active RHO-1 specifically at the site of the pseudocleavage furrow. Rather, this furrow forms at the boundary between the portion of the embryo where RHO-1 is active and the posterior of the embryo where RHO-1 is far less active (Figure 1 Supplement 2). We suggest that aster-directed furrowing during cytokinesis likewise results from asymmetric accumulation of the same components, without them necessarily being specifically enriched solely at the furrow.
While cytokinesis generally involves an equatorial contractile ring, furrow formation can be driven by an asymmetric - i.e. non-equatorial - accumulation of actomyosin. This behavior is exemplified during pseudocleavage during which the entire anterior cortex is enriched for actomyosin and the posterior is depleted of myosin (Figure 1 Supplement 2). Several published studies provide evidence that the asymmetric pattern of myosin accumulation contributes to cytokinesis (PMID 22918944, 17669650).
Minor comments
R1j *Can the authors explain why the quantification of Ant/Post Ect2 ratio in control embryos differs in different figures? For example: in Fig. 1D i) a slight increase of Ect2 asymmetry ratio is seen at around 80 s after anaphase onset. In comparison, in Fig. 2C (i) this increase is not obvious. Are those different genetic backgrounds? *
In figure 1 D, time 0 begins at anaphase onset, whereas in 2C, time 0 is specified at the time of nuclear envelope breakdown (NEBD). The duration between NEBD and anaphase onset is ~130 sec and an increase in ECT-2 polarization is observed at 220 s post NEBD, ie 90 sec post anaphase onset comparable to that seen in Fig 1D.
R1k *One key point of the paper is that myosin-dependent cortical flows amplify Ect2 asymmetry during polarization and cytokinesis. During polarization the data is convincing, however during cytokinesis Ect2 ratio is only slightly decreased after nmy-2 depletion, again is this decrease even significant? *
Figure 3 supplement 1 shows a significant difference in ECT-2 asymmetry between control and myosin-depleted embryos.
R1l *In the introduction: "Centralspindlin both induces relief of ECT-2 auto-inhibition and promotes Ect2 recruitment to the plasma membrane" it should be added 'Equatorial' membrane, since Ect2 membrane binding is, to my knowledge, not compromised in centralspindlin mutants or in Ect2 mutants that cannot bind centralspindlin. *
Generally speaking, the reviewer is correct that cortical accumulation of ECT-2 globally is centralspindlin independent. However, as seen in e.g. ZYG-9 depleted embryos, ECT-2 is recruited to the posterior cortex in a centralspindlin-dependent manner. Thus centralspindlin can promote ECT-2 accumulation to the cortex and the site of that accumulation will be dictated by the position of the spindle midzone.
R1m *Labels in the figures are often very small eg Fig. 1 ii-v) and difficult to read. In addition it is easier for the reader if the proteins shown in the fluorescent images is also labeled in the figure (eg Fig. 2B add NG-Ect2). *
These useful suggestions have been incorporated.
R1n *Material and methods it should be mentioned which IPTG concentration was used. *
The IPTG concentration (1 mM) has been added to the revised text.
R1o *The authors speculate that the Air1 phosphorylation sites in Ect2 PH domain prevent binding to phospholipid due the negative charge. At the same time, the authors propose that the PH domain binds to a more stable protein on the membrane, which is swept along with the cortical flows and they propose anillin could be that additional binding partner. I might miss something, but do the authors suggest Ect2 has two binding partners: anillin and the phospholipids? It would be necessary to explain this better. *
*The authors should test if anillin represents the suggested myosin II dependent Ect2 anchor. For this they should check if Ect2 localization to the membrane is altered upon on anillin RNAi. *
This summary of our model is largely correct, though we do not know the identity of the more stable cortical anchor(s). While we suspect the PH domain binds to a phospholipid, ECT-2 cortical localization also requires ~100 residues C-terminal to the PH domain. It is likely that this domain interacts with a cortical component.
In preliminary experiments, ECT-2 accumulation is not strictly anillin-dependent. However, functional redundancy may obscure a contribution of anillin. Anillin was mentioned simply because of the evidence for a physical interaction between ECT-2 and anillin (Frenete PMID 22514687). In the revised manuscript we also include the possibility that ECT-2 accumulations involves one or more anterior PAR proteins. The identity of the cortical anchor(s) is an interesting question for future studies. We consider this question beyond the scope of the current manuscript.
R1p *The title of fig. 3 does not fit the statement the authors want to make, since the key point is how Ect2 polarization is affected and not membrane localization in general. *
Thank you for this suggestion. The title has been changed to “Cortical flows contribute to asymmetric cortical accumulation of ECT-2”
R1q *In Fig 4A/C. After air1 depletion the authors observe a reduction in Ect2 asymmetry. Why are the centrosomes not marked in the figures? Because they cannot be detected? The authors would also need to show that the mitotic spindle and centrosomes are no altered by air1 RNAi in the zyg9 mutant. Otherwise the observed effect might be indirect. *
Centrosomes are perturbed by depletion of AIR-1 (Hannak, PMID 11748251), but they are still detectable and their positions will be added to figure 4. As has been extensively demonstrated, AIR-1 depletion does lead to attenuated spindles and defects in spindle assembly, some of which are also seen TPXL-1 depleted embryos. These consequences of AIR-1 depletion does complicate the analysis, but this is typical of factors that regulate many processes. This is one of the key reasons why we used ZYG-9 depletion in combination with AIR-1 depletion to overcome these indirect effects.
R1r *The authors state that tpxl-1 depletion attenuates Ect2 asymmetry, this is not seen in the quantification ((Fig. 4B(i)). The main phenotype they observe is that Ect2 levels on the membrane increase (Fig. 4 (ii) and (iii). They go on testing the function of tpxl1 by depleting tpxl1 in the zyg9 mutant, where the centrosomes are close to the posterior cortex. Here they see no effect on Ect2 asymmetry. Based on that they conclude that tpxl1 has no role in this process. To me this finding is not surprising since the centrosome is close the cortex in zyg9 mutant embryos. Therefore sufficient amounts of active Air1 could reach the membrane and displace Ect2. Thus an amplification of the inhibitory signal by tpxl1 on astral microtubules might not be required. The authors need to mention this possibility and tone down their statment (also in the discussion) that tpxl1 is not required for this process. *
In the text, we state, “Cortical ECT-2 accumulation is enhanced by TPXL-1 depletion, though the degree of ECT-2 asymmetry is unaffected (Figure 4B).… we observed robust depletion of ECT-2 at the posterior pole in zyg-9 embryos depleted of TPXL-1, but not AIR-1 (Figure 4C). We conclude that while AIR-1 is a major regulator of the asymmetric accumulation of ECT-2, the TPXL-1/AIR-1 complex does not play a central role in this process.” We consider this to be an accurate description of the results. In sum, we have found no evidence that TPXL-1 contributes to generating ECT-2 asymmetry, beyond its well established role in regulating spindle length and position. The are several other processes that are known to be AIR-1 dependent and TPXL-1 independent; these primarily involve the centrosome (Ozlu, PMID 16054030). Given that TPXL-1 associates with astral microtubules, the fact that microtubule depletion can enhance ECT-2 asymmetry also argues against a requirement for TPXL-1.
R1s *It was shown that the C-terminus of Ect2 is sufficient and the PH domain is required for Ect2 membrane localization in C. elegans (Chan and Nance, 2013; Gomez-Cavazos et al., 2020). Papers should be cited. *
Thank you for this helpful comment. Chan and Nance 2013 indeed shows that the ECT-2 C-term is sufficient to localize to the cell cortex. In contrast, the Gomez-Cavasos paper (PMID 32619481) shows in figure S2 that the PH domain is required for cortical localization of ECT-2; this paper does not focus extensively on cortical accumulation of ECT-2. We have cited Chan and Nance in the revised manuscript.
R1t *The authors find that nmy-2 depletion results in loss of asymmetry for the Ect2 C-term and Ect2 3A fragment during polarization. Why is the same experiment not shown for cytokinesis? *
Strong depletion of NMY-2 prevents polarity establishment, resulting in symmetric spindles, which in turn results in symmetric ECT-2 accumulation. Thus, the requested experiment would not provide significant additional information.
R1u *Air1 is targeted to GFP-C-term Ect2 fragment via GFP-binding to determine the influence on GFP-C-term Ect2 localization (Fig. 5F). They state that they see a reduction of Ect2 C-term but not of C-term 3A after targeting. The reader has to compare Fig. 5D with F. Since the differences are not big, they need to compare the Ect2 C-term and Ect2 C-term 3A with and without Air1 targeting in the same graph (plus statistics). Otherwise this statement is not convincing. *
It is not straightforward to directly compare ECT-2C in the presence and absence of GBP-mCherry-AIR-1, because the GBP:AIR-1 fusion protein recruits a large fraction of ECT-2C to the centrosome. For this reason we think it is best to compare the behavior over time of ECT-2C and ECT-2C3A in the presence of GBP-mCherry-AIR-1. At the onset of anaphase, these two fragments localize similarly, but they then diverge over time.
R1v *In Fig. 6A the authors determine the contribution of air1 to furrowing. For this they deplete air1 in the nop1 mutant. According to previous studies, air1 mutants have a monopolar spindle. How can the authors analyze the function of air1 in cytokinesis when the spindle is monopolar? Did the authors do partial air1 depletion? They authors need to show that there is not major effect on the spindle and centrosome for their conditions. For comparison air1(RNAi) alone has to be included, otherwise the experiment is not conclusive. *
AIR-1 depletion does not result in a monopolar spindle in C. elegans embryos, though the spindle is attenuated and disorganized (PMID 9778499). TPXL-1 depletion also results in short, well organized spindles (PMID 19889842). The concerns are the reason we performed the ZYG-9 depletion experiments in Figure 4C to ensure the centrosomes are proximal to the cortex.
R1w *Upon air1(RNAi) in the nop1 mutant NMY2 intensity seems decreased and not increased. Can the authors comment on that, since that is opposite of what is expected. *
This is expected as previous studies have shown that NOP-1 contributes to RHO-1 activation during polarization and cytokinesis (Tse, PMID 22918944). (NOP stands for No Pseudocleavage).
R1x *In Fig 6B they introduce a phosphomimetic point mutation in S634 [sic, T634] in the endogenous Ect2 locus. It not clear why the authors chose this site out of the six putative sites and why they only chose one and not 3 or 6 sites? This needs some explanation. *
In our early work with ECT-2 transgenes, we found that a T634E mutation strongly affected cortical ECT-2C, so we decided to assess its affect on the function and localization of endogenous ECT-2. While we were able to recover a T634E variant, we were not able to recover a T634A variant, despite considerable effort. Based on these experiences, we anticipated that we would be unable to recover a mutant version of ECT-2 in which all sites were changed to phosphomimetic.
R1y *In the model (fig. 7) no astral microtubules are shown during pronuclear meeting and metaphase. Astral microtubules are present at this stage and should be added to the schematic. *
MTs will be added to the figure.
Reviewer #1 (Significance (Required)):
R1z *The centrosomes inhibit cortical contractility during polarization and cytokinesis in the one-cell C. elegans embryo. Centrosome localized Air1 was proposed to be part of this inhibitory signal, however the phosphorylation target of Air1 is not known. The identification of Ect2 as a phosphorylation target of Air1 would be a great advancement in the field. However, the presented manuscript lacks convincing data that Ect2 is the phosphorylation target of Air1 during polarization and cytokinesis. *
We explicitly acknowledge that we have not directly shown that AIR-1 phosphorylates ECT-2. However, we have shown that (i) AIR-1 inhibits cortical ECT-2 localization, (ii) the negative regulator of AIR-1, SAPS-1, promotes AIR-1 cortical accumulation, (iii) that the cortical localization domain of ECT-2 has putative AIR-1 sites, which, when mutated to non-phosphorylatable residues leads to increased cortical accumulation of ECT-2 (and (iv) phosphomimetic residues reduce its cortical accumulation), and (v) that these AIR-1 sites are required to render GFP-ECT-2C responsive to GBP-AIR-1. For these reasons we feel that our data makes a strong, albeit indirect, case that AIR-1 regulates ECT-2, even though we clearly acknowledge that we do not directly show that AIR-1 directly phosphorylates ECT-2.
Direct proof would require the demonstration that AIR-1 phosphorylates ECT-2 in vivo. This would be difficult to show as ECT-2 phosphorylation is likely transient, it likely affects only a subset of the total ECT-2 pool, and it likely results in loss of membrane association of ECT-2. As it it not possible to synchronize C. elegans embryos, biochemical analysis would be very difficult. Even a phosphospecific antibody for the putative ECT-2 phosphosites might not be particularly informative, as it would be predicted to give a diffuse cytoplasmic signal.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
R2a* In this work, Longhini and Glotzer investigate the localization of an essential regulator of polarity and cytokinesis, RhoGEF ECT-2, in the one-cell C. elegans embryo. The authors show that centrosome localized Aurora A kinase (AIR-1 in C. elegans) and myosin-dependent cortical flows are critical in asymmetric ECT-2 accumulation at the membrane. Since membrane interaction of ECT-2 is dependent on the Pleckstrin homology domain present at the C-terminus of ECT-2, they further analyzed the importance of putative AIR-1 consensus sites present in this domain. The authors linked the relevance of these sites in controlling ECT-2 localization and its significance on cytokinesis. The manuscript is well written, the work is interesting, and the data quality is high. *
We thank the reviewer for their critique.
Major comments:
R2b - In Fig. 2, the authors claim that the centrosomes and the position of the mitotic spindle are critical in regulating the asymmetric enrichment of ECT-2 at the membrane. To test the relevance of spindle positioning on ECT-2 localization, the authors depleted PAR-3 and PAR-2. The authors observed that the ECT-2 asymmetry is affected in these settings. However, PAR-3 or PAR-2 depletion impacts polarity, which is critical for many cellular processes, including spindle positioning. Can the authors try to specifically misposition the spindle without affecting polarity? For instance, by depleting Galpha/GPR-1/2 and assessing the impact of such depletion on ECT-2 localization.
Thank reviewer for good suggestion. We have performed the suggested experiment (presented in Figure 2, supplement 2). As one might predict, ECT-2 starts out polarized as Gα is not required for polarity establishment. During anaphase, ECT-2 becomes more symmetric in Gα depleted embryos as compared to wild-type.
R2c *-I wonder why the intensity of ECT-2 at the anterior and posterior membrane decreases in air-1(RNAi) post anaphase onset (Fig. 4A)? Moreover, I fail to observe a significant asymmetric distribution of ECT-2 in embryos depleted for PERM-1. Therefore it appears that the difference between DMSO and MLN8237-treated embryos is not substantial (at least in the images)? *
We do not have a complete or rigorous explanation for all the changes in cortical ECT-2, but they are highly reproducible. We speculate that there are cell cycle regulated changes in ECT-2 accumulation, in addition to its regulation by AIR-1. For example, in figure 1, a strong reduction in both anterior and posterior cortical ECT-2 is evident beginning at approximately -350 sec, which may reflect the initial stages of Cdk1 activation. This may result from cell cycle regulated modulation of ECT-2, as there is evidence that mammalian ECT-2 is subject to a very potent inhibition membrane association by Cdk1 (PMID 22172673). Alternatively, there could be cell cycle modulation of the cortical factor that serves as the “co-anchor” of ECT-2. The ability of GBP-AIR-1 to induce GFP-ECT-2C dissociation also appears cell cycle regulated.
Consistent with a cell cycle regulated component, note that NEBD is delayed in AIR-1 depleted embryos (PMID 17669650, 17419991, 30861375). This delay results in a shorter interval between NEBD and e.g. the peak in Cdk1 activation, explaining the earlier decrease in AIR-1(RNAi) embryos vs. control, relative to NEBD.
Our quantitative analysis indicates a significant increase of cortical ECT-2 upon treatment with MLN8237. In addition, the quantitation in the previous version did show a significant polarization of ECT-2 in PERM-1-depleted embryos prior to treatment. We have revised this figure to simply show an acute increase in cortical ECT-2 upon drug treatment, as the focus of this experiment was solely to show that ECT-2 cortical accumulation is acutely responsive to chemical inhibition during cytokinesis in otherwise normal embryos.
*-The data in Fig. 5 and 6 are exciting but raise a few concerns: *
R2d *a). The authors show that ECT-2C localization mimics the localization of endogenous tagged ECT-2. However, all these analyses with ECT-2C and various mutants are performed in the presence of endogenous ECT-2. Can the author check the localization of these mutant strains in conditions where the endogenous proteins are depleted? I understand that the cortical flow would be perturbed in conditions where endogenous ECT-2 is depleted. However, I suspect that one can analyze the anaphase-specific distribution. *
We have examined ECT-2C localization in embryos depleted of ECT-2. Cortical localization of ECT-2C is not dependent upon endogenous ECT-2. This result is now shown in figure 5 supplement 1. However, as the reviewer suggested, embryos depleted of ECT-2 do not show a high degree of ECT-2C asymmetry as ECT-2 is required for the cortical flows that amplify the symmetry breaking during polarization. During cytokinesis, ECT-2C does show a modest change in localization at the poles; the extent of the polar reduction is limited and the changes are symmetric as ECT-2 displacement causes spindles to be symmetrically positioned and limits their elongation during anaphase.
R2e *b). Can the author comment on why ECT-2C does not accumulate at a similar level as ECT-2C(3A or 6A) at the cell membrane when AIR-1 is depleted (compare Fig. 5D with Supplemental Fig. 5)? *
When ECT-2C(3A or 6A) are expressed in otherwise wild-type embryos, embryo polarization occurs, resulting in anterior-directed flows that concentrate the factor(s) that enables the anterior enrichment of ECT-2 (and ECT-2C 3A/6A). By contrast, when AIR-1 is depleted, most embryos exhibit a “bipolar” phenotype in which PAR-2 is recruited to both anterior and posterior poles, and the actomyosin network becomes somewhat concentrated laterally (PMID 30801250, 30861375, 31636075). The differential positioning of the actomyosin network in AIR-1 depleted embryos is likely responsible for the interesting difference that the reviewer points out. This section of the results states. “Nevertheless, these variants accumulated in an asymmetric manner. ECT-2C asymmetry temporally correlated with anteriorly-directed cortical flows (Figure 5 D,E), raising the possibility that asymmetric accumulation of endogenous ECT-2 drives flows that cause asymmetry of the transgene, irrespective of its phosphorylation status.”
R2f *c). Does the cortical localization of the ECT-2C(6A) mutant become symmetric upon further depletion of AIR-1? Of course, if the asymmetric distribution of ECT-2C(6A) is dependent on the presence of endogenous protein in the cellular milieu, the point raised earlier will help address this concern. *
We have not performed this exact experiment with ECT-2C-3A though we have performed it with a longer ECT-2 C-terminal fragment (aa 559-924). As expected, due to the considerations described above, the asymmetry of ECT-2C-3A is reduced when AIR-1 is depleted. Likewise, ECT-2C-6A is becomes symmetric when endogenous ECT-2 is depleted due to the dependence of its asymmetry on cortical flows, as discussed above.
In the revised manuscript, we provide additional explanation of the AIR-1 depletion phenotype which will explain the origin of the asymmetric distribution of ECT-2.
R2g *d). The authors predict that the AIR-1 mediated phosphorylation delocalizes ECT-2 from the polar region of the cell cortex. Since the posterior spindle pole is much closer to the posterior cortical region, the delocalization is much more robust at the posterior cell membrane. I wonder why targetting AIR-1 at the membrane (GBP-mCherry-AIR-1) does not entirely abolish GFP-ECT-2C membrane localization? Can the author include the localization of GBP-mCherry-AIR-1 in the data? Also, do we know for sure if GBP-mCherry-AIR-1 is kinase active? *
The GBP-mCherry-AIR-1 transgene was obtained from the Gönczy lab which demonstrated that it has some activity (PMID 30801250). Given that centrosomal AIR-1 (as compared to astral AIR-1) is the primary pool of AIR-1 responsible for modulating cortical ECT-2 levels, it is a not clear that the GBP-fused form of AIR-1 is as active as the centrosomal pool of AIR-1; indeed we suspect it is significantly less active, similar to the manner in which TPXL-1/AIR-1 appears less active towards ECT-2 than centrosomal AIR-1. Indeed as the reviewer suggests, were this pool of AIR-1 highly active, we would expect that its cortical recruitment would preclude embryo polarization, and this transgene would cause lethality when expressed with a GFP-tagged cortical protein. These concerns notwithstanding, we do observe a specific reduction in the anterior accumulation of ECT-2C as compared to ECT-2C3A, suggesting that this form of the kinase has some ability to modulate ECT-2C.
Co-expression of GFP-ECT-2C with GBP-mCherry-AIR-1 induces the centrosomal/astral accumulation of GFP-ECT-2C, which is highly visible in the figure and not seen in the absence of GBP-mCherry-AIR-1. Not surprisingly, the co-expression also induces a cortical pool of GBP-mCherry-AIR-1 that is not seen in the absence of GFP-ECT-2C. These redistributions indicate formation of the complex between GFP-ECT-2C and GBP-mCherry-AIR-1. The mCherry-AIR-1 images could be added as insets to the figure, but in our opinion, they would not make a substantive contribution, given the dramatic accumulation of centrosomal GFP-ECT-2C.
R2h *e). The authors show that centrosomal enriched AIR-1 [spd-5(RNAi)], but not the astral microtubules localized AIR-1 [tpxl-1(RNAi)], is vital for ECT-2 membrane localization. Interestingly, the authors showed that AIR-1 acts in the centralspindlin-directed furrowing pathway (Fig. 6A). I wonder if the authors can combine NOP-1 depletion with TPXL-1 depletion? I guess this will further help to exclude the function of TPXL-1 in the centralspindlin-directed furrowing pathway. *
We would like to clarify that our data indicates that AIR-1 acts on both the centralspindlin-independent furrowing (e.g. the anterior furrow in 4C), as well as centralspindlin-dependent furrowing (Figure 6).
While the experiment the reviewer proposes appears simple in theory, the interpretation is potentially a bit more complex, due to the role of TPXL-1 in spindle elongation, which can affect centralspindlin-directed furrowing. That said, there are two published experiments and one experiment in the manuscript that indicate that centralspindlin dependent furrowing can occur in TPXL-1 depleted embryos. First, Lewellyn et. al. showed that while tpxl-1(RNAi) embryos furrow, tpxl-1(RNAi); zen-4(RNAi) embryos do not, suggesting centralspindlin can function in the absence of TPXL-1. Second, the same paper shows that embryos doubly depleted of TPXL-1 and GPR-1/2 exhibit multiple furrows. Our previous work has shown that furrowing in Galpha-depleted embryos is centralspindlin dependent (Dechant and Glotzer). Furthermore, in the current manuscript we found that embryos depleted of both TPXL-1 and ZYG-9 form posterior furrows (8/8 embryos, 6/8 furrows were strong furrows) although the appearance of these furrows is delayed, presumably due to the reduction in spindle elongation due to TPXL-1-depletion. As described in the manuscript, these posterior furrows have been previously shown to be centralspindlin dependent and NOP-1 independent.
In accordance with these results, and in direct response to the reviewer’s specific suggestion, we do observe furrowing in nop-1(it142); TPXL-1(RNAi) embryos (10/10 embryos furrow, 9/10 complete cytokinesis) . Thus, all of the available results indicate that TPXL-1 is largely dispensable for centralspindlin dependent furrowing. However, the role of TPXL-1 in centralspindlin-dependent furrowing is not a focus of the manuscript, thus we do not favor including this result, as it distracts from the primary focus of the study.
R2i *f). Why do NMY-2-GFP cortical levels appear lower in 30% of the embryos that show various degrees of cytokinesis defects (Fig. 6A)? *
There are a number of possible origins of the variability. As shown in (Reich 2019, Kapoor 2019, Zhao 2019, Klinkert 2019, PMID 31155349, 31636075, 30861375, 30801250), AIR-1 depletion results in variable polarization (unpolarized PAR-2, bipolarized PAR-2, anterior PAR-2, posterior PAR-2). Furthermore, spindles in AIR-1 depleted embryos exhibit somewhat variable positioning. While we were unable to correlate these sources of variability with furrow formation, these results demonstrate that AIR-1 depletion impairs furrowing directed by centralspindlin, which was not entirely expected, given that (i) AIR-1 depletion potently suppresses NOP-1 dependent flows of cortical myosin, as evidenced by the loss of an anterior furrow in AIR-1(RNAi); nop-1(it142) embryos and (ii) centralspindlin directed furrowing can occur in the posterior in ZYG-9 depleted embryos both in the presence or absence of AIR-1 (Figure 4C).
R2j *g). The authors report that phosphomimetic mutation at the phospho-acceptor residue in ECT-2 impacts its cortical accumulation. This strain, together with NOP-1 depletion, affects furrow ingression. One explanation for this phenotype is that phosphomimetic mutant weakly accumulates at the membrane. However, one interesting observation is that ECT-2T634E enriches at the central spindle (Fig. 6B, panel 120 sec), which somehow I could not find in the text. Could this additional localization of ECT2 at the central spindle contribute to the cytokinesis defects that the authors have observed? The microscopy images the authors have included show that ECT-2T634E significantly localizes at the equator at the time of furrow initiation. Can the authors add the localization of ECT2 wild-type and ECT-2T634E in NOP-1 depleted conditions where they see an apparent impact on the cytokinesis? Similarly, if the authors include the localization of NMY-2 in these conditions-it will further add more weightage to the data. *
We regularly detect trace amounts of ECT-2 on the central spindle and this is slightly enhanced at in the ECT-2T634E mutant. However, given the large cytoplasmic pool of ECT-2, it seems unlikely that the slight enrichment of ECT-2 on the central spindle significantly affects the cortical pool of ECT-2, though the reduction in cortical ECT-2 may facilitate its enrichment on the central spindle.
As shown in figure 3B, depletion of NOP-1 does not dramatically affect cortical ECT-2 levels in wild-type embryos. Likewise, we did not observe a significant effect of NOP-1 depletion in ECT-2 T634E, thus we decided not to include this negative result.
As discussed in general point 8, we suggest the modest reduction in the membrane pool of ECT-2 is unlikely to be the primary cause of the T634E, but rather the ability of AIR-1 to modulate induce its relocalization. Consistent with this interpretation, the embryos that failed ingression tended to have more symmetric spindles, which could limit the residual cortical flows that facilitate furrow ingression.
Minor comments:
R2k -An explanation of how the timing of NEBD was analyzed in multiple settings would be helpful.
Depending on the experiment, we used either ECT-2:mNG fluorescence (it is excluded from the nucleus until NEBD) and/or the Nomarski images to score NEBD.
R2l ____-*The authors mentioned on p. 6-'Despite significant depletion of tubulin.....during anaphase'. These experiments are performed in the near complete depolymerization of microtubules; thus, regular anaphase will not establish. I understand that the authors are monitoring localization wrt the timing similar to anaphase in the non-perturbed condition, and thus a bit of change in the sentence is required. *
Thank you for highlighting this point. We have substituted “following mitotic exit” for “anaphase”. In these images, mitotic exit can be scored by the emergence of contractility.
R2m*-After testing the relevance of SPD-5 (that primarily acts on PCM and not on centrioles)-the authors write on p. 6 that 'two classes of explanation...early embryo'. I did not understand the importance of this sentence here. *
To clarify, we deleted the words “classes of” from the sentence in question and following that sentence we added the word, “first” indicating that we were explaining the first of the two possible explanations
R2n*-The observed impact of spd-5 (RNAi) on ECT-2 localization could be because of the effects of SPD-5 depletion on centrosomal AIR-1? The authors can link the impact of SPD-5 depletion not only with the centrosome but also with AIR-1 in the discussion. *
Indeed, it is well established that SPD-5 is required for centrosomal AIR-1 (Hamill DR, et. Al Dev Cell (2002). The revised discussion now states, “Specifically, during both processes, ECT-2 displacement requires the core centrosomal component SPD-5, which is required to recruit AIR-1 to centrosomes{Hamill et al., 2002, #1201}, but ECT-2 displacement is not inhibited by depolymerization of microtubules and it does not require the AIR-1 activator TPXL-1 (see below).”
R2o-In the various Figure legends, sometimes the authors mention time '0' as anaphase, and other time as anaphase onset.
In all cases, anaphase onset was intended and the legends will be corrected.
Reviewer #2 (Significance (Required)):
R2p *The manuscript is well written, the work is interesting, and the data quality is of good quality. *
We thank the reviewer for their encouragement as well as for their thoughtful critique!
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
R3a* Symmetry breaking is the process by which uniformity of the system is broken. Many biological systems, such as the body axes establishment and cell divisions in embryos, undergo symmetry breaking to pattern cellular interior design. C. elegans zygote has been a classic model system to study the molecular mechanism of symmetry breaking. Previous studies demonstrated critical roles of centrosomes and microtubules in breaking symmetry in the actin cytoskeleton during anterior-posterior polarization and cytokinesis. It, however, remains elusive how centrosomes and/or microtubules regulate the assembly and contractility of the actin cytoskeleton. Recent reports identified Aurora-A AIR-1 as the key centrosomal kinase that suppresses the function of the actin cytoskeleton, but little is known about a substrate of the kinase during symmetry breaking events. *
Longhini and Glotzer proposed in this manuscript that RhoGEF ECT-2 plays a critical role in symmetry breaking of the actin cytoskeleton under the control of AIR-1 kinase. Kapoor and Kotak (2019) previously proposed the same GEF as a downstream effector of centrosomes, but this work did not provide direct evidence for ECT-2 as the AIR-1 effector. This manuscript identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. Although this manuscript lacks direct in vivo and in vitro evidence for phosphorylation of ECT-2 by AIR-1 kinase, the above findings reasonably support a model where in AIR-1 promotes the local inhibition of ECT-2 on the cortex. Design of the experiments, the quality of images, and data analysis are reasonable, and the main text was written very well. The main conclusion of this work will attract many readers in cell and developmental biology fields. I basically support its publication in the journals supported by Review Commons with minor revisions (see below).
We thank the reviewer for their encouraging remarks and helpful comments.
Minor comments
R3b 1) In Figures 2A and 2B, the authors claimed apparent correlation between spindle rocking and ECT-2 displacement. However, because both MTs and ECT-2 in Fig2AB images are blur, I cannot convince myself whether ECT-2 intensities on the cortex showed negative correlation with the distance between the posterior centrosome and the cortex. The authors may want to provide quantitative data set and use a statistical test to support this conclusion.
Only figure 2A focuses on the rocking. The important structure to assess is the position of the centrosome, as the astral arrays of microtubules are largely radially symmetric (except towards the spindle midzone). As this point in the manuscript were were not discriminating between the astral microtubules and the centrosomes, rather focusing on the overall position of the aster as a whole. Figures 2B, 2D, Fig 2 Supplements 1 and 2, Fig 3C, and Fig 4B, summarized in figure 7A provide quantitive evidence that the centrosome-cortex distance is an important determinant of ECT-2 cortical accumulation.
R3c *2) Figure 2D would [sic; presumably should] show a ratio between the anterior/posterior pole and the lateral cortex. *
The reviewer is presumably noticing that the lateral cortex is brighter than the poles when PAR-3 is depleted. While we agree with this assessment, the point of this experiment was to evaluate whether both centrosomes are equally capable of regulating cortical ECT-2 at the respective poles. It appears to us that comparing the anterior and posterior poles is the appropriate measurement to make to address this point and comparison of the poles to the lateral cortex in par-3(RNAi) vs control would be confusing to readers.
R3d *3) In Figure 3D, the authors need to clarify why they measured ECT-2 dynamics only within the "anterior pole". It would be reasonable to measure ECT-2 dynamics by FRAP and cortical high-speed live imaging on the posterior and the lateral cortex during symmetry breaking. *
We measured ECT-2 recovery at a variety of sites with similar recovery kinetics. The comparison of ECT-2 dynamics on anterior and posterior furrows were shown in order to compare ECT-2 dynamics on centralspindlin-dependent and -independent furrows.
We now provide additional supplemental data on ECT-2 dynamics during symmetry breaking. When ECT-2 is polarized, the residual signal is too low to obtain a measure of its recovery.
R3e 4) In Figure 4 supplement, a difference between with or without ML8237 seems marginal. The authors need to show a statistical test to claim "rapid enhancement of cortical ECT-2 after ML8237 treatment".
We will provide a statistical analysis. As the inhibitor affects ECT-2 globally, the anterior/posterior ratio doesn’t change significantly. To avoid confusion, we now present total cortical ECT-2 levels upon anaphase onset in this experiment as this is the most relevant parameter.
R3f *5) I would strongly suggest the authors to clearly state in the first paragraph of discussion that "this working hypothesis is not supported by direct evidence for phosphorylation of ECT-2 by AIR-1 kinase in vitro and in vivo." It should be reasonable to weaken the statement "by Aurora A-dependent phosphorylation of the ECT-2 PH domain" in p13. *
We agree with the underlying sentiment (as indicated by the “limitations” section that was present in the original version) and we have revised these sentences accordingly: “Our studies suggest that asymmetric, posteriorly-shifted, spindle triggers an initial focal displacement of ECT-2 from the posterior cortex by Aurora A-dependent phosphorylation of the ECT-2 PH domain, though the evidence for this phosphorylation event is indirect.”
Reviewer #3 (Significance (Required)):
*See the second paragraph of the Evidence, Reproducibility, and Clarity section. *
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Referee #3
Evidence, reproducibility and clarity
Symmetry breaking is the process by which uniformity of the system is broken. Many biological systems, such as the body axes establishment and cell divisions in embryos, undergo symmetry breaking to pattern cellular interior design. C. elegans zygote has been a classic model system to study the molecular mechanism of symmetry breaking. Previous studies demonstrated critical roles of centrosomes and microtubules in breaking symmetry in the actin cytoskeleton during anterior-posterior polarization and cytokinesis. It, however, remains elusive how centrosomes and/or microtubules regulate the assembly and contractility of the actin cytoskeleton. Recent reports identified Aurora-A AIR-1 as the key centrosomal kinase that suppresses the function of the actin cytoskeleton, but little is known about a substrate of the kinase during symmetry breaking events.
Longhini and Glotzer proposed in this manuscript that RhoGEF ECT-2 plays a critical role in symmetry breaking of the actin cytoskeleton under the control of AIR-1 kinase. Kapoor and Kotak (2019) previously proposed the same GEF as a downstream effector of centrosomes, but this work did not provide direct evidence for ECT-2 as the AIR-1 effector. This manuscript identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. Although this manuscript lacks direct in vivo and in vitro evidence for phosphorylation of ECT-2 by AIR-1 kinase, the above findings reasonably support a model where in AIR-1 promotes the local inhibition of ECT-2 on the cortex. Design of the experiments, the quality of images, and data analysis are reasonable, and the main text was written very well. The main conclusion of this work will attract many readers in cell and developmental biology fields. I basically support its publication in the journals supported by Review Commons with minor revisions (see below).
Minor comments
- In Figures 2A and 2B, the authors claimed apparent correlation between spindle rocking and ECT-2 displacement. However, because both MTs and ECT-2 in Fig2AB images are blur, I cannot convince myself whether ECT-2 intensities on the cortex showed negative correlation with the distance between the posterior centrosome and the cortex. The authors may want to provide quantitative data set and use a statistical test to support this conclusion.
- Figure 2D would show a ratio between the anterior/posterior pole and the lateral cortex.
- In Figure 3D, the authors need to clarify why they measured ECT-2 dynamics only within the "anterior pole". It would be reasonable to measure ECT-2 dynamics by FRAP and cortical high-speed live imaging on the posterior and the lateral cortex during symmetry breaking.
- In Figure 4 supplement, a difference between with or without ML8237 seems marginal. The authors need to show a statistical test to claim "rapid enhancement of cortical ECT-2 after ML8237 treatment".
- I would strongly suggest the authors to clearly state in the first paragraph of discussion that "this working hypothesis is not supported by direct evidence for phosphorylation of ECT-2 by AIR-1 kinase in vitro and in vivo." It should be reasonable to weaken the statement "by Aurora A-dependent phosphorylation of the ECT-2 PH domain" in p13.
Significance
See the second paragraph of the Evidence, Reproducibility, and Clarity section.
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Referee #2
Evidence, reproducibility and clarity
In this work, Longhini and Glotzer investigate the localization of an essential regulator of polarity and cytokinesis, RhoGEF ECT-2, in the one-cell C. elegans embryo. The authors show that centrosome localized Aurora A kinase (AIR-1 in C. elegans) and myosin-dependent cortical flows are critical in asymmetric ECT-2 accumulation at the membrane. Since membrane interaction of ECT-2 is dependent on the Pleckstrin homology domain present at the C-terminus of ECT-2, they further analyzed the importance of putative AIR-1 consensus sites present in this domain. The authors linked the relevance of these sites in controlling ECT-2 localization and its significance on cytokinesis. The manuscript is well written, the work is interesting, and the data quality is high.
Major comments:
- In Fig. 2, the authors claim that the centrosomes and the position of the mitotic spindle are critical in regulating the asymmetric enrichment of ECT-2 at the membrane. To test the relevance of spindle positioning on ECT-2 localization, the authors depleted PAR-3 and PAR-2. The authors observed that the ECT-2 asymmetry is affected in these settings. However, PAR-3 or PAR-2 depletion impacts polarity, which is critical for many cellular processes, including spindle positioning. Can the authors try to specifically misposition the spindle without affecting polarity? For instance, by depleting Galpha/GPR-1/2 and assessing the impact of such depletion on ECT-2 localization.
- I wonder why the intensity of ECT-2 at the anterior and posterior membrane decreases in air-1 (RNAi) post anaphase onset (Fig. 4A)? Moreover, I fail to observe a significant asymmetric distribution of ECT-2 in embryos depleted for PERM-1. Therefore it appears that the difference between DMSO and MLN8237-treated embryos is not substantial (at least in the images)?
- The data in Fig. 5 and 6 are exciting but raise a few concerns:
- a). The authors show that ECT-2C localization mimics the localization of endogenous tagged ECT-2. However, all these analyses with ECT-2C and various mutants are performed in the presence of endogenous ECT-2. Can the author check the localization of these mutant strains in conditions where the endogenous proteins are depleted? I understand that the cortical flow would be perturbed in conditions where endogenous ECT-2 is depleted. However, I suspect that one can analyze the anaphase-specific distribution.
- b). Can the author comment on why ECT-2C does not accumulate at a similar level as ECT-2C(3A or 6A) at the cell membrane when AIR-1 is depleted (compare Fig. 5D with Supplemental Fig. 5)?
- c). Does the cortical localization of the ECT-2C(6A) mutant become symmetric upon further depletion of AIR-1? Of course, if the asymmetric distribution of ECT-2C(6A) is dependent on the presence of endogenous protein in the cellular milieu, the point raised earlier will help address this concern.
- d). The authors predict that the AIR-1 mediated phosphorylation delocalizes ECT-2 from the polar region of the cell cortex. Since the posterior spindle pole is much closer to the posterior cortical region, the delocalization is much more robust at the posterior cell membrane. I wonder why targetting AIR-1 at the membrane (GBP-mCherry-AIR-1) does not entirely abolish GFP-ECT-2C membrane localization? Can the author include the localization of GBP-mCherry-AIR-1 in the data? Also, do we know for sure if GBP-mCherry-AIR-1 is kinase active?
- e). The authors show that centrosomal enriched AIR-1 [spd-5(RNAi)], but not the astral microtubules localized AIR-1 [tpxl-1(RNAi)], is vital for ECT-2 membrane localization. Interestingly, the authors showed that AIR-1 acts in the centralspindlin-directed furrowing pathway (Fig. 6A). I wonder if the authors can combine NOP-1 depletion with TPXL-1 depletion? I guess this will further help to exclude the function of TPXL-1 in the centralspindlin-directed furrowing pathway.
- f). Why do NMY-2-GFP cortical levels appear lower in 30% of the embryos that show various degrees of cytokinesis defects (Fig. 6A)?
- g). The authors report that phosphomimetic mutation at the phospho-acceptor residue in ECT-2 impacts its cortical accumulation. This strain, together with NOP-1 depletion, affects furrow ingression. One explanation for this phenotype is that phosphomimetic mutant weakly accumulates at the membrane. However, one interesting observation is that ECT-2T634E enriches at the central spindle (Fig. 6B, panel 120 sec), which somehow I could not find in the text. Could this additional localization of ECT2 at the central spindle contribute to the cytokinesis defects that the authors have observed? The microscopy images the authors have included show that ECT-2T634E significantly localizes at the equator at the time of furrow initiation. Can the authors add the localization of ECT2 wild-type and ECT-2T634E in NOP-1 depleted conditions where they see an apparent impact on the cytokinesis? Similarly, if the authors include the localization of NMY-2 in these conditions-it will further add more weightage to the data.
Minor comments:
- An explanation of how the timing of NEBD was analyzed in multiple settings would be helpful.
- The authors mentioned on p. 6-'Despite significant depletion of tubulin.....during anaphase'. These experiments are performed in the near complete depolymerization of microtubules; thus, regular anaphase will not establish. I understand that the authors are monitoring localization wrt the timing similar to anaphase in the non-perturbed condition, and thus a bit of change in the sentence is required.
- After testing the relevance of SPD-5 (that primarily acts on PCM and not on centrioles)-the authors write on p. 6 that 'two classes of explanation...early embryo'. I did not understand the importance of this sentence here.
- The observed impact of spd-5 (RNAi) on ECT-2 localization could be because of the effects of SPD-5 depletion on centrosomal AIR-1? The authors can link the impact of SPD-5 depletion not only with the centrosome but also with AIR-1 in the discussion.
- In the various Figure legends, sometimes the authors mention time '0' as anaphase, and other time as anaphase onset.
Significance
The manuscript is well written, the work is interesting, and the data quality is of good quality.
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Referee #1
Evidence, reproducibility and clarity
Summary
In this study the authors addressed how Ect2 localization is controlled during polarization and cytokinesis in the one-cell C. elegans embryo. Ect2 is a central regulator of cortical contractility and its spatial and temporal regulation is of uttermost importance. After fertilization, the centrosome induces removal of Ect2 from the posterior plasma membrane. During cytokinesis Ect2 activity is expected to be high at the cell equator and low at the cell poles. Similarly to polarization, the centrosome provides an inhibitory signal during cytokinesis that clears contractile ring components from the cell poles. Whether and how the centrosomes regulate Ect2 localization is not know and investigated in the study.
The authors start by filming endogenously-tagged Ect2 and find that Ect2 localizes asymmetrically, with high anterior and low posterior membrane levels during polarization and cytokinesis. They reveal that the centrosome together with myosin-dependent flows results in asymmetric Ect2 localization. Previous studies had suggested that Air1, clears Ect2 from the posterior during polarization and the authors expand those finding by showing that Air1 function is also required to displace Ect2 from the posterior membrane during cytokinesis. To elucidate if Ect2 displacement is induced by phosphorylation of Ect2 by Air1, the authors investigate the localization of a C-terminal Ect2 fragment containing the membrane binding PH domain. When the predicted Air1 phosphorylation sites are mutated to alanine, the Ect2 fragment still localizes asymmetrically but exhibits increased membrane accumulation.
Finally, they investigate the functional role of Air-1 during furrow ingression. They demonstrate that embryos deficient of Air1 and NOP1 have impaired furrow ingression. Lastly, the authors sought to confirm that there is a direct effect of Air1 on Ect2 function by generating a phosphomimetic point mutation of Ect2 using Crispr. They find that the membrane localization of phosphomimetic Ect2 is reduced and consequently furrow ingression is impaired.
Major comments
It is not convincing that the six putative phosphorylation sites are targeted by the Air1. If Air1 phosphorylation displaces Ect2 from the membrane, a reduction in Ant/Post Ect2 ratio is expected in the phosphodeficient mutants, like after air1 RNAi. However this is not observed for cytokinesis or polarization (Fig. 5D(i); E). This suggests that phosphorylation of those sites is not essential for the asymmetric Ect2 localization.
The authors aim to demonstrate that phosphorylation of the identified sites is important for cytokinesis. For this they investigate contractile ring ingression in the phosphomimetic point mutation. Since ring ingression is slower and fails in nop1 mutant they authors conclude that this demonstrates a functional importance of this site. I am not surprised that embryos ingress slower in this mutant since Ect2 localization to the membrane is reduced. This however does not show that this phosphorylation site is the target of the centrosome signal. Importantly, authors would need to demonstrate that Rho signaling and thus Ect2 activity, is increased at the poles, when phosphodeficient Ect2 is the only Ect2 in the embryo.
The authors use the Aurora A inhibitor MLN8237: It was shown prior (De Groot et al., 2015) that this inhibitor is not highly specific for Aurora A, and that it also inhibits Aurora B. Thus experiments need to be repeated with MK5108 or MK8745. They should also be conducted during polarization. Why does Aurora A inhibition not abolish asymmetry? That would be expected?
There is no statistical analysis of the results in the entire study. For all claims stating a change in Ant/Post Ect2 ratio or Ect2 membrane localization selected time points should be statistically compared: for example the main point of Fig.1 is that Ect2 becomes more asymmetric during anaphase. Thus a statistical analysis of the Ect2 ratio at anaphase onset (t=0s) and eg. t=90 s after anaphase onset should be performed; or Fig. 3A nop-1 mutant Ant/Post Ect2 ratio during polarization: again statistical analysis of control and nop-1 mutant embryos is needed at a particular time point.
The aim of Fig. 2B is to demonstrate that Ect2 localization is independent of microtubules, however they still observe some microtubules with the Cherry-tubulin marker and those are even very close to the membrane and therefore could very well influence Ect2 on the membrane. Therefore I am not convinced that this experiment rules out that microtubules have no role in regulating Ect2 localization.
Throughout the paper the authors should tone down their statement that Air1 breaks symmetry by phosphorylating Ect2, since phosphorylation of Ect2 by Air2 is not shown.
I understand that the establishment of Ect2 asymmetry is important for polarization. However, how does asymmetric Ect2 localization result in more active Ect2 at the cell equator, which is required for the formation of the active RhoA zone? Would we not expect an accumulation of Ect2 at the cell equator, or if that is not the case more active Ect2 at the equator versus the poles?
Minor comments
Can the authors explain why the quantification of Ant/Post Ect2 ratio in control embryos differs in different figures? For example: in Fig. 1D i) a slight increase of Ect2 asymmetry ratio is seen at around 80 s after anaphase onset. In comparison, in Fig. 2C (i) this increase is not obvious. Are those different genetic backgrounds?
One key point of the paper is that myosin-dependent cortical flows amplify Ect2 asymmetry during polarization and cytokinesis. During polarization the data is convincing, however during cytokinesis Ect2 ratio is only slightly decreased after nmy-2 depletion, again is this decrease even significant?
In the introduction: "Centralspindlin both induces relief of ECT-2 auto-inhibition and promotes Ect2 recruitment to the plasma membrane" it should be added 'Equatorial' membrane, since Ect2 membrane binding is, to my knowledge, not compromised in centralspindlin mutants or in Ect2 mutants that cannot bind centralspindlin.
Labels in the figures are often very small eg Fig. 1 ii-v) and difficult to read. In addition it is easier for the reader if the proteins shown in the fluorescent images is also labeled in the figure (eg Fig. 2B add NG-Ect2).
Material and methods it should be mentioned which IPTG concentration was used.
The authors speculate that the Air1 phosphorylation sites in Ect2 PH domain prevent binding to phospholipid due the negative charge. At the same time, the authors propose that the PH domain binds to a more stable protein on the membrane, which is swept along with the cortical flows and they propose anillin could be that additional binding partner. I might miss something, but do the authors suggest Ect2 has two binding partners: anillin and the phospholipids? It would be necessary to explain this better. The authors should test if anillin represents the suggested myosin II dependent Ect2 anchor. For this they should check if Ect2 localization to the membrane is altered upon on anillin RNAi.
The title of fig. 3 does not fit the statement the authors want to make, since the key point is how Ect2 polarization is affected and not membrane localization in general.
In Fig 4A/C. After air1 depletion the authors observe a reduction in Ect2 asymmetry. Why are the centrosomes not marked in the figures? Because they cannot be detected? The authors would also need to show that the mitotic spindle and centrosomes are no altered by air1 RNAi in the zyg9 mutant. Otherwise the observed effect might be indirect.
The authors state that tpxl-1 depletion attenuates Ect2 asymmetry, this is not seen in the quantification ((Fig. 4B(i)). The main phenotype they observe is that Ect2 levels on the membrane increase (Fig. 4 (ii) and (iii). They go on testing the function of tpxl1 by depleting tpxl1 in the zyg9 mutant, where the centrosomes are close to the posterior cortex. Here they see no effect on Ect2 asymmetry. Based on that they conclude that tpxl1 has no role in this process. To me this finding is not surprising since the centrosome is close the cortex in zyg9 mutant embryos. Therefore sufficient amounts of active Air1 could reach the membrane and displace Ect2. Thus an amplification of the inhibitory signal by tpxl1 on astral microtubules might not be required. The authors need to mention this possibility and tone down their statment (also in the discussion) that tpxl1 is not required for this process.
It was shown that the C-terminus of Ect2 is sufficient and the PH domain is required for Ect2 membrane localization in C. elegans (Chan and Nance, 2013; Gomez-Cavazos et al., 2020). Papers should be cited.
The authors find that nmy-2 depletion results in loss of asymmetry for the Ect2 C-term and Ect2 3A fragment during polarization. Why is the same experiment not shown for cytokinesis?
Air1 is targeted to GFP-C-term Ect2 fragment via GFP-binding to determine the influence on GFP-C-term Ect2 localization (Fig. 5F). They state that they see a reduction of Ect2 C-term but not of C-term 3A after targeting. The reader has to compare Fig. 5D with F. Since the differences are not big, they need to compare the Ect2 C-term and Ect2 C-term 3A with and without Air1 targeting in the same graph (plus statistics). Otherwise this statement is not convincing.
In Fig. 6A the authors determine the contribution of air1 to furrowing. For this they deplete air1 in the nop1 mutant. According to previous studies, air1 mutants have a monopolar spindle. How can the authors analyze the function of air1 in cytokinesis when the spindle is monopolar? Did the authors do partial air1 depletion? They authors need to show that there is not major effect on the spindle and centrosome for their conditions. For comparison air1(RNAi) alone has to be included, otherwise the experiment is not conclusive.
Upon air1(RNAi) in the nop1 mutant NMY2 intensity seems decreased and not increased. Can the authors comment on that, since that is opposite of what is expected.
In Fig 6B they introduce a phosphomimetic point mutation in S634 in the endogenous Ect2 locus. It not clear why the authors chose this site out of the six putative sites and why they only chose one and not 3 or 6 sites? This needs some explanation.
In the model (fig. 7) no astral microtubules are shown during pronuclear meeting and metaphase. Astral microtubules are present at this stage and should be added to the schematic.
Significance
The centrosomes inhibit cortical contractility during polarization and cytokinesis in the one-cell C. elegans embryo. Centrosome localized Air1 was proposed to be part of this inhibitory signal, however the phosphorylation target of Air1 is not known. The identification of Ect2 as a phosphorylation target of Air1 would be a great advancement in the field. However, the presented manuscript lacks convincing data that Ect2 is the phosphorylation target of Air1 during polarization and cytokinesis.
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Reply to the reviewers
The authors do not wish to provide a response at this time.
We will provide the point-by-point response when we submit the full revision of our manuscript
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Referee #3
Evidence, reproducibility and clarity
This paper presents an investigation of the mechanisms of how chitin is synthesized in Drosophila by investigating the chitin synthetase Kkv and two proteins related/redundant proteins that are required for chitin production Exp and Reb.
The authors show that synthesis of nascent chitin polymers is separable from the secretion of chitin and that Ex/Reb is specifically required for chitin translocation/secretion. To understand the functions of Exp/Reb, the authors perform structure/function analyses and examine the localization of the proteins. They find that Na-MH2 domain in Exp/Reb is required for chitin translocation, and that a motif the authors name CM2 is required for Exp localization. For Kkv, they show the WGTRE domain is required for ER exit and that a coiled-coiled domain is required for KKV localization and full Kkv activity. By using live imaging and mutations that disrupt membrane trafficking, the authors show that Kkv, which is a transmembrane protein, cycles to the membrane, and like most membrane proteins, is endocytosed and transits through the endocytic system and is returned to the apical surface. Interestingly, despite being dynamically moved around the cell, chitin synthesis produces highly organized extracellular matrixes. Considering that constitutive production of chitin by Kkv everywhere in the cell would create a mess, these results underscore that regulated organized secretion/translocation of chitin is central to generating patterned extracellular matrixes (as the saying goes, "location, location, location"). Consistent with Exp/Reb being important regulators in extracellular matrix patterning, Exp/Reb not only are required for export of chitin, in the absence of Exp/Reb, the pattern of Kkv localization at the apical surface is altered. Unexpectedly however, by using super resolution microscopy the authors show that Kkv and Exp/Reb have complementary rather than matching localizations. Thus, while it is not clear exactly how Exp/Reb are regulating Kkv, they are doing something very interesting.<br /> Overall, this paper will be of broad interest to the cell biology and developmental biology communities, and to the translational community working to develop chitin as a commercial biopolymer. It is also generally clearly written, although I think there are some inaccuracies in the how some points are phrased. The experiments are well done, and subject to the revisions out lined below.
Major concerns:
- A major conclusion of the paper is that Exp/Reb are not required for chitin synthesis. On the most basic level this statement is well supported, because chitin grains are made in the cytoplasm in the Exp/Reb mutants. However, I think the field would be better served with a more nuanced consideration or the role of Reb/Exp. From the data presented, it seems that in the absence of Reb/Exp, the total amount of chitin produced is greatly reduced. I think it would be worth considering Exp/Reb, or the synthesis process in general, as having processivity or duty cycle or quality control such that in the absence of Exp/Reb while Kkv may make short chitin polymers, or occasional long polymers, the major production of chitin doesn't get going without Exp/Reb. Thinking of Reb/Exp as processivity factors in addition to export factors dramatically changes how one thinks of the proteins and the process of chitin synthesis. While these considerations can be handed with some discussion, it would be very interesting to look at the length of the chitin polymers in the Reb/Exp mutants and see if the average chain length is much reduced. This would help distinguish between Exp/Reb reving up the total number of Kkv molecules that produce chitin and Exp/Reb allowing the same number of Kkv molecules to stay active and produce much longer chitin chains. A caveat here is that I have no idea how hard this is to do, so I won't put this at the level of a required revision, but this result would significantly deepen the analysis in the paper.
- In looking that the subcellular localization of the Kkv and Reb in regular and super resolution, the authors I think the authors missed an important, but straight forward way to gain insight into the apparent complementary distribution of Kkv and Exp/Reb. In stage 16 WT embryos, Kkv has a distinct ringed pattern that corresponds to the tanedial ridges (e.g. clearly visible in Fig. 6A and 6G). How those ridges are set up is unclear, although there are some interesting Turing-pattern models out there. One prediction might be that Exp/Reb should be in between the Kkv rings. If so, maybe Exp/Reb are key components of patterning chitin secretion to make this 3D patterned matrix? Alternatively, maybe Exp/Reb act on a smaller length scale and will match the Kkv ring pattern, just not overlapping with Kkv at the very fine scale. These are straightforward experiments and again could provide key insights into the function of Exp/Reb.
- In general, most of the figures do not include WT or a control for comparison. This makes it hard for non-experts to assess what the effect of a mutation or condition is. For example, there are no examples of WT or Df(exp reb) in Figures 1-4. I realize this would increase the number of panels, but the paper would be more accessible if comparisons were within figures instead of comparing between main and supplementary figures and other papers.
- To bolster the case the Exp/Reb directly regulate Kkv distribution, the authors should examine the distribution of Kkv in a catalytically null Kkv mutant, or drugs that block Kkv, or mutations in other genes required for Kkv activity to show that the altered distribution of Kkv in Exp/Reb mutants is a direct consequence of the lack of Exp/Reb rather than in indirect consequence of lack of extracellular chitin, which causes gross perturbations in the trachea. Also, are there differences in the distributions of Kkv in salivary glands with or without the presence of Exp/Reb? If Exp/Reb change the distribution of Kkv in the salivary glands, which normally do not express Kkv and presumably many other components of the chitin ECM system, this would be a powerful argument that there is a direct effect.
Minor concerns.
- Page 5 "These intracellular chitin punctae disappeared from stage 14, when chitin is then deposited extracellularly (Fig 1B')." Fig. 1B' is stage 15 embryos.
- Page 5 "lead to tracheal morphogenetic defects". It would be helpful to the reader if the text or legend told the reader what they were looking for? Broken tubes? Inflated tubes? Variable tubes?
- Fig. 1H. Main text says "co-expression of Kkv and expMH2/rebMH2 did not lead to tracheal morphogenetic defects (Fig 1H, ...". The tracheal dorsal trunk in Fig. 1H does not look WT. The legend does not state the stage, but the DT looks to have an enlarged diameter and it might be too long. Please present measurements on stage 16 trachea to confirm that there is no effect on tracheal morphology.
- Fig. 3E there is a lot of GFP-Kkv that is not in co-localized with the KDEL marker. Can the authors clarify what compartment all the other staining is? ER?
- Section 3.1. The authors imply that the WGTRE domain is specifically required for ER exit. However, an alternative is that absent the WGTRE domain, the protein just does not fold correctly, which would also preclude ER exit, but would be a different problem for the protein to make chitin if it isn't folded.
- Page 15. I disagree with statement "At stage 16, control embryos showed a highly homogeneous apical distribution of Kkv in stripes, corresponding to the taenidial folds, and Kkv vesicles were largely absent (Fig 6G)." In Fig. 6G, the tandeal ring pattern is clearly visible, as are the fusion cells. If Kkv distribution were "highly homogeneous" these structures/pattern would not be visible.
- Page 15. I also disagree with the characterization of the apical Kkv distribution in st 15 embryos. "In control embryos we detected a very uniform and homogenous pattern of apical Kkv (Fig 6I).". To my eye, the pattern is punctate and random for the clumps of stain, with the underlying beginnings of the tanidial pattern starting to be visible. The pattern appears neither uniform nor homogenous.
- P16. The degree of order in the distribution of Kkv is overstated. The authors state that "The results of this analysis, showed that Kkv on the apical membrane, is evenly distributed following a regular pattern (Fig. 6L,L',L',M)." However, given that there is barely a visibly perceptible difference between the actual distribution of Kkv in 6L' and a calculated random distribution in 6L", and that the pattern is neither visibly even or regular, it would be more representative to say something to the effect that the analysis shows there is "underlying order" or "some degree of order" or a "non-random pattern". Visually, the key difference between 6L ' and L" is that there are fewer closely clustered Kkv dots. You could still have an uneven distribution of Kkv that maintains minimum spacing, which is a kind of ordered organization, but not one that would be assumed from the description. It would be helpful if the authors instead of just saying a "regular pattern" also stated the nature of the pattern they observe, i.e. Grid? Stripes? Minimum spacing?
- Discussion. Another model for the role of Exp/Reb could be to bind and neutralize an inhibitor of Kkv activity. This would account complementary distribution of Kkv and Exp/Reb.
- Fig. 6L. what tissue is being analyzed? Presumably trachea, but this should be specified as salivary glands are also mentioned in the legend.
- Fig. 7 C models. I believe that the super resolution data is not accurately accounted for in the models. In both model 1 and model 2, Kkv and Exp/Reb are shown to be in close proximity, but the super resolution data suggests that most Kkv and Exp/Reb are separated hundreds of nanometers. Further, showing Kkv and Exp/Reb as touching was not supported by the coIP experiments, which failed to detect an interaction. It is possible that only a small fraction of Exp/Reb that is in close proximity to Kkv is active, but if so, this should be explicitly mentioned in the models to reconcile the data showing that Kkv and Exp/Reb are mostly not anywhere near each other.
- -Image analysis. Please detail the criteria for "apical" and "basal" regions were the basis for freehand segmentation. What was counted as apical and what was basal?
- Abstract and Introduction: The authors state that "We find that Kkv activity in chitin translocation, but not in polymerization, requires the activity of Exp/Reb, and in particular of its conserved Na-MH2 domain.", but then follow that with the statement that "Furthermore, we find that Kkv and Exp/Reb display a largely complementary pattern at the apical domain, and that Exp/Reb activity regulates the topological distribution of Kkv at the apical membrane." Many readers, will find the use of "furthermore" confusing because they will take furthermore as the about to be described data logically following the previous data, but then run headlong into the fact the Kkv and Exp/Reb show a complementary distribution, which does not obviously follow from Kkv activity requiring Exp/Reb. The authors could clarify this and highlight the interesting, unexpected and exciting nature of their results by replacing "Furthermore" with "Unexpectedly" or "Surprisingly", and emphasizing the important role of Exp/Reb in Kkv organization. Maybe something like: Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane.
Significance
The topic is interesting from the aspect of cell biology in terms of how a long polymer is created intracellularly, secreted and spatially organized to create a sophisticated extracellular matrix. The topic is also of general interest because chitin is central to the body plan of all insects, crustaceans and many other species, and chitin is of increasing interest as a biopolymer that could have extensive commercial uses.
In addition to an informative structure/function analysis of the Kvv and Exp/Reb, the results identify what is, to my knowledge, the first regulator of the spatial organization of chitin sythase in insects and it unexpectedly shows a complementary pattern to the the synthase. This highlights just how little we understand about how complex extracellular matrixes are synthesized.
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Referee #2
Evidence, reproducibility and clarity
This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.
The authors initially detected unusual intracellular chitin by overexpression of the chitin synthase kkv in tracheal cells before regular chitin deposition occurs. In addition, they recognized that the kkv gain of function mediated unusual intracellular chitin vesicles and in later stages in exp/reb mutants. These findings were the starting point of further experiments, suggesting that Kkv synthesizes chitin and that Kkv-mediated chitin deposition requires Exp/Reb activity to translocate and release chitin. Their genetic studies further show that chitin polymerization and translocation are uncoupled.
All primary studies were tested in embryonic tracheal cells and as a proof of principle control in salivary glands which do not express chitin. Elegant rescue experiments in the mutant background showed that the Exp/Reb Nα-MH2 domains are required but not sufficient for chitin translocation and deposition and are dispensable for protein localization. Additionally, they identified another conserved domain (CM2) which is required Exp/Reb localization but not for chitin translocation. Similarly, they investigated Kkv domains by rescue experiments in kkv mutant embryos. They identified the WGTRE domain as essential for ER exit and the coiled-coil region for proper apical Kkv localization. Altogether they provide evidence that Kkv requires proper localization at the apical membrane, which is likely coordinated by Exp/Reb. This precise Kkv localization is linked with Kkv activity and chitin deposition. Therefore, this work is new and fundamental to many disciplines such as insect biology, chitin biology, cell & developmental biology, and others. Thus, the work is worth publishing but requires some changes from my point of view.
Major Comments
- The authors nicely show that Kkv is able to synthesize chitin in a constitutive manner and that it can accumulate intracellularly. However, this needs some more input to understand the underlying biological sense. For example, what are the chitin vesicles' nature of early vesicles (st 13) and the unusual late (st15) chitin vesicles? Exocytosis, endocytosis or recycling? This can be clarified to understand chitin translocation and that synthesis and translocation are uncoupled. The authors tested rab5DN mutant salivary glands to exclude endocytosis. However, the chitin-positive vesicle size and amount in the rab5 mutant appear different from the control experiment, where much more intracellular chitin accumulates. Thus, it may suggest that some chitin vesicles are independent of Rab5-mediated endocytosis, others probably not. Indeed, the authors identified some KKv and some other chitin vesicles in all discussed intracellular processes; however, additionally, chitin appears to accumulate also in the cytoplasm. The authors conclude that Kkv protein might be able to polymerize chitin at all different intercellular stages, including endocytosis and degradation pathway. First, I wonder why chitin was found within membranous vesicles and, at the same time, within the cytoplasm. Second, does it make sense in the biological context when tracheal cells or other chitin-producing organs want to secrete chitin at the apical membrane while Kkv has the ability to produce chitin in all cellular areas, even in endosomes? In this context, another fundamental question concerning chitin secretion and subsequent organization could be investigated with the author's tools. Are the chitin vesicles loaded with chitin binding proteins or deacetylases that organize the formation of the nano and makro fibrillar chitin matrix in tracheal tubes? For example, previous research showed a reduced luminal accumulation of the 2A12 antigen in kkv mutants and expRNAi knockdown embryos.
- Observation of Extracellular kkv-GFP: does extracellular anti-GFP staining co-localize with the anti-Kkv antibody?
- Putative Kkv microvesicles: the authors state that extracellular GFP staining could be Kkv located in microvesicles. I wonder whether the observed extracellular GFP puncta contain a membrane or other membranous proteins.
- Fig.1:
- general remarks: some images of this figure could be improved by showing the single channels of CBP to judge whether chitin is secreted and/or vesicles appear. -In addition, some images show higher magnifications, others overview only. It would be beneficial to visualize the small vesicles additionally with higher magnifications.
- Fig.1M: This image is problematic due to the epidermal background staining. The tracheal system is hard to recognize. A single channel of Cbp ist not indicated.
- Fig. 1P: Apical membrane marker or any cytoplasmic marker would be extremely useful to judge subcellular Exp localization in this experiment - this image is hard to compare with Exp localization in Fig S2D.
Fig. 2O: Apical/Basal accumulation, what are the numbers at the Y-axis?
Fig. 3F: The authors state that the WGTRE domain is required for ER exit of Kkv based on colocalization studies with KDEL and FK2. However, the study with FK2 is not convincing as immunostainings are of poor quality. The GFP construct appears not to be expressed in all tracheal cells, and moreover, the FK2 staining is faint. Thus, judging whether the protein is not ubiquitinated from the presented image is challenging. However, it does not change the key message, Kkv does not exit ER. By the way, there is a new paper showing Serca to be essential for ER exit of Kkv, which would fit the discussion of the kkv domains.
Fig. 7 - model: First, Since the authors do not show that Kkv is part of a membranous microvesicle, I'm skeptical whether this should be part of a model that explains the shown data. Therefore, I'm asking the authors to delete it or to show it more clearly. Second, the meaning of the yellow arrowheads is not indicated. Third, the explanation in the legend is sound, but showing the two options could be improved.
Minor comments:
- Missing reference: Chirin has been recognized importance in physiology (Zhao et al., 2019; Zhu et al., 2016) but also as a biomaterial (? Reference). Suggestion: DOI: 10.3390/ma15031041 (Improving Polysaccharide-Based Chitin/Chitosan-Aerogel Materials by Learning from Genetics and Molecular Biology) This paper discusses the current usage and potential of chitin as a biomaterial in many disciplines.)
- 3.1, second paragraph final sentence: double point
Referees cross-commenting
Rev#1 asks the same questions as I do. Technical questions and the idea to compare endogenous Kkv.The same is true with Rev#3. Overlapping questions concerning technical things about figure illustration and clarity of presented stainings. Altogether, the criticism will improve the manuscript
Significance
This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.
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Referee #1
Evidence, reproducibility and clarity
In this manuscript, the authors provide data on the function of exp/reb and Kkv in chitin deposition. They show chitin polymerization and deposition are uncoupled and exp/reb are required for the deposition of the chitin by regulating the distribution of kkv at the apical membrane. However, there is no direct interaction between Kkv and Exp/reb. The functional analysis of Kkv and Exp/reb is interesting.
The overexpression lines are used throughout the manuscript to analyze protein functions. Since ectopic expression of kkv and exp leads to chitin synthesis and deposition. Authors use this overexpression system to analyze the functional domain of kkv and Exp/reb. It is reasonable. However overexpression line might not represent the endogenous protein perfectly, it might cause some issues to answer certain questions.
Major comments
- Fig. 4 Does ectopic overexpression of Kkv-GFP have the same expression pattern as the endogenous Kkv? The overexpression line may lead to ectopic expression. the colocalization of endogenous Kkv and intracellular vesicles would be more accurate.
- Are Kkv and Exp/reb expressed at the same time endogenously? If kkv is expressed earlier than Exp, can intracellular chitin be detected in wild-type embryos at early stages? Fig. 1b shows overexpression of Kkv at S13 has intracellular chitin (exp is not expressed at this stage).
- Fig. 1B no intracellular chitin is detected. Fig. 1H intracellular chitin is detected. Does Overexpression of exp-MH2 interfere with the endogenous Exp function?
- For measurement, some detailed info is needed, for example, what is your area of interest?
Minor comments:
- Fig. 2 and Fig. 3. how do you define the region of apical and basal? An apical marker is needed here. N is the total number of embryos or the number of sections in the same embryo?
- Fig. 5H What is your area of interest to measure vesicles? Which tracheal segment do you measure? Some details need to be provided here.
- Fig. 5 what is your area of interest when you measure Kkv?
Significance
This work further advance the knowledge about chitin synthesis and deposition
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Reply to the reviewers
1. General Statements
All three reviewers demonstrated similar concerns and provided clear guidance on potential revisions. All three reviewers recognized the importance of our study to the mitosis, genome instability and DNA damage fields, and identify limitations regarding potential therapeutic implications of the study. To mitigate these limitations and extend the breadth of our study, the revised manuscript now includes colony formation assays to more directly evaluate the impact of mitotic DNA damage therapies in cancer cell proliferation. It also includes several of the experimental suggestions/clarifications proposed by the reviewers. Lastly, we include here a clear plan of additional experiments that we agree to conduct.
2. Description of the planned revisions
Reviewer #1
Major comments:
1) In the opinion of the reviewer, the study is somewhat unbalanced as it starts with a high throughput analysis of a large number of compounds but only etoposide treatment is investigated in detail in the key experiments shown in Figures 5 and 6. The effect of topoisomerase II inhibitor on kinetochore-MT stability has already been demonstrated by Bakhoum et al, 2014. If authors wish to generalize that similar phenotypes are observed after various types of DNA damage, they should test additional compounds (such as Lomustine, Mitomycin C, and Carboplatin). In addition, measuring of the kinetochore-MT half-life in figure 5 should be performed with better time resolution within the first 5 minutes. This would allow better comparison of the measured half lives that are much shorter than 5 minutes.
R: We agree with the reviewer and we will perform additional measurements of kinetochore microtubule half-life with these different compounds and include shorter time points to increase the temporal resolution within the first 5 min.
3) Involvement of Kid and Kif4A in arm ejection of the polar chromosomes is an interesting observation in context of mitotic DNA damage. However, it is unclear how the distribution of the chromokinesines was evaluated in Figure 6A-D. Was the signal quantified at the metaphase plate or at polar chromosomes? It seems that Kif4A localizes to the polar chromosome caused by etoposide treatment whereas no signal is visible in DMSO control (Fig. 6C).
R: Since whole cells were exposed to DNA damage, we had previously quantified total fluorescence intensity of Kid and Kif4a on all chromosomes (aligned and unaligned). We nevertheless concede that some chromosomes might have been more exposed (or are more susceptible) to DNA damage and we will therefore provide a quantification of the fluorescence intensity ratio of Kid and Kif4A levels on polar chromosomes relative to the metaphase plate, with and without mitotic DNA damage.
4) Authors convincingly showed that SAC is activated by mitotic damage and this is also consistent with previous reports. However they did not address if DDR pathways contribute to the activation of SAC. This would be interesting especially in context of a recent report that showed Bub3 as a direct substrate of ATM (Xiao et al. 2022, JBC). I wonder if the polar chromosomes are formed and missegregate also in the absence of ATM activity.
R: This is an interesting suggestion and we have already obtained data from one experiment regarding the formation of polar chromosomes upon ATM inhibition. We will perform additional independent validation of these data and include our findings in the fully revised manuscript.
Reviewer #2
Major comments:
…They need to present the % of cells with polar chromosomes, and it would also be informative to understand the rate of cells with lagging chromosomes, or that underwent anaphase with polar chromosomes with and without chromokinesin depletion.
R: We will quantify the frequency of the different segregation errors upon DNA damage, with and without Chromokinesin depletion.
It would be very helpful for them to provide a schematic model between DNA damage, overstable microtubules, satisfied SAC, monotelic attached chromosomes, and the role of chromokinesins. At present these connections are very unclear.
R: We thank the reviewer for drawing attention to this point. We will provide a step-by-step model in the fully revised manuscript.
Reviewer #3
Major comments:
According to Figure 2C, the ratio of "Exit with micronuclei (from misaligned chromosome(s))" is relatively low compared to other phenotypes such as "Mitotic arrest" or "Cell death." I wonder if polar chromosome phenotype is also correlated with these other cell fates. Please clarify which fate is correlated with polar chromosome formation after DNA damage.
R: We will provide these correlations. We already know that those cells that arrest in mitosis is due to misaligned chromosomes. We will also perform the correlation between cells that died and the presence of misaligned chromosomes.
In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.
R: We now clarify that the observed mitotic delay in the presence of DNA damaging compounds occurred after nocodazole washout. As so, nocodazole was no longer present in the system. We also draw the attention that DNA damage in the presence of nocodazole, a condition that promotes maximal SAC activity, was fully dependent on MPS1 activity (Figure 4A). We have also obtained data from one experiment regarding the formation of polar chromosomes after nocodazole wash-out and ATM inhibition. We will perform additional independent validation of these data and include our findings in the fully revised manuscript.
Figure 6E-G: I wonder whether siKid+siKif4a affected %polar chromosomes or not.
R: We will perform this experiment and include the results addressing this point in the fully revised manuscript.
Page 10, line 287: the authors claim that "we show that long-term mitotic DNA damage..., causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesisns,...." However, only Mad1 localization data is provided in Figure 6E-G, and whether siKid + siKif4a rescues the missegregation of polar chromosomes is not clear. The authors should either provide supporting evidence or revise this sentence for clarity.
R: We will determine whether Kid+Kif4a depletion rescues the missegregation of polar chromosomes (i.e. reduces the frequency of cells that exit with polar chromosomes in the presence of mitotic DNA damage).
3. Description of the revisions that have already been incorporated in the transferred manuscript
Reviewer #1
Major comments:
2) Authors claim that the observed phenotype of the chromosomal missegregation following the mitotic DNA damage occurs specifically in cancer cells but the data supporting this statement is poor. They need to show more data in non-transformed cells or remove the statement.
R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.
Minor comments:
1) Specificity of Aurora-B pT232 antibody should be validated to exclude cross-reactivity with Aurora-A at spindle pole (Fig S8C)
R: Antibody specificity against active Aurora A and B was validated with specific kinase inhibitors. These data are now included in the revised manuscript.
Reviewer #2
Major comments:
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The term 'long-term DNA damage during mitosis' is confusing. DNA damage occurs when? (before, or during mitosis?). Their live cell data shows they are following cells that underwent mitosis within a certain time window after damage occurred, but it is not clear if it occurs only before, or sometimes during mitosis.
R: We realize that this was indeed confusing. The only experiment where mitotic DNA damage might have been introduced before mitosis is in Figure 1A-C. All other experiments were directly or indirectly based on live-cell data in which only cells that were already in mitosis when they were exposed to DNA damage were analysed. Because these live-cell experiments revealed that DNA damage before mitosis prevented mitotic entry at the used concentration of the DNA-damaging agents, mitotic cells scored upon DNA damage in fixed cell experiments must have been already in mitosis when DNA damage was applied. We also now include a scheme of each experimental set up in the respective figures to clearly indicate how the data was obtained and to facilitate the respective interpretations. This is now clarified in the main text.
All damage occurred in cells treated with nocodazole - could this have impacted the results? Was similar DNA damage induced if cells were arrested in monastrol/STLC, or MG132 for example? They show the distal Mad2 data in STLC but not the damage. Also Figure S4A/B appears to be from one experiment only which makes it difficult to interpret.
R: We have additionally induced DNA damage when the cells were arrested in STLC. yH2AX levels as inferred by western blot analysis were indistinguishable from cells treated with nocodazole. These data are now included in the revised manuscript. We also clarify that data presented in previous Figure S4 are from 2 independent experiments.
Why not show the RPE1 data for polar chromosomes?
R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.
Confusing interpretation - monotelic attachments, yet also stable attachments,..? Please can they clarify what is meant by these terms.
R: Monotelic attachments in which a single chromatid is attached to microtubules are intrinsically unstable due to the lack of centromeric tension. However, mitotic DNA damage alters this scenario and stabilizes monotelic attachments. We have now clarified this point in the main text.
They focus most of the study on understanding why polar chromosomes arise after DNA damage. However, this phenotype seems to be a relatively minor effect. Eg. In Figure 2b only a few cells exhibit very extended mitosis, and in Figure 2c only a small percentage exit mitosis with misaligned chromosomes. Furthermore, in Figure 4B the percentage of polar chromosomes with only distal Mad2 is low. In Figure 4A the images are not clear whether a 'both' or 'distal' is being shown. It does not seem as if any are distal only, and an example of this would be helpful.
R: We thank the reviewer for alluding to this important point. The frequency of cells with polar chromosomes with/without DNA damage is indicated in Figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments on polar chromosomes only (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes. We also draw the reviewer’s attention to the B&W panels where the different types of attachments are clearly highlighted.
It is also not clear what 'other' means in Figure 2C.
R: The phenotypes classified as ‘other’ in Figure 2C are detailed in Figure S1C. This was indicated in the figure 2 legend, but we have now clarified it also in the main text.
They conclude that chromokinesin depletion rescues the polar chromosomes phenotype. However they do not directly assess the rate of polar chromosome formation, only the % of polar chromosomes that are only distal Mad2 as far as I can see?
R: Similar to reviewer 1, we thank this reviewer for alluding to this important point. The frequency of cells with polar chromosomes with/without DNA damage is indicated in figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes.
It is not clear from how many experiments data are shown for the Mad1 distal experiments in Figures 4 and S4 (there are no error bars, so is this one experiment only?). This should be indicated in figure legends, and repeated if performed only once.
R: Data presented in Figure 4 is a pool from 3 independent experiments and data presented in Figure S4 is a pool from 2 independent experiments. For these reasons, there are no error bars to include. This is now clarified in the figure legends.
Reviewer #3
Major comments:
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Page 6, line 155: the authors claim that "In contrast, among other defects, treatment with any of the DNA-damaging compounds caused a significant mitotic delay due to the presence of misaligned chromosomes near the spindle poles." Although Figure 2A shows a representative image of polar chromosomes, I do not find quantitative data that analyze %polar chromosomes in mitosis treated with DNA-damaging compounds. I also do not find the data supporting the claim that polar chromosomes caused a mitotic delay. Because most subsequent analyses were performed based on this result, the quantitative data should be provided here. For the latter, I suggest showing "time in mitosis (Fig 2B)" separately with or without polar chromosomes.
R: The frequency of cells with polar chromosomes with/without DNA damage is indicated in figure 2C. The respective duration of mitosis due to polar chromosomes is clearly and significantly increased as shown in Figure 2B. In fixed material we chose 70 min after nocodazole washout in these experiments because there is no difference in the frequency of cells with polar chromosomes between DMSO and DNA-damage-treated cells, allowing a direct comparison of the types of kinetochore-microtubule attachments (please see new Figure 5). We now clarify that beyond this time frame, only DNA-damage-treated cells show polar chromosomes. We now highlight in figure 2C what fraction of cells underwent mitotic arrest due to polar chromosomes, as well as those that exited mitosis with polar chromosomes.
In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.
R: We now clarify that the observed mitotic delay in the presence of DNA damaging compounds occurred after nocodazole washout. As so, nocodazole was no longer present in the system. We also draw the attention that DNA damage in the presence of nocodazole, a condition that promotes maximal SAC activity, was fully dependent on MPS1 activity (Figure 4A).
Line 226, (our unpublished observations): because the authors claim that "the formation of polar chromosomes due to the stabilization of kinetochore-microtubule attachments upon long-term mitotic DNA damage is likely exclusive to cancer cells," the authors should present data on RPE-1 cells at least for %polar chromosome formation (as suggested in comment 1) and Mad1 localization. Plus, even though the data is provided, the statement "exclusive to cancer cells (page 8, line 230)" is speculative and should be toned down. Mad1 localization data is also important because the authors claim that "long-term mitotic NA damage specifically stabilized kinetochore-microtubule attachments in cancer cells (page 10, line 288)" in the discussion.
R: Data from non-transformed RPE-1 cells are now included in the revised manuscript.
For the Mad1 assay, such as in Fig. 4A, the authors analyzed the CENP-C pair with two or one Mad1 foci formation. However, in some representative pictures, for example, Fig S4A-Etoposide, I found pairs of CENP-C signals on the polar chromosome without any Mad1 foci (the one next to the pairs shown in the square). As the authors argue, these kinetochores may represent polar chromosomes that eventually satisfy SAC and may be important. I, therefore, wonder why those kinetochores are omitted from the assay. Please explain this point in the manuscript if there is any reason.
R: We have now provided a clearer example and clarified in the main text that only chromosomes outside the spindle area were considered polar chromosomes.
Minor comments:
Page 7, line 168: the authors claim that "regardless of the type of DNA lesion, long-term mitotic DNA damage persists throughout mitosis and promotes micronuclei formation from polar chromosomes." However, the former claim is not fully supported by Figure S3, which addressed the effect of Etoposide only; the latter claim is not fully supported by Figure 2C, which lacks clarity (as pointed out in comment 2) and statistical analysis. Please revise this sentence.
R: We now present the levels of yH2AX after treatment with Lomustine, Mitomycin C, and Carboplatin and compared it with DMSO-treated controls and Etoposide. We also include statistics for the cell fate and respective chromosome segregations errors after treatment with the different DNA damaging agents.
Line 182: it would be helpful for readers to explain why MG132 was used.
R: This is now explained in the main text.
Line 210: it would be helpful for readers to explain briefly what PA-GFP means and how the assay works.
R: This is now explained in the main text.
Figure 1E: some color codes for each compound are difficult to distinguish. I also found it challenging to locate some lines on the graph. I recommend separating this graph, for example, by types of DNA lesions caused by compounds, and color codes that are easy to distinguish should be used.
R: We have now changed the most confusing colors and provide a higher temporal resolution chart in the low yH2AX region to facilitate visualization.
4. Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
R: None
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Referee #3
Evidence, reproducibility and clarity
In the manuscript entitled "Long-term mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells," the authors propose that DNA damage on mitotic chromosomes causes chromokinesin-mediated polar chromosomes, which eventually results in missegregation and micronuclei formation. They first performed screening of compounds that cause DNA damage on mitotic chromosomes and found that DNA damage delayed mitosis in the nocodazole wash-out experiment. The authors found that several DNA damage-inducing compounds all caused an increase of asymmetric Mad1 localization on polar chromosomes. Using photoactivatable GFP-a-tubulin, the authors showed that a-tubulin stabilizes after Etoposide treatment. They finally showed that chromokinesin Kid and Kif4a knockdown rescues the asymmetric Mad1 localization.
Major comments:
- Page 6, line 155: the authors claim that "In contrast, among other defects, treatment with any of the DNA-damaging compounds caused a significant mitotic delay due to the presence of misaligned chromosomes near the spindle poles." Although Figure 2A shows a representative image of polar chromosomes, I do not find quantitative data that analyze %polar chromosomes in mitosis treated with DNA-damaging compounds. I also do not find the data supporting the claim that polar chromosomes caused a mitotic delay. Because most subsequent analyses were performed based on this result, the quantitative data should be provided here. For the latter, I suggest showing "time in mitosis (Fig 2B)" separately with or without polar chromosomes.
- According to Figure 2C, the ratio of "Exit with micronuclei (from misaligned chromosome(s))" is relatively low compared to other phenotypes such as "Mitotic arrest" or "Cell death." I wonder if polar chromosome phenotype is also correlated with these other cell fates. Please clarify which fate is correlated with polar chromosome formation after DNA damage.
- In Figure 3, the authors used Nocodazole-treated background to assess the involvement of SAC in DNA-damaging compound-induced mitotic delay. However, as shown in Figure 2B, DNA-damaging compounds cause a minor delay in mitosis, which might be challenging to analyze in the presence of Nocodazole. There is also a possibility that DNA damage response (DDR) works independently and adjunctly to delay mitosis. Because one of the major claims of the authors is that "the SAC is the only mechanism that is required to delay mitosis in the presence of long-term mitotic DNA damage (page 10, line278)", I recommend Nocodazole wash-out (as in Figure 2B) to examine the effect of MPS1-IN-1 (and ideally an inhibitor of the DDR pathway, such as ATMi) on mitotic delay induced by DNA-damaging compounds.
- Line 226, (our unpublished observations): because the authors claim that "the formation of polar chromosomes due to the stabilization of kinetochore-microtubule attachments upon long-term mitotic DNA damage is likely exclusive to cancer cells," the authors should present data on RPE-1 cells at least for %polar chromosome formation (as suggested in comment 1) and Mad1 localization. Plus, even though the data is provided, the statement "exclusive to cancer cells (page 8, line 230)" is speculative and should be toned down. Mad1 localization data is also important because the authors claim that "long-term mitotic NA damage specifically stabilized kinetochore-microtubule attachments in cancer cells (page 10, line 288)" in the discussion.
- For the Mad1 assay, such as in Fig. 4A, the authors analyzed the CENP-C pair with two or one Mad1 foci formation. However, in some representative pictures, for example, Fig S4A-Etoposide, I found pairs of CENP-C signals on the polar chromosome without any Mad1 foci (the one next to the pairs shown in the square). As the authors argue, these kinetochores may represent polar chromosomes that eventually satisfy SAC and may be important. I, therefore, wonder why those kinetochores are omitted from the assay. Please explain this point in the manuscript if there is any reason.
Minor comments:
- Page 7, line 168: the authors claim that "regardless of the type of DNA lesion, long-term mitotic DNA damage persists throughout mitosis and promotes micronuclei formation from polar chromosomes." However, the former claim is not fully supported by Figure S3, which addressed the effect of Etoposide only; the latter claim is not fully supported by Figure 2C, which lacks clarity (as pointed out in comment 2) and statistical analysis. Please revise this sentence.
- Line 182: it would be helpful for readers to explain why MG132 was used.
- Line 210: it would be helpful for readers to explain briefly what PA-GFP means and how the assay works.
- Figure 6E-G: I wonder whether siKid+siKif4a affected %polar chromosomes or not.
- Page 10, line 287: the authors claim that "we show that long-term mitotic DNA damage..., causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesisns,...." However, only Mad1 localization data is provided in Figure 6E-G, and whether siKid + siKif4a rescues the missegregation of polar chromosomes is not clear. The authors should either provide supporting evidence or revise this sentence for clarity.
- Figure 1E: some color codes for each compound are difficult to distinguish. I also found it challenging to locate some lines on the graph. I recommend separating this graph, for example, by types of DNA lesions caused by compounds, and color codes that are easy to distinguish should be used.
Referees cross-commenting
I generally agree with other reviewers' comments and confirmed that they raised similar concerns.
Significance
It has been described previously that mitotic arrest induces DNA damage and that the DDR pathway during mitosis is attenuated. The data presented in this manuscript provide a potentially novel cellular response against DNA damage during mitosis. The manuscript will be of interest to those in the field of the cell cycle (especially mitosis), the DDR, and tumor chemotherapies. While the finding that DNA damage during mitosis causes polar chromosomes is potentially interesting, the manuscript is still rather descriptive, and data that address the molecular mechanism is insufficient for the level that the authors conclude. Although the data quality is high, I think some essential data supporting their conclusion and clarity of the description are missing from the manuscript, which can be addressed before publication.
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Referee #2
Evidence, reproducibility and clarity
Novais-Cruz et al present an interesting and generally well-performed study on the impact of DNA damage just prior to mitosis on chromosome segregation fidelity. Overall, the experiments are performed and presented to a high standard, and the key findings are potentially of interest. However, in its present form the manuscript is overall quite confusing and it is difficult to assess the robustness of their conclusions. Their observations and mechanistic model connecting the observations is not clear at all in the current form. Several key pieces of data are missing that would help craft the story, and more explanation is needed to connect their overarching hypothesis to the data better. The below points serve as an illustration of the missing information that would be needed in order to make a proper judgement of whether the data support their main conclusions.
- The term 'long-term DNA damage during mitosis' is confusing. DNA damage occurs when? (before, or during mitosis?). Their live cell data shows they are following cells that underwent mitosis within a certain time window after damage occurred, but it is not clear if it occurs only before, or sometimes during mitosis.
- All damage occurred in cells treated with nocodazole - could this have impacted the results? Was similar DNA damage induced if cells were arrested in monastrol/STLC, or MG132 for example? They show the distal Mad2 data in STLC but not the damage. Also Figure S4A/B appears to be from one experiment only which makes it difficult to interpret.
- Why not show the RPE1 data for polar chromosomes?
- Confusing interpretation - monotelic attachments, yet also stable attachments,..? Please can they clarify what is meant by these terms.
- They focus most of the study on understanding why polar chromosomes arise after DNA damage. However, this phenotype seems to be a relatively minor effect. Eg. In Figure 2b only a few cells exhibit very extended mitosis, and in Figure 2c only a small percentage exit mitosis with misaligned chromosomes. Furthermore, in Figure 4B the percentage of polar chromosomes with only distal Mad2 is low. In Figure 4A the images are not clear whether a 'both' or 'distal' is being shown. It does not seem as if any are distal only, and an example of this would be helpful.
- It is also not clear what 'other' means in Figure 2C.
- They conclude that chromokinesin depletion rescues the polar chromosomes phenotype. However they do not directly assess the rate of polar chromosome formation, only the % of polar chromosomes that are only distal Mad2 as far as I can see? They need to present the % of cells with polar chromosomes, and it would also be informative to understand the rate of cells with lagging chromosomes, or that underwent anaphase with polar chromosomes with and without chromokinesin depletion.
- It would be very helpful for them to provide a schematic model between DNA damage, overstable microtubules, satisfied SAC, monotelic attached chromosomes, and the role of chromokinesins. At present these connections are very unclear.
- It is not clear from how many experiments data are shown for the Mad1 distal experiments in Figures 4 and S4 (there are no error bars, so is this one experiment only?). This should be indicated in figure legends, and repeated if performed only once.
Significance
Novais-Cruz et al present an interesting and generally well-performed study on the impact of DNA damage just prior to mitosis on chromosome segregation fidelity. Overall, the experiments are performed and presented to a high standard, and the key findings are potentially of interest to the mitosis, and genomic instability fields.
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Referee #1
Evidence, reproducibility and clarity
Summary:
In the presented manuscript, authors investigated consequences of DNA damage on progression through mitosis. In agreement with previous reports, they observed a mitotic delay that was dependent on activation of the spindle assembly checkpoint (SAC). Starting with a high throughput screening authors concluded that the SAC-dependent mitotic delay is a common feature and is not limited to a specific type of DNA damage. They followed with a more detailed analysis using selected compounds and showed that mitotic DNA damage promotes formation of polar chromosomes with stable kinetochore-microtubule attachments. The live-cell imaging revealed that the cells carrying DNA damage eventually exited mitosis with the misaligned chromosomes forming micronuclei in the daughter cells. Most of the conclusions are supported by the experimental data with some exceptions detailed below.
Major comments:
- In the opinion of the reviewer, the study is somewhat unbalanced as it starts with a high throughput analysis of a large number of compounds but only etoposide treatment is investigated in detail in the key experiments shown in Figures 5 and 6. The effect of topoisomerase II inhibitor on kinetochore-MT stability has already been demonstrated by Bakhoum et al, 2014. If authors wish to generalize that similar phenotypes are observed after various types of DNA damage, they should test additional compounds (such as Lomustine, Mitomycin C, and Carboplatin). In addition, measuring of the kinetochore-MT half-life in figure 5 should be performed with better time resolution within the first 5 minutes. This would allow better comparison of the measured half lives that are much shorter than 5 minutes.
- Authors claim that the observed phenotype of the chromosomal missegregation following the mitotic DNA damage occurs specifically in cancer cells but the data supporting this statement is poor. They need to show more data in non-transformed cells or remove the statement.
- Involvement of Kid and Kif4A in arm ejection of the polar chromosomes is an interesting observation in context of mitotic DNA damage. However, it is unclear how the distribution of the chromokinesines was evaluated in Figure 6A-D. Was the signal quantified at the metaphase plate or at polar chromosomes? It seems that Kif4A localizes to the polar chromosome caused by etoposide treatment whereas no signal is visible in DMSO control (Fig. 6C).
- Authors convincingly showed that SAC is activated by mitotic damage and this is also consistent with previous reports. However they did not address if DDR pathways contribute to the activation of SAC. This would be interesting especially in context of a recent report that showed Bub3 as a direct substrate of ATM (Xiao et al. 2022, JBC). I wonder if the polar chromosomes are formed and missegregate also in the absence of ATM activity.
Minor comments:
- Specificity of Aurora-B pT232 antibody should be validated to exclude cross-reactivity with Aurora-A at spindle pole (Fig S8C)
Significance
This study addresses the impact of mitotic DNA damage on chromosome segregation which is an important but largely unexplored topic. Authors extend earlier observations by Bakhoum et al, 2014 and demonstrate that also the misaligned polar chromosomes result in formation of micronuclei and may promote chromosomal instability. The study will be of interest mainly for the mitosis filed. The possibility that the described phenotype may have implications for cancer therapies is interesting but will surely require more detailed studies.
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Reply to the reviewers
Manuscript number: RC-2022-01406
Corresponding author(s): Harris, Reuben
1. General Statements [optional]
We would like to thank all three reviewers for their time and thorough assessment of our manuscript. We appreciate their constructive feedback and believe our work has been considerably strengthened by addressing the comments, suggestions, and concerns raised during peer review. In the following responses, we address the reviewer’s critiques point-by-point.
2. Point-by-point description of the revisions
Reviewer 1
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Moraes et al. build upon their recent studies of APOBEC3 antagonism by EBV BORF2 by showing that additional RNR subunits encoded by other herpesviruses share this activity, suggesting that the host-virus arms races involving APOBEC3 proteins is more widespread than previously thought. Furthermore, the authors show that herpesviruses infecting primates that lack A3B (New World Monkeys) do not apparently exhibit a capacity to antagonize human A3B, suggesting that this function was not required during the evolution of those viruses (while it seemingly was important for viruses infecting hosts that encode A3B). Overall, this is a technically sound submission that combines confocal immunofluorescence, co-immunoprecipitation, and enzymatic assays to comprehensively test the sensitivity of different A3s to counteraction by viral RNRs. The enzymatic (deamination) assays performed prove to be the most insightful, since co-IP and colocalization microscopy was not entirely sufficient to reveal which domains of A3 are important for targeting by RNRs. It is well-written, well-organized, and well-referenced, and will be of interest to readers who study APOBEC3s, herpesviruses, and host-virus arms races more generally.
Response: Thank you for appreciating the technical aspects and broader impact of our studies.
Major points:
- Figure 4B: the fluorescence microscopy data does not match well with the Co-IP (in Figure 4A). For example, L1B in A3A enhances the A3A-BORF2 co-IP but no clear differences are observed in colocalization. Or are the authors claiming the presence of L1B results in greater colocalization between A3A and BORF2, because there is slightly less diffuse BORF2 in the cytoplasm under these conditions? If that is the case, then quantitative colocalization analysis will need to be performed. In general, virtually none of the colocalization analysis in Figure 4B matches well with the co-IP results of Figure 4A. The authors take this to suggest that L7, and not L1, is most important determinant for BORF2 binding to A3s, but in that case, then the colocalization data is disconnected functionally from co-IP results. This is not necessarily a large problem, since the authors ultimately test the enzymatic activity of A3s in the presence of different RNRs. These latter functional experiments more objectively define what regions of A3s are important for antagonism by RNRs.
Response: Thank you for giving us an opportunity to clarify these important points. We understand how the fluorescence microscopy data in Figure 4B may at first appear to disagree with the co-IP results in Figure 4A. However, we would like to point out that WT A3A—which has a shorter L1 region and binds less strongly to BORF2 compared to A3B—is nevertheless efficiently relocalized by BORF2 (PMID 35476445, 31534038, 31493648). We believe that this observation can be explained by compensatory avidity interactions during cytoplasmic aggregate formation in living cells, a process mediated by the formation of a non-canonical BORF2-BORF2 dimer as detailed in our recent cryo-EM studies (PMID 35476445). These avidity interactions explain how a weaker interaction (as indicated by weaker co-IP levels) can still result in the formation of large cytoplasmic aggregates. We have therefore revised our text to explain this apparent incongruity (page 11, lines 10-13).
Can the authors discuss/cite more about the actual subcellular compartments that the A3s are being relocated towards by the RNPs? In general, the authors' comments are limited to whether the A3 is predominantly in the nucleus, or not.
Response: Previous imaging studies with markers for cytoplasmic organelles by our lab suggested that BORF2-A3B aggregates accumulate within the endoplasmic reticulum (ER) (PMID 30420783). However, in our recent cryo-EM studies of the BORF2-A3B complex (PMID 35476445), we discovered that disrupting BORF2-BORF2 dimerization prevents aggregate formation but does not affect EBV BORF2’s ability to bind to A3B and relocalize the complex to the cytoplasmic compartment. In other words, dimerization-deficient mutants of BORF2 clearly cause A3B-BORF2 heterodimers to appear diffusely cytoplasmic. Therefore, we no longer have a reason to implicate the ER and we aim to clarify this in future studies aiming to define the full molecular composition of the large cytoplasmic aggregates.
Since the authors draw a connection between the absence of A3B in New World Monkeys and the fact that New World Monkey-specific viruses don't seem to counteract A3s, can the authors discuss what could be learned by studying human individuals who lack A3B and the evolution of herpesviruses in those individuals?
Response: This is a very interesting point, but we would prefer not to speculate on this in our manuscript. Although there is indeed an A3B deletion allele in the human population (predominantly southeast Asia), its worldwide allele frequency is quite low and most people still have 1 or 2 copies of this antiviral gene. Thus, the deletion allele frequency is not high enough to remove the selective pressure on the virus to maintain A3B counteraction activity through its RNR.
However, we did discover one Old World monkey species that completely lacks *A3B* (*Colobus angolensis*). We showed that the RNR from the gamma-herpesvirus that infect these monkeys (ColHV-1) lacks the ability to antagonize human A3B, ancestral A3B, human A3A, or the endogenous A3A of its natural host (__Figure 8__ and __Figure 8—figure supplement 3__). Thus, as you predicted, relieving the selective pressure within a species over an evolutionary period of time likely resulted in loss of A3B-antagonism activity by the viral RNR (page 19, lines 8-16).
Minor points:
- I'm not sure it makes sense to call out Figures 1A-D in the Introduction section, rather than the Results section.
Response: We have changed the Introduction and removed the original Figure 1 completely. We have however added a new Figure 1, which provides a structural rationale for our overall experimental approach.
Reviewer #1 (Significance (Required)):
This work represents a step-wise advance from the authors' previous work on herpesvirus RNPs and counteraction of host APOBEC3s. I study host-virus molecular arms race on evolutionary scales and this article is of interest and significance to me, and I assume to others in the field as well. The findings found within the submission are interesting but not necessarily informative about human health and disease. However, the subsequent work that this manuscript inspires is likely to tell us more about herpesvirus evolution in human patients and the mechanisms by which APOBEC3s promote cancer.
Response: We thank you again for appreciating the broader significance of our work and how the results present here may inspire important future studies.
Reviewer 2
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: Building off the groups prior work on A3B and EBV BORF2 interactions, here they have expanded their studies to examine additional herpesvirus RNRs, demonstrating which features are conserved. Using a combination of IP experiments and IF, they have included KSHV ORF61 and HSV-1 ICP6 RNRs, and demonstrated that the A3 loop structures, L1, L3, and L7 from A3A, A3B, and A3G play varying roles in determining the ability to interact with the different RNRs. They then go on to demonstrate that the ability of BORF2 to block the deaminase activity of A3B is dependent on the tyrosine at position 481. Lastly, and most interestingly, they show that RNRs from Old World monkeys, but not New World monkeys, can bind to A3A and A3B, lead to their re-localization, and block deaminase activity.
Response: We thank you for appreciating the molecular details and broader impact of our studies. Please note that we have revised the paper to focus on gamma-herpesviruses by removing the less informative results with HSV-1 and adding new studies on Old/New World viral RNRs including comparisons with ancestral A3B.
Major comments: The vast majority of this work is very convincing. The authors claims are clearly reflected in the data presented for the most part. However, the work done with HSV-1 ICP6 co-IP is not very convincing. The authors claim that L7 and L3 swaps from A3Bctd to A3Gctd decreases pulldown (lines 5-12, p.7; lines 18-21, p.8; line 17, p.16). The figures (2A, 3A, 4A) however show only A3A being pulled down with ICP6. The re-localization data however does seem more consistent with the above claims. The authors note this in line 9, p.8. However, they come to a different conclusion in line 2, p.8, regarding the discrepancy between IP and IF data.
Response: As mentioned above, we have removed the less convincing results with HSV-1 ICP6. We believe uncovering the mechanistic details of HSV-1 ICP6 interaction with A3B will require significant additional work and, therefore, would prefer to address this question in future studies.
The data and methods are clearly presented, with the exception of the supplemental figures, where it is unclear how the predicted modeling was conducted.
Response: We apologize for the brief description in our earlier submission. We have revised our Methods section and included a more detailed description regarding the generation of protein structural models (page 29, lines 20-23; page 30, lines 1-6).
Experiments all seem to be sufficiently replicated.
Response: Thank you.
Minor comments:
The references to prior studies seem comprehensive. Text and figures were all very clear. Introducing the supplemental figure 1 earlier, may provide clarity to the argument about degree of relatedness (line 2, p.7).
Response: We agree with this suggestion and have made changes to introduce the structural model of KSHV ORF61 in our new Figure 1.
The suggestion of ORF61 interaction with L3 as an anchor region (line 10-12, p.9) was not very clear/could benefit from a bit more elaboration.
Response: We agree with this comment and have placed the predicted structural model of KSHV ORF61 bound to A3Bctd in our new Figure 1 and we have changed the text to clarify the role of A3B L3 in binding to KSHV ORF61 (page 10, lines 4-8).
Reviewer #2 (Significance (Required)):
This work builds on the conceptual framework of host-pathogen interactions and co-evolution, adding new examples of co-divergence of primate herpesviruses with their respective host restriction factors. Following up on past findings (Cheng et al., 2019; Shaban et al., 2021), and reports from others (Stewart et al., 2019), they outline the degree to which their initial findings (BORF2 and A3B interactions) are conserved across other herpesvirus RNRs, and place them in the context of the evolution of the A3 gene locus and expansion.
This work will be of great interest to virologists. Especially those that work in the field of host pathogen evolution and the molecular arms race.
My background is in host-pathogen interactions and herpesvirus evolution. I lack the sufficient expertise to evaluate the predicted modeling.
Response: We thank you again for appreciating the novelty and significance of our work. We are also hopeful that it will be of great interest to virologists.
Reviewer 3
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this manuscript, Moraes and colleagues build upon previous publications from this group to 1) characterize the variation in the ability of orthologs of BORF2 from six different herpesviruses to bind and/or relocalize and/or inhibit the deaminase activity of A3A and A3B; 2) use swaps and other mutagenesis to measure whether various regions and amino acids in A3A, A3B, and A3G contribute to the observed variation in the ability of RNR subunits from different viruses to bind these A3s.
The data convincingly show that different regions of different A3s contribute differently to binding of RNRs from different viruses. These same regions also have variable effects on RNR-mediated relocalization and inhibition of the deamination activity of A3A, A3B, and A3G. In the last set of experiments presented in the manuscript, the authors show that the RNR from the four viruses isolated from humans and rhesus macaques are able to bind human A3B, while the RNRs from two New World monkey viruses are unable to bind human A3B. Finally, the authors suggest a correlation between the timing of the birth of A3B in the branch leading to the last common ancestor of hominoids/Old World monkeys and the gain of A3 binding/antagonism by herpesvirus RNRs. However, these evolutionary implications are not convincingly supported by the current datasets and would require a significant burden of initial experiments to test.
Response: We thank the reviewer for the nice summary of our work and for appreciating the loop swaps experiments showing differential RNR binding to APOBEC3s. In our original submission, we compared the RNRs of 4 viruses infecting Catarrhini primates and 2 viruses infecting New World primate species. We found that only the RNRs from viruses that infect Catarrhini primates bind, relocalize, and inhibit human A3B. We have now performed additional experiments to further investigate this remarkable association (Figures 7, 8, and associated supplementary material) which are detailed in our revised manuscript and summarized below:
First, we have expanded the scope of our experiments to include all publicly available RNR sequences from primate gamma-herpesviruses (i.e., 11 RNRs in contrast to our initial 6 RNRs). Second, we tested this whole panel against human A3B and found that only the RNRs from viruses that infect Old World primates that encode A3B are able to bind, relocalize, and inhibit human A3B (Figures 6 & 8). In comparison, binding to human A3A in co-IP experiments is invariably weaker and/or not detectable, relocalization phenotypes are less pronounced, and DNA deaminase activity is not inhibited (Figure 6 and Figure 8—figure supplement 2B). __Third, a subset of this RNR panel was tested against the A3A enzymes of their natural host species (__Figure 7) and, again, only the RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. Fourth, as an addition test of this idea, we grafted a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enabled the chimeric protein to bind to both human A3B and A3A (likely through L7), though not to the natural marmoset A3A protein. Fifth, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral A3B sequence and showed that this enzyme is nuclear and as active (if not more active) than human A3B (Figure 8). This ancestral A3B protein is also bound, relocalized, and inhibited by most present day RNRs from gamma-herpesviruses that infect species with A3B, but not by the RNRs of any of the NWM-infecting viruses tested. The only exception to this association between A3B and Catarrhini-infecting gamma-herpesviruses is the RNR of the African Colobus virus ColHV-1, which we found can likely be explained by the loss of A3B in its host species due to a deletion that occurred approximately 10-14 mya after the split of the Colobinae subfamily into African and Asian tribes, which further supports the idea that A3-antagonism by gamma-herpesvirus RNRs is maintained by the selective pressure imposed by the antiviral activity of A3B.
Major Comments
1) The use of only the human orthologs of A3A and A3B limit the inferences that can be made regarding the ability of RNRs from various viruses to bind the A3s from the host species of that virus. For example, human A3A (and other hominoid A3As) have a rather distinct Loop 1 sequence, where that same loop in rhesus A3A is a much more similar to A3B. It follows that the RNRs from rhesus-tropic viruses could very well bind and inhibit A3A from rhesus. Likewise, the A3B-RNR interactions within and between species could differ markedly. Indeed, we know that the loops of A3s are some of the most rapidly evolving regions of these genes.
Response: We agree fully with these points and have addressed them through several new experiments. We have now tested the RNRs from rhesus macaque and NWM gamma-herpesviruses against the A3A enzymes of their natural host species (Figure 7) and found that only RNRs form viruses that infect Old World primates bind and relocalize the A3A enzymes tested. In addition, we used all available present-day primate A3B sequences to reconstruct the most likely ancestral sequence and showed that this ancestral A3B enzyme is antagonized exclusively by the RNRs of present-day gamma-herpesviruses that infect A3B-encoding primates.
2) If RNR's ability to bind A3s correlated or was driven by the birth of A3B in catarrhine primates, the evolution of the binding/antagonism trait would be highly unparsimonious. The most parsimonious scenario would be emergence of A3 antagonism in the LCA of alpha and gammaherpesviruses (since the authors show A3 binding in HSV-1 and several gammaherpesviruses) with a loss of the trait in NWM-infecting viruses; alternatively, the trait could have been horizontally transferred gained 3 independent times, but this is certainly unlikely and not supported by any data. However, it is also possible that the RNRs from NWM infecting viruses do, in fact, bind/antagonize the A3 orthologs from NWMs. This needs to be tested before addressing the complexities of the birth of the antagonism trait.
Response: Please see our responses above. All of our results support a model in which the birth of A3B in an ancestral primate selected for a gamma-herpesvirus with A3B binding and neutralization activity and that this activity has been maintained through evolution and still manifests today in all of the tested present-day RNRs of gamma-herpesviruses that infect species with A3B.
Minor Comments
1) The authors should state more discreetly what is new to this paper and what was shown in previous paper and in some cases repeated here. For example, figure 1 is all repeated experiments from previous papers which is unusual for a manuscript.
Response: This is a fair point and we have removed the original Figure 1 and replaced it with a structural model that provides a strong rationale for the rest of our studies. For the sake of clarity, we have also revised our text and made the necessary changes to ensure a clear distinction between new and repeated results.
2) The authors conclude that RNRs bind to A3s via partially distinct surfaces, but they don't actually test binding. Swaps and mutations do not show that the site of mutation is a site of interaction. but they do test the requirement of these AAs or regions for binding. Formally, these mutations could be exerting an allosteric effect on the binding interface of RNR and A3. In combination with the CryoEM data, these new data do support the model that these are different surfaces of interaction, but the wording should be more precise to present this.
Response: In our revised manuscript we use a combination of in silico protein structure prediction and docking to model the binding interface between human A3Bctd and KSHV ORF61 (new Figure 1). This approach predicts an interaction with the L3 region of A3B, which we validate through co-IP and co-localization experiments (Figure 3). In contrast, EBV BORF2 requires the L7 region to bind to A3Bctd and this interaction is additionally strengthened by L1 residues (Figures 2 & 4). Taken together with our prior cryo-EM data, these results point to a model in which EBV BORF2 and KSHV ORF61 bind to different surfaces of A3B (albeit near the active site and likely due to evolutionary “wobbling”). We therefore believe it to be unlikely that this mechanism is allosteric given our prior structural studies and the likely common evolutionary origin of this A3B antagonism mechanism.
3) Similar to point 1, the authors repeatedly discuss the "most critical determinant of EBV BORF2 binding" and other "most critical" interactions. This is not supported by the data and should be changed to something along the lines of 'the site of largest effect among the sites we analyzed'.
Response: We have endeavored to change this text as suggested except in cases referring to the interactions between EBV BORF2 and A3Bctd, since the results presented here together with our cryo-EM structure of the BORF2-A3Bctd complex (PMID 35476445) allow us to confidently say that L7 and L1 are the most critical determinants.
4) All microscopy figures need an A3 only panel (no RNR) to be able to judge relocalization.
Response: Changed as suggested.
5) The matrix of labels above each IP blot is excessive since each lane only has one component that differs from the other lanes. A single label for each lane would make the plot easier to discern. These figures would also benefit from clearer labels including which virus each blot panel corresponds to (these could be along the left side of each blot; currently, the RNR gene name is provided, but this is a bit hard to find within the figure). Figures 2-4 would benefit from a label above each panel A indicating "L1" "L3" "L7".
Response: Changed as suggested.
6) If the authors comment on pg9 ln 8 about intermediate relocalization effect, they should also mention 1C A3B L7G against BORF2
Response: Changed as suggested (page 8, lines 16-19).
7) Why is there no quantification of 6D relocalization? Could be supplemental if needed.
Response: We have performed quantification of the relocalization phenotypes in Figures 2, 3 & 4 in order to allow direct comparison between WT and chimeric A3 enzymes in the presence of the same viral RNR (EBV BORF2 or KSHV ORF61). On the other hand, the images in Figure 6D are representative of the A3B/A relocalization phenotypes elicited by a larger panel of different viral RNRs. These representative images should be interpreted together with the co-IP and ssDNA deaminase activity assay data in Figures 6C & 6E, respectively.
8) Pg 6 ln 13 and Pg 8 ln2-4, ICP6 doesn't coIP w A3B; this should be clarified.
Response: A similar concern was also raised by reviewer #2, and we agree that the ICP6 data present in the original version of this manuscript are not as easily interpretable compared to results with the RNRs from gamma-herpesviruses such as EBV and KSHV. For the sake of clarity and cohesion, we decided to remove all of the HSV-1 ICP6 data from the revised version of our manuscript and focus on the A3B interactions with gamma-herpesviruses.
9) Pg 8 ln21-23, the authors assume loss of function for A3G, but this swap could be functionally equivalent, but necessary for binding; it should be clarified that this is different than changing sequence and still binding.
Response: Rephrased (page 9, lines 13-16).
10) Pg 9, ln 11, what is an anchor region?
Response: We have removed the term “anchor region” and rephrased our text to more clearly describe the importance of L3 in KSHV ORF61 binding (page 10, lines 4-8).
11) Pg 9, ln 23, speculative - this might be explained by this 3 AA motif but it has not been tested.
Response: Changed wording (page 10, lines 17-20).
12) Pg 10 ln 8, this doesn't show that this region is dispensable for binding, only that there is equivalent contribution or lack of contribution of function by the A and B loops, again assuming that the G loop is LOF
Response: Removed the phrase “indicating that this region may be dispensable for the interaction” (page 11, 1-2).
13) Pg 10 ln 10 - "can be explained by presence of bulky tryp" - this should be reworded to 'could or is likely caused by'.
Response: Changed as suggested (page 11, lines 4-6).
14) Pg 11 ln 21, "can be explained by our cryo-EM" should be reworded to 'is supported by these contacts in cryo-EM'
Response: Changed as suggested (page 12, lines 15-18).
15) Pg 13 ln 10 (and other places) dissociation rates are only part of affinity, Ka is equally important (pg 18 ln 8 also)
Response: We agree and have revised our text account for this suggestion (page 13, lines 22-23; page 14, lines 1-2; page 22, lines 12-15).
16) Pg 14, ln 15 should be reworded to 'relocalize HUMAN cellular A3s'
Response: Changed as suggested (page 15, line 11).
17) Pg 16 Ln 16, this should be reworded as the data can't say it is completely dispensable without deletion of the loop.
Response: Changed as suggested (page 21, lines 10-11).
18) New World monkeys have high activity of transposable elements of distinct types relative to catarrhines. It would be useful to mention that A3s restrict endogenous elements as well and how this might be a factor in the proposed evolutionary model.
Response: This is a very interesting point that we plan to discuss in a future review. In addition, many future experiments will be needed to test the potential relationship between the birth of A3B and its potential impact on different classes of endogenous transposable elements.
19) Are the New World monkey viruses pathogenic in their native hosts? Perhaps not based on previous literature (reviewed in PMID: 11313011). This should be included in the discussion as it could certainly effect the evolutionary model for the birth/retention of A3 antagonism in these viruses.
Response: While interesting, the observation that NWM herpesviruses do not cause disease in their native hosts is not unusual. In fact, most gamma-herpesviruses (including human viruses like EBV and KSHV) have limited pathogenic potential when infecting their natural hosts (Fleckenstein B, Ensser A. Gammaherpesviruses of new world primates. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. 2007). Additionally, although the mentioned study (PMID: 11313011) reports asymptomatic infection of squirrel monkeys with SaHV-2, pathogenic infection/ oncogenic transformation have been reported following natural infection of marmosets with CalHV-3 (PMID: 11158621).
20) In previous papers on this topic, the lab has tested the effect of mutations on viral titers. While this may be beyond the scope of this paper, this would certainly elevate the paper and should be more clearly discussed.
Response: As noted by the reviewer, we have previously demonstrated that A3B restricts EBV replication though a mutation-dependent mechanism and that this is counteracted by EBV BORF2 (PMID 30420783). While we completely agree that investigating the effect of A3B-catalyzed mutations on the titers of different gamma-herpesviruses would be interesting, this would be technically challenging as we are currently not equipped to work with KSHV or any non-human primate herpesvirus.
21) What is the degree of sequence similarity among these and other RNRs? Is there any sense of what region of RNR binds A3s from the CryoEM structures and differences within these regions that might explain the functional differences?
Response: We thank the reviewer for raising this important point. We have now included a new Figure 1 where we leverage the cryo-EM structure of the EBV BORF2-A3Bctd complex to make inferences about which regions of KSHV ORF61 may be involved in binding A3B/A. As described above, we also graft a short helical loop structure (HLS) from EBV BORF2 into the marmoset CalHV-3 RNR and showed that this small change enables the chimeric protein to bind to bind both human A3B and A3A (likely through L7), though not to the natural host marmoset A3A protein (Figure 7). Many additional interspecies chimeras could be constructed but we feel these are better suited for future studies (and specially to accompany future structural work in this area).
Reviewer #3 (Significance (Required)):
Significance
Previous work from the Harris lab showed that a subunit of the ribonucleotide reductase of some herpesviruses acts as an antagonist of several human APOBEC3s. Mechanistically, these viral protein block A3 inhibition by relocalizing nuclear A3s as well as inhibiting A3 deamination by binding and occluding the A3 active site. For Epstein-Barr virus, deletion of the antagonist (BORF2) results in a decrease in viral replication and accumulation of mutations likely introduced by host A3B that is no longer inhibited. However, deletion of the A3 antagonist from herpes simplex virus-1 (ICP6) had no effect on viral titers. Most recently, this group published a cryoEM structure of BORF2 in complex with the c-terminal half of A3B. This structure showed extensive contacts between BORF2 and two loops of A3B - L1 and L7.
The manuscript under review focuses on the previously suggested differences in the ability of different RNRs to bind A3A and A3B. This work provides an important contribution to this topic in defining specific regions of A3A and A3B and A3G that are necessary for viral RNRs to bind them. The variability in these interactions is surprising and likely testament to the impactful coevolution of herpesviruses and primate A3s. This manuscript will be of particular interest to virologists studying A3s or herpesviruses as well as evolutionary biologists interested in the rules of engagement between host restriction factors and viruses.
Response: We thank you again for these thoughtful comments and for appreciating the overall significance of our work.
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Referee #3
Evidence, reproducibility and clarity
Summary
In this manuscript, Moraes and colleagues build upon previous publications from this group to 1) characterize the variation in the ability of orthologs of BORF2 from six different herpesviruses to bind and/or relocalize and/or inhibit the deaminase activity of A3A and A3B; 2) use swaps and other mutagenesis to measure whether various regions and amino acids in A3A, A3B, and A3G contribute to the observed variation in the ability of RNR subunits from different viruses to bind these A3s.
The data convincingly show that different regions of different A3s contribute differently to binding of RNRs from different viruses. These same regions also have variable effects on RNR-mediated relocalization and inhibition of the deamination activity of A3A, A3B, and A3G. In the last set of experiments presented in the manuscript, the authors show that the RNR from the four viruses isolated from humans and rhesus macaques are able to bind human A3B, while the RNRs from two New World monkey viruses are unable to bind human A3B. Finally, the authors suggest a correlation between the timing of the birth of A3B in the branch leading to the last common ancestor of hominoids/Old World monkeys and the gain of A3 binding/antagonism by herpesvirus RNRs. However, these evolutionary implications are not convincingly supported by the current datasets and would require a significant burden of initial experiments to test.
Major Comments
- The use of only the human orthologs of A3A and A3B limit the inferences that can be made regarding the ability of RNRs from various viruses to bind the A3s from the host species of that virus. For example, human A3A (and other hominoid A3As) have a rather distinct Loop 1 sequence, where that same loop in rhesus A3A is a much more similar to A3B. It follows that the RNRs from rhesus-tropic viruses could very well bind and inhibit A3A from rhesus. Likewise, the A3B-RNR interactions within and between species could differ markedly. Indeed, we know that the loops of A3s are some of the most rapidly evolving regions of these genes.
- If RNR's ability to bind A3s correlated or was driven by the birth of A3B in catarrhine primates, the evolution of the binding/antagonism trait would be highly unparsimonious. The most parsimonious scenario would be emergence of A3 antagonism in the LCA of alpha and gammaherpesviruses (since the authors show A3 binding in HSV-1 and several gammaherpesviruses) with a loss of the trait in NWM-infecting viruses; alternatively, the trait could have been horizontally transferred gained 3 independent times, but this is certainly unlikely and not supported by any data. However, it is also possible that the RNRs from NWM infecting viruses do, in fact, bind/antagonize the A3 orthologs from NWMs. This needs to be tested before addressing the complexities of the birth of the antagonism trait.
Minor Comments
- The authors should state more discreetly what is new to this paper and what was shown in previous paper and in some cases repeated here. For example, figure 1 is all repeated experiments from previous papers which is unusual for a manuscript.
- The authors conclude that RNRs bind to A3s via partially distinct surfaces, but they don't actually test binding. Swaps and mutations do not show that the site of mutation is a site of interaction. but they do test the requirement of these AAs or regions for binding. Formally, these mutations could be exerting an allosteric effect on the binding interface of RNR and A3. In combination with the CroEM data, these new data do support the model that these are different surfaces of interaction, but the wording should be more precise to present this.
- Similar to point 1, the authors repeatedly discuss the "most critical determinant of EBV BORF2 binding" and other "most critical" interactions. This is not supported by the data and should be changed to something along the lines of 'the site of largest effect among the sites we analyzed'.
- All microscopy figures need an A3 only panel (no RNR) to be able to judge relocalization.
- The matrix of labels above each IP blot is excessive since each lane only has one component that differs from the other lanes. A single label for each lane would make the plot easier to discern. These figures would also benefit from clearer labels including which virus each blot panel corresponds to (these could be along the left side of each blot; currently, the RNR gene name is provided, but this is a bit hard to find within the figure). Figures 2-4 would benefit from a label above each panel A indicating "L1" "L3" "L7".
- If the authors comment on pg9 ln 8 about intermediate relocalization effect, they should also mention 1C A3B L7G against BORF2
- Why is there no quantification of 6D relocalization? Could be supplemental if needed.
- Pg 6 ln 13 and Pg 8 ln2-4, ICP6 doesn't coIP w A3B; this should be clarified.
- Pg 8 ln21-23, the authors assume loss of function for A3G, but this swap could be functionally equivalent, but necessary for binding; it should be clarified that this is different than changing sequence and still binding.
- Pg 9, ln 11, what is an anchor region?
- Pg 9, ln 23, speculative - this might be explained by this 3 AA motif but it has not been tested.
- Pg 10 ln 8, this doesn't show that this region is dispensable for binding, only that there is equivalent contribution or lack of contribution of function by the A and B loops, again assuming that the G loop is LOF
- Pg 10 ln 10 - "can be explained by presence of bulky tryp" - this should be reworded to 'could or is likely caused by'.
- Pg 11 ln 21, "can be explained by our cryo-EM" should be reworded to 'is supported by these contacts in cryo-EM'
- Pg 13 ln 10 (and other places) dissociation rates are only part of affinity, Ka is equally important (pg 18 ln 8 also)
- Pg 14, ln 15 should be reworded to 'relocalize HUMAN cellular A3s'
- Pg 16 Ln 16, this should be reworded as the data can't say it is completely dispensable without deletion of the loop.
- New World monkeys have high activity of transposable elements of distinct types relative to catarrhines. It would be useful to mention that A3s restrict endogenous elements as well and how this might be a factor in the proposed evolutionary model.
- Are the New World monkey viruses pathogenic in their native hosts? Perhaps not based on previous literature (reviewed in PMID: 11313011). This should be included in the discussion as it could certainly effect the evolutionary model for the birth/retention of A3 antagonism in these viruses.
- In previous papers on this topic, the lab has tested the effect of mutations on viral titers. While this may be beyond the scope of this paper, this would certainly elevate the paper and should be more clearly discussed. What is the degree of sequence similarity among these and other RNRs? Is there any sense of what region of RNR binds A3s from the CryoEM structures and differences within these regions that might explain the functional differences?
Significance
Previous work from the Harris lab showed that a subunit of the ribonucleotide reductase of some herpesviruses acts as an antagonist of several human APOBEC3s. Mechanistically, these viral protein block A3 inhibition by relocalizing nuclear A3s as well as inhibiting A3 deamination by binding and occluding the A3 active site. For Epstein-Barr virus, deletion of the antagonist (BORF2) results in a decrease in viral replication and accumulation of mutations likely introduced by host A3B that is no longer inhibited. However, deletion of the A3 antagonist from herpes simplex virus-1 (ICP6) had no effect on viral titers. Most recently, this group published a cryoEM structure of BORF2 in complex with the c-terminal half of A3B. This structure showed extensive contacts between BORF2 and two loops of A3B - L1 and L7.
The manuscript under review focuses on the previously suggested differences in the ability of different RNRs to bind A3A and A3B. This work provides an important contribution to this topic in defining specific regions of A3A and A3B and A3G that are necessary for viral RNRs to bind them. The variability in these interactions is surprising and likely testament to the impactful coevolution of herpesviruses and primate A3s. This manuscript will be of particular interest to virologists studying A3s or herpesviruses as well as evolutionary biologists interested in the rules of engagement between host restriction factors and viruses.
Expertise keywords: restriction factors, APOBEC3 evolution, evolutionary genomics, genetic conflict
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Referee #2
Evidence, reproducibility and clarity
Summary:
Building off the groups prior work on A3B and EBV BORF2 interactions, here they have expanded their studies to examine additional herpesvirus RNRs, demonstrating which features are conserved. Using a combination of IP experiments and IF, they have included KSHV ORF61 and HSV-1 ICP6 RNRs, and demonstrated that the A3 loop structures, L1, L3, and L7 from A3A, A3B, and A3G play varying roles in determining the ability to interact with the different RNRs. They then go on to demonstrate that the ability of BORF2 to block the deaminase activity of A3B is dependent on the tyrosine at position 481. Lastly, and most interestingly, they show that RNRs from Old World monkeys, but not New World monkeys, can bind to A3A and A3B, lead to their re-localization, and block deaminase activity.
Major comments:
The vast majority of this work is very convincing. The authors claims are clearly reflected in the data presented for the most part. However, the work done with HSV-1 ICP6 co-IP is not very convincing. The authors claim that L7 and L3 swaps from A3Bctd to A3Gctd decreases pulldown (lines 5-12, p.7; lines 18-21, p.8; line 17, p.16). The figures (2A, 3A, 4A) however show only A3A being pulled down with ICP6. The re-localization data however does seem more consistent with the above claims. The authors note this in line 9, p.8. However, they come to a different conclusion in line 2, p.8, regarding the discrepancy between IP and IF data.
The data and methods are clearly presented, with the exception of the supplemental figures, where it is unclear how the predicted modeling was conducted.
Experiments all seem to be sufficiently replicated.
Minor comments:
The references to prior studies seem comprehensive. Text and figures were all very clear. Introducing the supplemental figure 1 earlier, may provide clarity to the argument about degree of relatedness (line 2, p.7).
The suggestion of ORF61 interaction with L3 as an anchor region (line 10-12, p.9) was not very clear/could benefit from a bit more elaboration.
Significance
This work builds on the conceptual framework of host-pathogen interactions and co-evolution, adding new examples of co-divergence of primate herpesviruses with their respective host restriction factors. Following up on past findings (Cheng et al., 2019; Shaban et al., 2021), and reports from others (Stewart et al., 2019), they outline the degree to which their initial findings (BORF2 and A3B interactions) are conserved across other herpesvirus RNRs, and place them in the context of the evolution of the A3 gene locus and expansion.
This work will be of great interest to virologists. Especially those that work in the field of host pathogen evolution and the molecular arms race.
My background is in host-pathogen interactions and herpesvirus evolution. I lack the sufficient expertise to evaluate the predicted modeling.
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Referee #1
Evidence, reproducibility and clarity
Moraes et al. build upon their recent studies of APOBEC3 antagonism by EBV BORF2 by showing that additional RNR subunits encoded by other herpesviruses share this activity, suggesting that the host-virus arms races involving APOBEC3 proteins is more widespread than previously thought. Furthermore, the authors show that herpesviruses infecting primates that lack A3B (New World Monkeys) do not apparently exhibit a capacity to antagonize human A3B, suggesting that this function was not required during the evolution of those viruses (while it seemingly was important for viruses infecting hosts that encode A3B). Overall, this is a technically sound submission that combines confocal immunofluorescence, co-immunoprecipitation, and enzymatic assays to comprehensively test the sensitivity of different A3s to counteraction by viral RNRs. The enzymatic (deamination) assays performed prove to be the most insightful, since co-IP and colocalization microscopy was not entirely sufficient to reveal which domains of A3 are important for targeting by RNRs. It is well-written, well-organized, and well-referenced, and will be of interest to readers who study APOBEC3s, herpesviruses, and host-virus arms races more generally.
Major points:
- Figure 4B: the fluorescence microscopy data does not match well with the Co-IP (in Figure 4A). For example, L1B in A3A enhances the A3A-BORF2 co-IP but no clear differences are observed in colocalization. Or are the authors claiming the presence of L1B results in greater colocalization between A3A and BORF2, because there is slightly less diffuse BORF2 in the cytoplasm under these conditions? If that is the case, then quantitative colocalization analysis will need to be performed. In general, virtually none of the colocalization analysis in Figure 4B matches well with the co-IP results of Figure 4A. The authors take this to suggest that L7, and not L1, is most important determinant for BORF2 binding to A3s, but in that case, then the colocalization data is disconnected functionally from co-IP results. This is not necessarily a large problem, since the authors ultimately test the enzymatic activity of A3s in the presence of different RNRs. These latter functional experiments more objectively define what regions of A3s are important for antagonism by RNRs.
- Can the authors discuss/cite more about the actual subcellular compartments that the A3s are being relocated towards by the RNPs? In general, the authors' comments are limited to whether the A3 is predominantly in the nucleus, or not.
- Since the authors draw a connection between the absence of A3B in New World Monkeys and the fact that New World Monkey-specific viruses don't seem to counteract A3s, can the authors discuss what could be learned by studying human individuals who lack A3B and the evolution of herpesviruses in those individuals?
Minor points:
- I'm not sure it makes sense to call out Figures 1A-D in the Introduction section, rather than the Results section.
Significance
This work represents a step-wise advance from the authors' previous work on herpesvirus RNPs and counteraction of host APOBEC3s. I study host-virus molecular arms race on evolutionary scales and this article is of interest and significance to me, and I assume to others in the field as well. The findings found within the submission are interesting but not necessarily informative about human health and disease. However, the subsequent work that this manuscript inspires is likely to tell us more about herpesvirus evolution in human patients and the mechanisms by which APOBEC3s promote cancer.
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- Sep 2022
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www.biorxiv.org www.biorxiv.org
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Reply to the reviewers
1. General Statements [optional]
See cover letter for more details.
Summary of response to reviewers:
We were immensely pleased that the reviewers considered our conclusions “well supported” and our study “beautifully executed”. Reviewers also recognized the significance of our work. Reviewer 1 stated that “building a model that describes one of these pathways will allow us to begin to test therapies to treat or prevent scoliosis” then noted that we “help to build a larger model of normal spine morphogenesis” and that this is “important”. Reviewer 2 called our work an “exciting advance in our understanding of one of the essential signaling pathways that help regulate body axis straightening and spine morphogenesis in zebrafish” and mentioned that our work “may also help to further our understanding of the etiology and pathophysiology of multiple forms of neuromuscular scoliosis in humans”. Reviewer 3 agreed, stating that our work “adds important information on the role of urotensin signaling in spine formation” and noted that it is timely: “findings are of special significance in the light of recent reports that mutations in UTS2R3 show association with spinal curvature in patients with adolescent idiopathic scoliosis”.
We thank the three reviewers for reading our research and providing feedback. In all cases, we have incorporated (or plan to incorporate) their suggestions, and we believe this has (will) make our manuscript much stronger. Indeed, reviewers had only a small number of “major points”, and all are easily addressed as summarized below. We have already addressed some of those “major points”, as well as the majority of “minor points” raised by reviewers, in our current draft. We expect that all comments can be fully addressed within around 1 month.
2. Description of the planned revisions
Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are plannedto address the points raised by the referees.
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We have divided our responses by whether the reviewers considered their points major or minor. All points have already been, or will soon be, fully addressed.
Major points
Reviewer 1
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The key conclusions are well supported, see below for my two major issues.
Please don't call this lordosis. Lordosis or hyperlordosis effects lumbar vertebra. The curve in the lumbar region shifts body weight so that human gait is more efficient that that in the great apes, or so the story goes. Zebrafish do not have lumbar vertebra equivalents or a natural curve in the caudal region. Similarly, fish do not have the equivalent vertebra to generate kyphosis, which is again a hyper flexion of a normal human spinal curve. Instead zebrafish have Weberian, precaudal and caudal vertebra. It would be so much more useful for the field if the authors used these terms and specified ranges, i.e. numbered vertebrae, that are effected so we can directly and accurately compare regions of defects between zebrafish mutants. It would help to make the point that the uts2r3 mutant has more caudally located curves than urp1/2 double mutants. We appreciate this point and agree with the reviewer. Lordosis (or hyperlordosis) is indeed the accentuation of a curve which naturally exists in humans but not zebrafish. We called the phenotype of Urotensin pathway mutants ‘lordosis’ or ‘lordosis-like’ because of the position of the curves — in caudal vertebrae, which are evolutionarily and positionally equivalent to lumbar vertebrae, though they are structurally different to human lumbar vertebrae. To address this comment, we will no longer refer to the phenotype as lordosis in our Introduction or Results sections and we will expand our Discussion to include this point raised by the reviewer.
- The observation that urp1/2 double mutants have curves only in the D/V plane and almost completely lack side-to-side curves is noteworthy. Does the urp1-/-urp2-/- mutant uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be very interesting. It is particularly important to describe how penetrant the phenotype is and how many times it was observed. See 9 minor comments. It would help the reader if the authors explicitly described the features that they see in the cfap298 mutant that constitute lateral curves and that are lacking in urp1/2 (e.g. in figure 4E).
We plan to expand the figure and analysis describing D/V curves and M/L curves. While our first draft included only cfap298 and urp1-∆P;urp2-∆P mutants, our next draft will also incorporate uts2r3 and pkd2l1 mutants. We have already scanned cohorts of all mutant fish, and so the remaining work to render and quantify the degree of lateral curvature will not take long. This will allow us to conclusively determine whether these different mutations indeed uncouple two systems controlling posture in different directions. As the reviewer requests, we will include all fish analyzed in either main or supplementary figures, include numbers in figure legends, and quantify the penetrance of M/L and D/V curves.
We have also generated cfap298;urp1-∆P;urp2-∆P triple mutants and are currently scanning them to reveal skeletal form. Preliminary data suggests triple mutants have three-dimensional curves but D/V curves are more severe in triple mutants than in cfap298 mutants alone. This makes sense if Urp1/Urp2 are important for controlling D/V spinal shape and, as our qPCR shows, Urp1/Urp2 are downregulated but not lost completely in cfap298 mutants. It also furthers the notion that cilia motility controls D/V and M/L curves by separable mechanisms. * *
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Reviewer 2
Need to show that the CRISPANT targeting was effective for mutagenesis at each loci screened in the work presented in Figure 1E. In Figure 1E, we presented the phenotypes of crispant embryos (i.e. embryos injected with four gRNAs targeting a specific gene alongside relatively high doses of Cas9 protein; see schematic in Figure 1G). In positive controls (cfap298 and sspo), crispants showed the expected phenotype in all cases (Figure 1E and see Figure 1H for quantitation). As for germline mutants, urp1 and urp2 crispants showed no early axial phenotypes (Figure 1E and 1H). As such, the reviewer requests that we perform molecular assays to determine whether mutagenesis was successful in these embryos. To do so, we will perform either T7 assays or next-generation/Sanger sequencing of mutated loci. This will allow us to determine and quantify the effectiveness of our mutagenesis. Results will be shared in a new supplementary figure. These assays are straightforward and we expect they will not take very long to complete. Indeed, we have performed these assays previously for other genes (e.g. Grimes et al., 2019 and several unpublished genes). We have achieved high levels of mutagenesis in all cases, making us very confident that we will achieve similarly high levels of mutagenesis in this case.
Reviewer 3
The addition of the F0 crispant experiment to show that the pro-peptide of urp1/2 does not have a function and is responsible for the difference between the observed morpholino and the crispr phenotype was important. However, since no phenotype was observed in crispants it is important to add evidence of induced cuts for all guide RNAs used in the crispant experiment. These control experiments might have been done already. If not, they can easily be done in a short period of time by performance of T7 assays on injected fish and would not require additional reagents. This is the same point raised by reviewer 2 and so we refer to the response above. In summary, we agree with the reviewer and we are currently performing these suggested experiments which are straightforward and working well.
The authors claim that there were no structural defects observed in urp1/2 double mutants. However, the hemal arch in figure 3 E seems to be deformed. This could be normal variance or a phenotype. This can be addressed by simple reinspection of the scans.
We believe there are no major vertebral structural defects that could be attributed to causing the spinal curves because vertebrae are well-formed in mutants and we see no defects in the initial patterning of vertebrae in our calcein experiments. However, since urp1-∆P;urp2-∆P and uts2r3 mutant spines are curved, the vertebrae are a little misshapen. We plan two revisions, one textual and one analytical.
First, we will make clear in our textual edits that some vertebrae are slightly misshapen, as occurs in non-congenital forms of human spinal curve disease (in congenital forms, the shape defects are more striking and likely causative in the curvature). We agree with the reviewer that stating that there is a lack of vertebral structural defects lacked nuance, so we will expand on this in our next draft.
Second, we will quantify vertebral shapes in spinal curve mutants and report these data in our next draft. After reinspection of the scans, as the reviewer suggested, we believe it would be informative for our readers to see quantitation of vertebral shape. We expect these data to more rigorously back up our statements about ‘minor structural differences’ of vertebrae between uncurved and curved individuals. We have already begun this work, and completing it should only take a few more weeks. As an example, we have measured the shape of centra by calculating aspect ratios in wild-type and urp1-∆P;urp2-∆P double mutants in curved regions of the spine:
These preliminary data already make clear that there are indeed subtle morphological differences between vertebrae in mutants and wild-type, as occurs in human spinal curve deformities. We will present completed versions of these data (several parameters that describe vertebral shape) in our next draft and provide comments about whether such changes could be causative in spinal curve etiology as occurs in congenital-type scoliosis.
Minor points
Reviewer 1
Supplementary FigS3B How to measure the Cobb Angle is unclear. Why is the first curve not counted? I count 3 curves. First a ventral displacement, then a dorsal to ventral return, then a sharp flex before the tail. How to measure Cobb angle might be easier to explain if the figure is expanded into steps. Identify the apical vertebra, then showing how the lines are drawn parallel to those vertebrae, then where the measured angle forms between the lines perpendicular to the drawn parallel lines.
We will more thoroughly explain how Cobb angle is measured in our next draft.
5a. I think we (zebrafish biologists) need be explicit about what we mean with "without vertebral defects." What do we count as defects? Vertebrae can be fused, bent, shortened or the growing edges can be slanted. In Figure 3E, and movie7, it is clear that the highlighted mutant vertebrae are shorter than WT. The growing ends of normal vertebra are perpendicular to the long axis of the vertebra. In the mutants the ends are slanted. Please define in the text what you consider a relevant vertebral defect, because these vertebrae have defects. Or are you only considering the calcein stained centra at 10dpf?
We strongly agree with the reviewer. As described more thoroughly above in response to Major Comment – Reviewer 3, we plan both textual edits and new quantitation of vertebral shape to address this comment. Our quantitation indeed shows some vertebrae are shorter in mutants as the reviewer noticed. We also plan a new paragraph in the Discussion section which will speak about the issue of what zebrafish biologists might mean by “without vertebral defects”.
5b. Do you want to base your patterning conclusion on primarily the calcein data as these are closer to the notochord patterning time window. Please anchor this conclusion to a specific time or standard length e.g. 10dpf/5.6mm.
When we edit our descriptions of vertebral defects, and include new quantitative data on the shape of vertebrae, we will be clear that the vertebrae are slightly structurally malformed. In addition, when we speak of the calcein data, we will anchor those conclusions to the specific timepoint best studied by this method, as the reviewer suggests.
"At 30 dpf... several mutants exhibited a significant curve in the pre-caudal vertebrae, in addition to a caudal curve (Fig. 3D and S3C). Since pre-caudal curves were rare in mutants at 3-months, this suggested that curve location is dynamic".The frequency of this observation is important. Does it effect all or a fraction of mutants? Can you provide some numbers to anchor these observations? Maybe fractions e.g.. 3 of 4 fish had precaudal curves at 30pdf, and 0 of 10 fish had precaudal curves by 3 mpf?
In our next draft, we will provide numbers of fish examined at 30 dpf and also show graphical summaries of curve position (as we did for younger fish). Last, all scans will be included in a new supplementary figure.
The description of the pkd2l1 mutant, instead of terming it kyphosis can you tell the reader the vertebra number at the peak of the curve. The authors say the pkd2l1 mutant is highly distinct from urp1/urp2-/-, but the reader needs to hear exactly what is distinct. For example, does this mutant have both lateral and D/V curves?
We have now scanned several pkd2l1 mutant fish and we will include images of pkd2l1 mutants at two different timepoints together with quantitation of curve position. Our results agreed with those previously published for this mutant line (Sternberg et al., 2018) but we believe it is important for our readers to see side-by-side images and quantitation so they can see the distinctions.
At 3-months of age, pkd2l1 mutants essentially appear wild-type but by around 12-months they have developed a D/V curve in the pre-caudal vertebrae. They do not exhibit M/L curves; we will quantify this and include these data in our Figure about M/L deviation.
We called the phenotype displayed by pkd2l1 mutants “kyphosis” to be in line with a previous publication describing these mutants (Sternberg et al., 2018). We will add new wording in the Discussion about whether or not zebrafish can truly model kyphosis and lordosis (see response to Reviewer 1 major comment above), and we make clear in our Results that the phenotype has “been argued to model kyphosis (Sternberg et al., 2018)” rather than “is kyphosis”.
It is intriguing that pkd2l1 mutants do not exhibit any curves until much later in life than urp1-∆P;urp2-∆P and uts2r3mutants. Inspired by this finding, we aged urp1-∆P and urp2-∆P single mutants and found that they go on to develop D/V curves by 12-months i.e.
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*3-months 12-months Position of curve
urp1-∆P no curves mild D/V curves Mostly caudal
urp2-∆P mild D/V curves intermediate D/V curves Mostly caudal
urp1-∆P;urp2-∆P severe D/V curves severe D/V curves Mostly caudal
uts2r3 severe D/V curves severe D/V curves Mostly caudal
cfap298 severe 3D curves severe 3D curves Caudal and pre-caudal
pkd2l1 no curves mild D/V curves Mostly pre-caudal
Phenotypes in urp1-∆P and urp2-∆P single mutants upon aging shows: 1) Urp1 and Urp2 are not entirely redundant in long-term spine maintenance and 2) proper Urp1/Urp2 dose is essential. We will include these new data in our next draft.
Does uts2r3-/- have no /minimal side-to-side curves like urp1/urp2-/-?
This is an interesting question. To address it, we will add images of uts2r3 mutant spines from the dorsal aspect and include them with our new quantitation of lateral curvature. To sum, the reviewer’s suggestion is correct – there are minimal side-to-side curves in uts2r3 mutants.
One finding that deserves more discussion is the observation that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? Please consider the following paper. It shows a proprioception system that maintains normal side-to-side posture. A spinal organ of proprioception for integrated motor action feedback. Picton LD, Bertuzzi M, Pallucchi I, Fontanel P, Dahlberg E, Björnfors ER, Iacoviello F, Shearing PR, El Manira A. Neuron. 2021 Apr 7;109(7):1188-1201.e7. doi: 10.1016/j.neuron.2021.01.018. Epub 2021 Feb 11. PMID: 33577748
Thank you for pointing out this manuscript. We will include it in our expanded Discussion.
Reviewer 2
Fig 3F: might be improved by making the images black and white and possibly inverted. It is not easy to clearly see the vertebrae as is. * *
Thanks for the suggestion, we will make this change.
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3. Description of the revisions that have already been incorporated in the transferred manuscript
Minor points
Reviewer 1
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Figure 1D legend says urp1 is expressed in dorsal while urp2 is express in all CSF-cNeurons, but the image for urp1 shows only ventral cells in WT, while the image for urp2 shows the same cells ...and more dorsal cells. Please replace image with one that matches the text. Apologies for this, we have now corrected it. The image was correct but we accidentally wrote “dorsal” instead of “ventral” when describing the CSF-cN sub-population harboring urp1 transcripts.
In Figure 2H, the position of curve apex graphic, how many fish were examined? In 2f it looks like n=8 and n=9. Can this info be added to the figure?
We have now included the number examined in the legend.
I did not find legends for the movies. The first call to the movies calls movies 1-3 without explaining what each shows. The labels on the downloaded files are not informative.
Apologies for forgetting to submit these. We have now added informative Movie legends.
Reviewer 3
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It would be helpful to the reader to add a little more information on urp1 and upr2 proteins and their processing to make it clear while only the 3' region of the protein was targeted to induce mutations. We have incorporated textual edits to make this more clear. We now state in the second sentence of the Results section:
Urp1 and Urp2 are encoded by 5-exon genes with the final exon coding for the 10-amino acid peptides that are released by cleavage from the pro-domain (Fig. 1A).
Together with Fig. 1A and Supplementary Fig. 1, we hope it is now clear to readers how Urp1 and Urp2 are generated from a 5-exon gene encoding the pro-domain and the peptide, which are separated by cleavage.
It would also be helpful to the reader to have a schematic indicating the guide target sites (they could be added to figure S1 C + D) in the protein to be able to interpret the result more easily.
Done!
Figure 5: Addition of a square to H would help understand were the pictures in D-F were taken.
Done!
4. Description of analyses that authors prefer not to carry out
N/A. We are performing all experiments/analyses requested by reviewers.
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Referee #3
Evidence, reproducibility and clarity
The presented work by Bearce et al. is based on the hypothesis that urp1 and urp2, and their receptor uts2r3 play a role during zebrafish spine development. Previously it had been shown that cilia function as well as Reissner fiber formation are important for spine development and that both cilia motility and the Reissner fiber influence urp1/2 expression. Further, morpholino knock-down of upr1/2 did show the typical curly down phenotype observed in cilia and RF mutants. The authors generate CRISPR mutants for urp1, urp2 by targeting the 10-amino acid secreted peptides and do not find an early phenotype in single, double or maternal zygotic mutants or cripants. However, they observe a late onset curvature of the spine in urp1/2 double mutants and in generated uts2r3 single mutant. Spinal curvature was assessed through measurement of the Cobb angel in microCT scans and compared with other scoliosis mutants. This analysis revealed similarities between urp1/2 and uts2r3 mutants and differences with curvatures observed in cilia motility (cfap298) or Reissner fiber (sspo) mutants, which did show decreased expression levels of urp1 and 2. These differences in spine curvature do indicate that the phenotypes are not caused by the same mechanism. Analysis of the Reissner fiber in transgenic animals did show no defects.
Major points:
The paper is generally well written and easy to follow. All experiments are described in sufficient detail and reagents are listed. However, there are two points that should be addressed to strengthen the conclusion of the paper.
- The addition of the F0 crispant experiment to show that the pro-peptide of urp1/2 does not have a function and is responsible for the difference between the observed morpholino and the crispr phenotype was important. However, since no phenotype was observed in crispants it is important to add evidence of induced cuts for all guide RNAs used in the crispant experiment. These control experiments might have been done already. If not, they can easily be done in a short period of time by performance of T7 assays on injected fish and would not require additional reagents.
- The authors claim that there were no structural defects observed in urp1/2 double mutants. However, the hemal arch in figure 3 E seems to be deformed. This could be normal variance or a phenotype. This can be addressed by simple reinspection of the scans.
Minor points:
- It would be helpful to the reader to add a little more information on urp1 and upr2 proteins and their processing to make it clear while only the 3' region of the protein was targeted to induce mutations.
- It would also be helpful to the reader to have a schematic indicating the guide target sites (they could be added to figure S1 C + D) in the protein to be able to interpret the result more easily.
- Figure 5: Addition of a square to H would help understand were the pictures in D-F were taken.
Significance
While scoliosis in human patients is very prevalent, our understanding on the mechanism that lead to the development of spinal curvature are very limited and so are the treatment strategies. The zebrafish has emerged as an important model to study spine development and formation of scoliosis. While not all findings in the presented work are novel, this work adds important information on the role of urotensin signaling in spine formation. These findings are of special significance in the light of recent reports that mutations in UTS2R, the human ortholog of uts2r3, show association with spinal curvature in patients with adolescent idiopathic scoliosis. As such, this work will be of interest not only to basic researches but also the medical field.
My field of expertise: zebrafish, CRISPR/Cas, genetics, skeletal development, spine formation
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Referee #2
Evidence, reproducibility and clarity
Major concern:
- Need to show that the CRISPANT targeting was effective for mutagenesis at each loci screened in the work presented in Figure 1E.
Minor Concern:
- Fig 3F : might be improved by making the images black and white and possibly inverted. It is not easy to clearly see the vertebrae as is.
Significance
Summary:
This is a beautifully executed study on the role of Urp signaling in spine morphogenesis in zebrafish. This work also challenges the model that Urp1/ 2 controls the extension and straightening of the body axis of the zebrafish embryos. Here, using a double mutant in urp1 and urp2, they show that urp1/2 are dispensable for axial straightening. Moreover, they provide redundant roles during larval development in particular for maintaining a straight spine. They go on to show that scoliosis observed in urp1/2 double mutant fish are distinct - showing only dorsal-ventral lordosis , whereas previously published scoliosis phenotypes _showing curvates in dorsal-ventral and medial-lateral axes as observed in cilia- and Reissner fiber-related scoliosis mutants. They provide clear evidence that loss of Urp signaling does not affect the stability of the Reissner fiber as it does in cilia-related scoliosis mutants. Underscoring the distinct regulation of Urp signaling on spine morphology during larval development. Altogether, this is an exciting advance in our understanding of one of the essential signaling pathways that help to regulate body axis straightening and spine morphogenesis in zebrafish. These studies may also help to further our understanding of the etiology and pathophysiology of multiple forms of neuromuscular scoliosis in humans. I recommend it for publication after revisions.
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Referee #1
Evidence, reproducibility and clarity
Summary
The authors investigate the role of Urotensin Related Peptides (Urp1 and Urp2) on zebrafish spine straightness. One model of normal spinal morphogenesis proposes that when the spine bends, material in the central canal of the spinal cord (the Reissner Fiber, RF, mostly composed of scospondin) stimulates surrounding Cerebral Spinal Fluid contacting neurons (CSF-cN), that in turn release Urotensin like peptides that cause dorsl muscles to contract and straighten the spine. It is clear that motile cilia in the central canal are responsible for forming/compacting the RF from monomers of scospondin. Mutations were generated that removed the peptide-coding portion of Urp1, Urp2 and that removed most of the Urp receptor Uts2r3 and made a missense scospondin gene. They used cfap298-/- as an immotile cilia control and scospondin-/- as a Reissner Fiber absent control. The authors show Urotensin peptides and receptor Uts2r3 function in juvenile but not embryonic axis straightening. They defined the timecourse of spinal curves onset and change during larval life, i.e., 9 dpf to 17 dpf and found that curves were dynamic between 30dpf and 3 mpf. Unlike cfap mutants, urotensin mutants show no sex bias in scoliosis expression. The authors used a temperature sensitive mutation in cfap298 and the GFP-tagged scospondin gene to show that active cilia are required for both initial formation of the RF before 28 hpf and to maintain the RF between 6 and 12 dpf. Finally the authors demonstrated that the receptor Uts2r3 is not required for establishment or maintenance of the RF at 28 hpf and 12 dpf.
Major comments
The key conclusions are well supported, see below for my two major issues.
- Please don't call this lordosis. Lordosis or hyperlordosis effects lumbar vertebra. The curve in the lumbar region shifts body weight so that human gait is more efficient that that in the great apes, or so the story goes. Zebrafish do not have lumbar vertebra equivalents or a natural curve in the caudal region. Similarly, fish do not have the equivalent vertebra to generate kyphosis, which is again a hyper flexion of a normal human spinal curve. Instead zebrafish have Weberian, precaudal and caudal vertebra. It would be so much more useful for the field if the authors used these terms and specified ranges, i.e. numbered vertebrae, that are effected so we can directly and accurately compare regions of defects between zebrafish mutants. It would help to make the point that the uts2r3 mutant has more caudally located curves than urp1/2 double mutants.
- The observation that urp1/2 double mutants have curves only in the D/V plane and almost completely lack side-to-side curves is noteworthy. Does the urp1-/-urp2-/- mutant uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be very interesting. It is particularly important to describe how penetrant the phenotype is and how many times it was observed. See 9 minor comments. It would help the reader if the authors explicitly described the features that they see in the cfap298 mutant that constitute lateral curves and that are lacking in urp1/2 (e.g. in figure 4E).
Minor comments
- Figure 1D legend says urp1 is expressed in dorsal while urp2 is express in all CSF-cNeurons, but the image for urp1 shows only ventral cells in WT, while the image for urp2 shows the same cells ...and more dorsal cells. Please replace image with one that matches the text.
- In Figure 2H, the position of curve apex graphic, how many fish were examined? In 2f it looks like n=8 and n=9. Can this info be added to the figure?
- Supplementary FigS3B How to measure the Cobb Angle is unclear. Why is the first curve not counted? I count 3 curves. First a ventral displacement, then a dorsal to ventral return, then a sharp flex before the tail. How to measure Cobb angle might be easier to explain if the figure is expanded into steps. Identify the apical vertebra, then showing how the lines are drawn parallel to those vertebrae, then where the measured angle forms between the lines perpendicular to the drawn parallel lines.
- I did not find legends for the movies. The first call to the movies calls movies 1-3 without explaining what each shows. The labels on the downloaded files are not informative.
- a. I think we (zebrafish biologists) need be explicit about what we mean with "without vertebral defects." What do we count as defects? Vertebrae can be fused, bent, shortened or the growing edges can be slanted. In Figure 3E, and movie7, it is clear that the highlighted mutant vertebrae are shorter than WT. The growing ends of normal vertebra are perpendicular to the long axis of the vertebra. In the mutants the ends are slanted. Please define in the text what you consider a relevant vertebral defect, because these vertebrae have defects. Or are you only considering the calcein stained centra at 10dpf?
5b. Do you want to base your patterning conclusion on primarily the calcein data as these are closer to the notochord patterning time window. Please anchor this conclusion to a specific time or standard length e.g. 10dpf/5.6mm. 6. "At 30 dpf... several mutants exhibited a significant curve in the pre-caudal vertebrae, in addition to a caudal curve (Fig. 3D and S3C). Since pre-caudal curves were rare in mutants at 3-months, this suggested that curve location is dynamic" The frequency of this observation is important. Does it effect all or a fraction of mutants? Can you provide some numbers to anchor these observations? Maybe fractions e.g.. 3 of 4 fish had precaudal curves at 30pdf, and 0 of 10 fish had precaudal curves by 3 mpf? 7. The description of the pkd2l1 mutant, instead of terming it kyphosis can you tell the reader the vertebra number at the peak of the curve. The authors say the pkd2l1 mutant is highly distinct from urp1/urp2-/-, but the reader needs to hear exactly what is distinct. For example, does this mutant have both lateral and D/V curves? 8. Does uts2r3-/- have no /minimal side-to-side curves like urp1/urp2-/-? 9. One finding that deserves more discussion is the observation that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? Please consider the following paper. It shows a proprioception system that maintains normal side-to-side posture. A spinal organ of proprioception for integrated motor action feedback. Picton LD, Bertuzzi M, Pallucchi I, Fontanel P, Dahlberg E, Björnfors ER, Iacoviello F, Shearing PR, El Manira A. Neuron. 2021 Apr 7;109(7):1188-1201.e7. doi: 10.1016/j.neuron.2021.01.018. Epub 2021 Feb 11. PMID: 33577748
Significance
Scoliosis effects about 3% of children worldwide. Mammals have not been good models for this condition. Zebrafish seem to have an intrinsic susceptibility to scoliosis, as well as several technical advantages. Scoliosis is likely caused by disruption of several different and independent pathways. Building a model that describes one of these pathways will allow us to begin to test for therapies to treat or prevent scoliosis.
- The authors demonstrate that urp1 and 2 are required for normal adult spine straightness. While loss of the uts2r3 receptor (A.K.A. uts2ra, Zhang et.al., Nat Genet, 2018) and the uts4 (receptor, Alejevski, et.al, Open Bio, 2021) lead to adult spinal bends or scoliosis, of the four described urotensin ligand paralogs, only urp, not uts2, urp1 or urp2 have been tested by deletion for a role in scoliosis (Quan et.al., Peptides 2021). In the current work, the authors help to build a larger model of normal spine morphogenesis and show that mutations effecting later steps do not have typical cilia associated phenotypes. Contributing a step to this model is important.
- The authors show that juvenile or adult scoliosis can be independent of the embryonic curves, Curly Tail Down phenotype. This result is somewhat in conflict with previous work from Zhang, in which Curly Tail Down phenotype from a cilia defective mutant (ZMYND10) was rescued by overexpression of urp1 peptide. It is possible that urp1 functions in place of the natural peptide for this function. As before there are four paralogs of urotensin peptides. The second conflicting observation from Zhang is that embryos injected with morpholino to urp1 shows Curly Tail Down phenotype. It is well known that morpholinos can have off-target effects.
- The authors observe that urp1/urp2 double mutants have almost no side-to-side defects and all the obvious bends are in the D/V plane. Does this uncouple two systems for posture? If this separate a DV from side-to-side postural control system, that would be amazing.
- The authors provide evidence that curves are dynamic and erasable between 30 dpf and 3 mpf. This could be a time window to apply therapeutics.
- The authors provide a new graphic tool, a chart that logs the location of the apical curve vertebra (Figure 2H and SFigure 3C). This will allow better comparison between various scoliosis mutants.
- The authors describe 3 different version of scoliosis in 3 mutants. In cfap298 mutants (immotile cilia) curves effect all 3 dimensions. In urp1/urp2-/- mutants, curves only appear in the D/V plane. In uts2r3 mutants, curves appear more caudal than those in urp1-/-,urp2-/- mutants, though it is not clear if these are 3D curves.
Audience: Biologists and physicians interested in 1) scoliosis, 2) normal morphogenesis, and 3) maintenance of the spine, 4)neurophysiologists interested in postural control and regulation of repetitive movements, like walking and swimming.
My expertise: zebrafish genetics, scoliosis, gastrulation
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Reply to the reviewers
Manuscript number: RC-2022-01574
Corresponding author(s): Casey, Greene
- General Statements [optional] We thank the reviewers for their thorough feedback. We have addressed all the points raised, revised the manuscript accordingly, and explained our changes below. To aid readability, the reviewers’ comments have been converted to italics, and our responses have been bolded.
Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The authors systematically evaluate the performance of linear and non-linear ML methods for making predictions from gene expression data. The results are interesting and timely, and the experiments are well designed.
I have a few minor comments:
- It was hard for me to understand Figure 1B. I think a figure like this would be very helpful however. What do the numbers represent? If sample ID, then I am not sure why x-axis label is also "samples"
- For analysis of GTEx data, not sure what "studywise splitting" would mean, since the GTEx dataset is one study? Do you leave out the same individuals from all tissues for evaluation?
We thank the reviewer for their input on these two points. To make Figure 1B clearer and to elaborate on our stratified splitting methods, we have amended its description to “We stratify the samples into cross-validation folds based on their study (in Recount3) or donor (in GTEx). We also evaluate the effects of sample-wise splitting and pretraining (B).”
- I found the sample size on x-axis of Fig 2a confusing. If I understand correctly, GTEx has a total of ~1000 subjects. So in some sense, effective sample size can not be bigger than 1000. If you are counting subjects x tissue as sample, then it can be misleading in terms of the effective sample size.
We thank the reviewer for this point. To incorporate it into the manuscript, we’ve added the following text to the description of Fig. 2: “It is worth noting that "Sample Count" in these figures refers to the total number of RNA-seq samples, some of which share donors. As a result, the effective sample size may be lower than the sample count. “
- Would be interesting to assess out-of-sample generalizability of linear and non-linear models. Have you tried training on GTEx and predicting on Recount3 or vice versa?
This question intrigued us. We reran the tissue prediction experiments from the manuscript on a subset of the GTEx and Recount3 datasets in which we performed an intersection over tissues and genes. We found that in the out-of-sample domain the logistic regression model and the three layer neural network performed similarly, while the five layer net generally had a lower accuracy despite having similar accuracy in the training domain. We also found (consistent with our results in the paper) that GTEx predictions are an easier task than their Recount counterparts. Below are plots demonstrating these findings:
[These plots appear in the PDF but do not appear to work in the ReviewCommons Form].
Reviewer #1 (Significance (Required)):
Important and timely study, evaluating linear vs non-linear methods for predicting phenotype from gene expression datasets.
We appreciate the reviewer’s positive comments on the timeliness of our manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary
The authors want to assess the presence of non-linear signal in gene expression values in the task of tissue and sex classification. They use logisitic regression classifiers and two types of neural networks, with 3 and 5 layers, and assess classification performance on two large expression datasets from Recount3 and GTEX and three simulated datasets.
The authors carefully construct their learning setup in such a way that one can reason about the removal of linear signal from the expression features. The interesting conclusion is, that although the linear approach works well on both datasets, and sometimes even better than the more complex models. The authors convingly show, that there is a significant non-linearity in the gene expression data. However, just because it is "there" does not imply that any non-linear methods performs better.
Major comments:
- Are the key conclusions convincing?
The authors did a good job in showing, that there is non-linear signal in gene expression features for the classification problems studied.
We thank the reviewer for their positive feedback.
- Should the authors qualify some of their claims as preliminary or speculative, or
remove them altogether?
The overall claims of the authors are justified, the discussion may be improved.
We appreciate the reviewer’s support for our overall claims and we have adjusted the manuscript as noted point by point below.
- Would additional experiments be essential to support the claims of the paper?
No, additional experiments are not essential. But the authors did not compare to other non-linear methods such as SVM or knn-classifiers in the resulst or conclusion section. It is unlikely that the main conclusion would change if those methods were tried. But it is possible that other "simpler" non-linear methods, such as knn for example, are able to outperform the logistic regression classifier on the GTEX and Recount3 data set. Thus, the authors should at least mention this as part of the conclusion and could extend their discussion on the implications of their study concerning other tasks or models.
We agree that there should be more discussion of other models in the conclusion section. We have updated the fifth paragraph of the conclusion accordingly:
“We are also unable to make claims about all problem domains or model classes. There are many potential transcriptomic prediction tasks and many datasets to perform them on. While we show that non-linear signal is not always helpful in tissue or sex prediction, and others have shown the same for various disease prediction tasks, there may be problems where non-linear signal is more important. It is also possible that other classes of models, be they simpler nonlinear models or different neural network topologies are more capable of taking advantage of the nonlinear signal present in the data.”
- Are the suggested experiments realistic in terms of time and resources?
Not applicable.
- Are the data and the methods presented in such a way that they can be reproduced?
There is a separate github repo which has the code to reproduce the analyses. This is good. However, would be nice to explain in more detail in the manuscript how the limma function was used for removing the linear signal, as they mention the "removeBatchEffect" function was used, but it would be good to tell the reader how that works, as this is their way for assessing the effect of linear-signal removal. Are there any limitations for the assessment of signal removal in this way?
We thank the reviewer for their input, and have updated the model training section on signal removal to read: “We also used Limma[24] to remove linear signal associated with tissues in the data. We ran the ‘removeBatchEffect’ function on the training and validation sets separately, using the tissue labels as batch labels. This function fits a linear model that learns to predict the training data from the batch labels, and uses that model to regress out the linear signal within the training data that is predictive of the batch labels.”
We have also elaborated on the limitations of signal removal by updating the sentence “This experiment supported our decision to perform signal removal on the training and validation sets separately, as removing the linear signal in the full dataset induced predictive signal (supp. fig. 6)” to read “This experiment supported our decision to perform signal removal on the training and validation sets separately. One potential failure state when using the signal removal method would be if it induced new signal as it removed the old. This state can be seen when removing the linear signal in the full dataset(supp. fig. 6).”
- Are the experiments adequately replicated and statistical analysis adequate?
Yes
Minor comments:
- Specific experimental issues that are easily addressable.
no
- Are prior studies referenced appropriately?
Yes
- Are the text and figures clear and accurate?
*Also, they conducted 3 different experiments in Figure 3. It would be useful to separate the figure into 3) A, 3) B, and 3) C and link that specifically in the text. Figure 4 is an extended version of Figure 2, just with the additional results of the signal removed performances. *
We appreciate the feedback. To make the figure and the text more clear, we have added A, B, and C subheadings to figure 3, and updated the subfigure’s references within the text accordingly.
First, the pairwise results in 4B are hard to read as the differences in colors and line type are difficult to see as some lines are short. Second, we did not find it helpful to reproduce the full signal approach in Figure 4. We would suggest to make Figure 4 as Figure 2, and simply only talk about the Full signal mode in the beginning, how it is in the text.
We agree. We have made Figure 4 our new Figure 2 and updated the references in the text.
Further, it would be nice to give better names in the legends of these plots. Pytorch_lr is not a nice name.
We thank the reviewer for pointing this out. We have updated the names in the legends to be “Five Layer Network”, “Three Layer Network”, and “Logistic Regression”
- Do you have suggestions that would help the authors improve the presentation of
their data and conclusions?
As the Recount3 dataset is different in quality and complexity it would be reasonable to show the results of the binary classifcation also in the main paper. In particular, as this behaves different to the GTEX binary classification.
We have now moved the Recount binary classification figure from the supplement to join the GTEx binary classification data as the new figure 4.
-The title is somewhat unprecise. It may induce the impression that the paper is about expression-prediction, although that is not the case. Further, in the abstract they don't mention what prediction problem they solve and that these are classification problems. After reading the paper it is clear why the authors choose that, but we are suggesting an alternative title that the authors may consider:
The effect of nonlinear signal in classification problems using gene expression values
We agree with the reviewer’s comment and have updated our title to “The effect of non-linear signal in classification problems using gene expression”
Further, they should give more details on the problem learned in the abstract.
We thank the reviewer for their feedback, and have added details to the abstract about the problem domains. The relevant sentence now reads “We verified the presence of non-linear signal when predicting tissue and metadata sex labels from expression data by removing the predictive linear signal with Limma, and showed the removal ablated the performance of linear methods but not non-linear ones.”
*-In addition, the conclusion section, which may be title as Disucssion and Conclusion, could contain additional points concerning the topology and training of the neural networks. *
We have updated the heading of the final section to Discussion and Conclusion. To expand on the potential drawbacks of our neural network topologies, we have also updated the limitation portion of Discussion and Conclusion to read “We are also unable to make claims about all problem domains or model classes. There are many potential transcriptomic prediction tasks and many datasets to perform them on. While we show that non-linear signal is not always helpful in tissue or sex prediction, and others have shown the same for various disease prediction tasks, there may be problems where non-linear signal is more important. It is also possible that other classes of models, be they simpler nonlinear models or different neural network topologies are more capable of taking advantage of the nonlinear signal present in the data.”
Obviously, it is possible that other simpler or more complex neural networks have a better performance on the GTEX and Recount3 data sets compared to logistic regression. In fact, the results from Figure4 suggest that, as there is clearly useful non-linear signal in those datasets for the classification problems studied. However, optimizing a non-linear model is inherently more complex and time-consuming, and thus may not be done thoroughly in previously published papers. Compared to a linear model that is easier and faster to optimize, this may be one reason why studies find that, despite non-linear signal, the linear model performs better. Other factors such as the samples size, which the authors already mention, of course also plays a big role, and if hundreds of thousands of datasets would be there , e.g. from single cell measurements, non-linear methods may have a better chance of outcompeting linear models.
We agree, which is why we consider the signal removal experiment to be so important. By demonstrating that the non-linear methods we used were in fact learning non-linear signal we were able to show that there was something that non-linear models were able to learn that logistic regression was unable to. That is to say that while the presence of non-linearity in the decision boundary is necessary for non-linear models to outperform linear ones, it is not by itself sufficient. Perhaps with more data or a different model non-linear methods would perform better, but there is certainly a class of models and problems where logistic regression is preferable.
Reviewer #2 (Significance (Required)):
The submitted manuscript adds to the discussion of the necessity of non-linear models when solving classification problems using gene expression data. The significance is mostly technically, as a comparison of logistic regression and two neural network topologies that are being compared on two large expression datasets. However, there is also a conceptual part of the contribution, which is with regards to the implications of their experiments.
Interested audience would be computer scientists and bioinformaticians or others, that are involved in creating or interpreting these or similar prediction models.
Our field of expertise is in the creation of machine learning models using different types of OMICs data. All aspects of the work could be assessed.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this manuscript, the authors discuss an interesting problem regarding the comparative performance of linear and non-linear machine learning models. The main conclusion is that logistic regression (linear model) and neural networks (non-linear model) have comparable performance if the data contain both linear and non-linear relations between the features (X) and the prediction target (Y), however, if the linear component in the X-Y relation is removed (e.g. regressed out) the neural networks will outperform logistic regression. This conclusion implies that linear models such as logistic regression mainly relies on the linearity in the X-Y relation.
However, whether X-Y relation has a linear component and whether the data (e.g. for different Y classes) are linearly separable are two different questions. For example, consider a data generating mechanism, y=x^2+x and label the data points using two classes (y1). Clearly, the data is linearly separable, and any machine learning algorithm should perform very well on this problem. Now remove the linear component form the X-Y relation and use y=x^2 to generate the data. The data is still linearly separable, and the performance of logistic regression should not be affected.
We agree that there is a difference between optimal linear decision boundaries and linear relationships between elements in the training data. Our use of the term “relationship” in place of “decision boundary” was imprecise. To make this more clear, we have made the following changes:
Introduction:
“Unlike purely linear models such as logistic regression, non-linear models should learn more sophisticated representations of the relationships between expression and phenotype.” -> “Unlike purely linear models such as logistic regression, non-linear models can learn non-linear decision boundaries to differentiate phenotypes.”
“However, upon removing the linear signals relating the phenotype to gene expression we find non-linear signal in the data even when the linear models outperform the non-linear ones.” -> “However, when we remove any linear separability from the data, we find non-linear models are still able to make useful predictions even when the linear models previously outperformed the nonlinear ones.”
Discussion and conclusion:
We removed the following paragraph: “Given that non-linear signal is present in our problem domains, why doesn’t that signal allow non-linear models to make better predictions? Perhaps the signal is simply drowned out. Recent work has shown that only a fraction of a percent of gene-gene relationships have strong non-linear correlation despite a weak linear one [23].”
The point is that the performance of linear models is mainly dependent on whether the data are linearly separable instead of the linearity in X-Y relation as the manuscript suggests.
We agree that this is the key point and appreciate the reviewer for helping us to more carefully hone the language to convey this point.
Reviewer #3 (Significance (Required)):
The performance comparison between linear and non-linear machine learning models is important.
We appreciate the reviewer’s recognition of the significance of the work.
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Referee #3
Evidence, reproducibility and clarity
In this manuscript, the authors discuss an interesting problem regarding the comparative performance of linear and non-linear machine learning models. The main conclusion is that logistic regression (linear model) and neural networks (non-linear model) have comparable performance if the data contain both linear and non-linear relations between the features (X) and the prediction target (Y), however, if the linear component in the X-Y relation is removed (e.g. regressed out) the neural networks will outperform logistic regression. This conclusion implies that linear models such as logistic regression mainly relies on the linearity in the X-Y relation. However, whether X-Y relation has a linear component and whether the data (e.g. for different Y classes) are linearly separable are two different questions. For example, consider a data generating mechanism, y=x^2+x and label the data points using two classes (y<=1 and y>1). Clearly, the data is linearly separable, and any machine learning algorithm should perform very well on this problem. Now remove the linear component form the X-Y relation and use y=x^2 to generate the data. The data is still linearly separable, and the performance of logistic regression should not be affected. <br /> The point is that the performance of linear models is mainly dependent on whether the data are linearly separable instead of the linearity in X-Y relation as the manuscript suggests.
Significance
The performance comparison between linear and non-linear machine learning models is important.
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Referee #2
Evidence, reproducibility and clarity
Summary
The authors want to assess the presence of non-linear signal in gene expression values in the task of tissue and sex classification. They use logisitic regression classifiers and two types of neural networks, with 3 and 5 layers, and assess classification performance on two large expression datasets from Recount3 and GTEX and three simulated datasets. The authors carefully construct their learning setup in such a way that one can reason about the removal of linear signal from the expression features. The interesting conclusion is, that although the linear approach works well on both datasets, and sometimes even better than the more complex models. The authors convingly show, that there is a significant non-linearity in the gene expression data. However, just because it is "there" does not imply that any non-linear methods performs better.
Major comments:
- Are the key conclusions convincing?
The authors did a good job in showing, that there is non-linear signal in gene expression features for the classification problems studied. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
The overall claims of the authors are justified, the discussion may be improved. - Would additional experiments be essential to support the claims of the paper?
No, additional experiments are not essential. But the authors did not compare to other non-linear methods such as SVM or knn-classifiers in the resulst or conclusion section. It is unlikely that the main conclusion would change if those methods were tried. But it is possible that other "simpler" non-linear methods, such as knn for example, are able to outperform the logistic regression classifier on the GTEX and Recount3 data set. Thus, the authors should at least mention this as part of the conclusion and could extend their discussion on the implications of their study concerning other tasks or models. - Are the suggested experiments realistic in terms of time and resources?
Not applicable. - Are the data and the methods presented in such a way that they can be reproduced?
There is a separate github repo which has the code to reproduce the analyses. This is good. However, would be nice to explain in more detail in the manuscript how the limma function was used for removing the linear signal, as they mention the "removeBatchEffect" function was used, but it would be good to tell the reader how that works, as this is their way for assessing the effect of linear-signal removal. Are there any limitations for the assessment of signal removal in this way? - Are the experiments adequately replicated and statistical analysis adequate?
Yes
Minor comments:
- Specific experimental issues that are easily addressable.
no - Are prior studies referenced appropriately?
Yes - Are the text and figures clear and accurate?
Also, they conducted 3 different experiments in Figure 3. It would be useful to separate the figure into 3) A, 3) B, and 3) C and link that specifically in the text. Figure 4 is an extended version of Figure 2, just with the additional results of the signal removed performances. First, the pairwise results in 4B are hard to read as the differences in colors and line type are difficult to see as some lines are short. Second, we did not find it helpful to reproduce the full signal approach in Figure 4. We would suggest to make Figure 4 as Figure 2, and simply only talk about the Full signal mode in the beginning, how it is in the text. Further, it would be nice to give better names in the legends of these plots. Pytorch_lr is not a nice name. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
As the Recount3 dataset is different in quality and complexity it would be reasonable to show the results of the binary classifcation also in the main paper. In particular, as this behaves different to the GTEX binary classification. - The title is somewhat unprecise. It may induce the impression that the paper is about expression-prediction, although that is not the case. Further, in the abstract they don't mention what prediction problem they solve and that these are classification problems. After reading the paper it is clear why the authors choose that, but we are suggesting an alternative title that the authors may consider:
The effect of nonlinear signal in classification problems using gene expression values
Further, they should give more details on the problem learned in the abstract. - In addition, the conclusion section, which may be title as Disucssion and Conclusion, could contain additional points concerning the topology and training of the neural networks. Obviously, it is possible that other simpler or more complex neural networks have a better performance on the GTEX and Recount3 data sets compared to logistic regression. In fact, the results from Figure4 suggest that, as there is clearly useful non-linear signal in those datasets for the classification problems studied. However, optimizing a non-linear model is inherently more complex and time-consuming, and thus may not be done thoroughly in previously published papers. Compared to a linear model that is easier and faster to optimize, this may be one reason why studies find that, despite non-linear signal, the linear model performs better. Other factors such as the samples size, which the authors already mention, of course also plays a big role, and if hundreds of thousands of datasets would be there , e.g. from single cell measurements, non-linear methods may have a better chance of outcompeting linear models.
Significance
The submitted manuscript adds to the discussion of the necessity of non-linear models when solving classification problems using gene expression data. The significance is mostly technically, as a comparison of logistic regression and two neural network topologies that are being compared on two large expression datasets. However, there is also a conceptual part of the contribution, which is with regards to the implications of their experiments.
Interested audience would be computer scientists and bioinformaticians or others, that are involved in creating or interpreting these or similar prediction models.
Our field of expertise is in the creation of machine learning models using different types of OMICs data. All aspects of the work could be assessed.
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Referee #1
Evidence, reproducibility and clarity
The authors systematically evaluate the performance of linear and non-linear ML methods for making predictions from gene expression data. The results are interesting and timely, and the experiments are well designed.
I have a few minor comments:
- It was hard for me to understand Figure 1B. I think a figure like this would be very helpful however. What do the numbers represent? If sample ID, then I am not sure why x-axis label is also "samples"
- For analysis of GTEx data, not sure what "studywise splitting" would mean, since the GTEx dataset is one study? Do you leave out the same individuals from all tissues for evaluation?
- I found the sample size on x-axis of Fig 2a confusing. If I understand correctly, GTEx has a total of ~1000 subjects. So in some sense, effective sample size can not be bigger than 1000. If you are counting subjects x tissue as sample, then it can be misleading in terms of the effective sample size.
- Would be interesting to assess out-of-sample generalizability of linear and non-linear models. Have you tried training on GTEx and predicting on Recount3 or vice versa?
Significance
Important and timely study, evaluating linear vs non-linear methods for predicting phenotype from gene expression datasets.
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Reply to the reviewers
Reply to the Reviewers
We thank the reviewers for their excellent suggestions and constructive comments. We now added new data on PE15/PPE20 binding to Ca2+, the PDIM status of mutant strains, additional controls, added to the discussion, added detail to the Methods, and provide all RNA-seq data. Please see replies to the comments in detail below:
Reviewer 1:
Major points
- Cellular localization:
- “The authors do not describe the cellular fractionation method…”, “The authors show some Western blot data in Fig. S3, though the legend is superficial (abbreviations not explained) and the controls with markers for cellular localization appear to be lacking”. “Further, the authors do not prove that FLAG-tagged PE20 is functional.”
We included a description of the fractionation method in Materials and Methods (lines 475-485). We also added detail to the legend of Fig. 4A to explain the abbreviations and controls used. The same cell fractions were used in Fig. 4A and Fig. S3A, as mentioned in the Figure S3 legend (“The same cell fractions as in Fig. 4A were used, see controls therein”). We know that the FLAG-tagged PPE20 is functional because the strain used in this experiment is the same we used for genetic complementation experiments in which FLAG-tagged PPE20 functionally complements ppe20 deletion in all three assays (ATP consumption, biofilm, Ca2+ influx, Fig.4 B,C,D,G).
- “The authors should extend discussion part of the manuscript. Several proteomic studies.” “Did authors analyze culture filtrate fraction by Western?
We thank the reviewer for the references and extended the Discussion to include results from existing proteomic studies on PE15/PPE20 (lines 229-234). We did not test for PE15/PPE20 in culture filtrate, and previous proteomic results are contradictory. Several PE/PPE proteins, including PE15/PPE20 have been detected in the cell wall and in the CFP, but not consistently. The functional significance of this dual localization is unclear.
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Is PE15/PPE20 a channel?
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“PPE20 purified alone from the cytosol of E. coli?”
We did not purify either protein by itself. As the reviewer correctly notes, PE/PPE proteins are refractory to individual purification. We clarified that we purified and used the complex for experiments even if only PPE20 is shown, as in Figure 3C,D, and E (Lines 124-127). See also Methods line 382 ff.
- “…a positive control of a mutant that is indeed deficient in Mg2+ import (and thus showing a phenotype) is lacking.”
Lacking a specific Mg2+ import mutant, and because it is a relatively minor point, we removed the statements about selectivity.
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Thermal melting assay
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It is surprising to see that the thermal melting assays was done for PPE20 and PE15 as separately purified proteins.
We co-purified PE15 and PPE20 for all biochemical experiments. We clarified that point (see also point 2 above).
- “the thermal melting assay only seemed to give some results for PPE20 alone, and not for PE15”
PE15 did not produce interpretable results in this assay, as mentioned in line 144. We clarified in the Fig. 3 legend that the complex was used although only PPE20 is detected by Western blot and shown in Figure 3C.
- “…the results are counter-intuitive… How can the authors be sure that the presence of Ca2+ does not simply lead to more protein precipitation (via rather unspecific interactions) at elevated temperatures? Some positive controls with bona fide calcium binding protein in the same thermal melting setup would have helped to clarify this.”
The effect of Ca2+ on PPE20 is somewhat counterintuitive, although not unprecedented. Proteins can be stabilized or destabilized by ligand binding, and a recent proteome-wide study on the basis of thermal shift analysis showed that ~17% of proteins were destabilized by ligand (ATP). For a channel in particular, ligand binding might be expected to be coupled to protein relaxation in the process of channel opening, which could well translate to lower thermal stability. We added the positive control showing the behavior of a known Ca2+ binding protein (new Fig. S2A). In addition, we included a negative control showing that Ca2+ does not generally increase protein denaturation (Fig. S2B). We think that this control addresses the reviewer’s concern more directly.
- If the authors want to stick to their claims regarding Ca2+ binding to PE15/PPE20, they have to perform additional assays (e.g. equilibrium dialysis or ITC) with the entire PE15/PPE20 complex. Further, they have to show that PE15/PPE20 forms a proper oligomeric protein that is membrane bound and reasonably behaved on size exclusion chromatography, when expressed in and purified from E. coli.
Detecting Ca2+ binding to proteins is not trivial, and we thank the reviewer for suggesting equilibrium dialysis as another, orthogonal assay. We now show an equilibrium dialysis experiment that confirms Ca2+ binding by the PE15/PPE20 complex. Please see the new Fig. 3F. and G. and lines 146-152 (Results) and 429-443 (Methods).
The PE/PPE proteins are generally difficult to express and purify recombinantly, likely due to the typically large unstructured regions. Also, the yield of PE15/PPE20 when expressed in E. coli was very low so that we were not able to detect the complex by SEC. However, data in Fig. 3 conclusively show that PE15 and PPE20 bind.
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RNA-seq data
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The authors should include a table with all other identified genes that are potentially involved in calcium homeostasis
We provided all other significant differentially expressed genes in the new Table S1.
Minor points:
- “what is the binding affinity of the Ca sensor?”
We added the Ca2+ binding affinity of Twitch-2B (KD: 200nM) in line 176.
- Figure 4D: “one would expect a drop in FRET signal after EGTA addition… Can the authors explain?”
We do see a clear drop in FRET signal after EGTA addition, in particular in 7H9 medium (black versus red line, Fig. 5B). Given the high affinity of Twitch-2B for Ca2+ (200nM), however, it is not surprising that the drop is not more pronounced, as intracellular Ca2+ is expected to be tightly bound to Twitch.
- The experiments showing outer membrane localization of PE15/PPE20 are very important, but results of these experiments (western-blot and FRET) are shown in supplementary figures. They should be transferred/integrated into the main Figures.
We agree and moved Figure S3A to the main Figures as Figure 4A.
- Line 166: the authors claim that the assay did not work in 7H9 due to low Ca2+ concentration in this medium. Why did the authors not just add a bit more calcium to show whether this claim holds true?
7H9 is not a suitable medium for these experiments because the baseline Ca2+ concentration is too high, not too low (see Fig. 5B, grey versus black line). Adding more Ca2+ to 7H9 medium resulted in precipitation, probably due to its interaction with phosphates. Our use of “low” in this context was confusing, we changed the wording of this sentence (line 180-181).
- Line 183: more detailed description on cellular fractionation and subsequent anti-FLAG Western needed here.
We added more detail in the Materials section (lines 475 ff).
Reviewer 2:
- A major concern regarding the importance of the data: there are considerable technical challenges in generating Ca2+ depleted media. This is clear in that M. tuberculosis seems to be unaffected by Ca2+ in the medium - similar growth seems in Ca2+-free media to media with up to 10mM Ca2+ (Fig. S1). This raises a concern about the physiological relevance of the data (mammalian cells have intracellular Ca2+ of 0.01-0.1mM, extracellular free Ca2+ is around 1mM).
If we correctly understand this comment, the reviewer is unconvinced that we fully and reproducibly depleted Ca2+ from medium based on a lack of an effect of Ca2+ on in vitro growth. We tested for baseline Ca2+ levels and depletion in media by inductively coupled plasma optical emission spectrometry and added these data showing precise quantitation of Ca2+ in medium (see new Fig. S1B).
- The role of PE15/PPE20 in Ca2+ acquisition may be clearer if the authors ensure that the PDIM layer is intact. Specifically, there is a technical issue: The authors use Tween80 as a detergent. Tween-80 partially strips the outer cell wall of M. tuberculosis resulting in shedding of PDIM and PE/PPE proteins. Tyloxapol is a somewhat milder detergent. Some of the experiments would possibly show clearer phenotypes by use of Tyloxapol.
We share the concern about PDIM, as PDIM loss is common in in vitro culture. We analyzed the total lipids by thin layer chromatography and confirmed the presence of PDIM in all three strains (Fig S3C, lines 198-201). We repeated experiments with Tyloxapol and did not see differences to Tween-80. We nonetheless now show the Tyloxapol data (Fig 5D).
- The authors could increase the impact of their work be exploring the role of PE15/PPE20 during pathogenesis of resting versus activated bone marrow macrophages where Ca2+ fluxes of the host cell play a role in host responses.
We agree. In vivo or macrophage experiments are a logical next step to fully characterize the function of PE15/PPE20, but we think it is beyond the scope of this manuscript. The main contribution of this paper is the identification of channel function of a PE/PPE protein pair that extends the novel channel paradigm for these proteins. These data support that transport might indeed be a shared function of the entire PE/PPE family with 169 members.
Minor:
- The authors should consider citing Sharma et al (2021)
We cited the paper.
- Are there Ca2+ binding motifs in PPE20?
We did not detect canonical Ca2+ binding motifs in PPE20.
- RNAseq data may need to be deposited in a public database.
RNA-seq data have been deposited to NCBI - GEO accession GSE214266
Link: https://urldefense.com/v3/https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE214266;!!NuzbfyPwt6ZyPHQ!tCf4MS_HRKJFn6qV2orkDAkXTWvx9IIU11fAV7TguYE2ietoMBpBgRC7rvfnM9bsoiVdIvDBUHdPmHZliDP2o5sRZR2ziK4$
Token: cvmhakcgbpmbfuz
- In its current state, the work is somewhat incremental
The function of the large PE/PPE protein family of Mtb has been one of the most longstanding and perplexing puzzles in Mtb biology. For more than 20 years, speculation about their potential role, for example in antigenic variation, abounded but no conclusive evidence for this or another shared function emerged. A recent landmark paper then conclusively showed that a subset of the PE/PPE proteins function as nutrient channels (Wang et al., Science 2020). However, whether transporter function is a general function of the family of 169 PE/PPE proteins remains untested. Our PE/PPE pair is associated with a different type VII secretion system (Esx-3) and belongs to a different subfamily than the previous examples, suggesting a shared function across families and perhaps even all of these proteins. Given the intense interest and many false leads that have plagued the identification of PE/PPE function in the last 20 years, the difficulty of working with them biochemically, as well as the almost complete absence of understanding of Ca2+ homeostasis in Mtb, we do not consider our work incremental.
Reviewer 3
- My only slight concern is the meaning attached to the "biofilm" assays. It is never very clear to me that this is anything more than formation of a surface pellicle and general hydrophobicity of the mycobacterial cells.
We fully agree that Mtb biofilms remain poorly defined. However, the term biofilm as used in our study has already found its way into the literature and we would rather not cause confusion by calling the same phenomenon by a different name. Whatever the term used, we do not suggest any other relevance other than it being a Ca2+-dependent phenotype that serves as one of several tests to parse PE15/PPE20’s role in Ca2+ homeostasis.
Cross-consultation comments:
- We agree with the concerns of reviewer#2 that the role of PDIM and use of detergent should be looked at more closely.
We tested the roles of PDIM and detergent, see reviewer 2.
- Likewise, the paper would strongly benefit from some further insights into the potential physiological role of PPE20/PE15 in calcium homeostasis.
We show PE15/PPE20 function in the transport of Ca2+ and the first Ca2+-related cellular phenotypes in Mtb. Testing the role of the complex in an infection model is outside of the scope of this manuscript and mouse infection experiments would take many months and would likely be intractable because of the expected extensive redundancy among the 169 PE/PPE proteins.
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Referee #3
Evidence, reproducibility and clarity
Review of Boradia et al, "Calcium transport by the Mycobacterium tuberculosis PE15/PPE20 proteins" This manuscript describes studies aimed at understanding the role of calcium in the pathogenesis of tuberculosis. The authors begin by showing that, analogous to the situation in other bacteria, ATP levels are directly (and rather dramatically) affected by extracellular calcium levels. The authors then look at the effect on "biofilm formation" and, again analogous to other bacteria, find a link. The authors then perform RNAseq on bacterial cells with and without 1mM Ca++ and identify a pair of genes that is strongly downregulated by calcium sufficiency. These genes are PE/PPE family members which have been recently associated with channel formation in the mycomembrane to allow transport of small molecule solutes across the outer cell envelope. The authors show these proteins are associated in a complex by reciprocal pull-down experiments in tagged proteins and show directly that they bind calcium by a thermal stability change of this complex in the presence of calcium. Finally, they show, using a calcium sensitive FRET reporter expressed in Mtb, that these two proteins allow calcium influx and that such an influx is blocked in a strain where they have been deleted.
Overall, the study is excellent and convincingly establishes the transport function of another pair of PE/PPE proteins. My only concern with this is that they stop just short of delving into the actual infection biology of calcium, but I suppose that will be next. The tools they developed in this study, specifically the knockout strain and the FRET reporter, put them in a strong position to explore the role of calcium during growth in macrophages and other in vivo studies that are surely planned.
My only slight concern is the meaning attached to the "biofilm" assays. It is never very clear to me that this is anything more than formation of a surface pellicle and general hydrophobicity of the mycobacterial cells. I wonder if the presence of calcium alters the aggregation state of the bacilli and or affects the surface in some more subtle manner. I am not convinced that the word "biofilm" as it is used commonly in other bacteria, has anything to do with the physical properties that are being observed in the case of Mtb.
Significance
The manuscript clearly establishes that this pair of PE/PPE proteins plays a direct role in calcium transport in MTB and provides several useful tools to begin to understand the role of calcium in TB pathogenesis. The work is outstanding and novel.
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Referee #2
Evidence, reproducibility and clarity
The authors demonstrate that M. tuberculosis responds to increasing Ca2+ concentrations by increasing ATP levels as well as increased ability to form biofilms. Culturing of M. tuberculosis with Ca2+ results in downregulation of pe15/ppe20. The proteins are recombinantly expressed and the results show that PE15 and PPE20 form a complex. In addition, PPE20 seems to be destabilized by Ca2+ suggesting that it interacts with this metal ion. A pe15/ppe20 knockout shows lower levels of ATP increase upon incubation with Ca2+ although the differences with would-type are very modest. Similarly, the knockout shows an impaired ability to form biofilms at 1mM and 10mM Ca2+. Finally, the authors make a FRET reporter of intracellular Ca2+ concentrations based on the Twitch system which nicely shows that intracellular Ca2+ levels are lower in the knockout mutant.
Overall, the data suggest that PE15/PPE20 are involved in Ca2+ uptake which contributes to our evolving understanding of the role of the different PE/PPE proteins in nutrient acquisition. The highlight of the paper is the Twitch bioreporter for Ca2+ which could be useful in exploring the role of Ca2+ in mycobacteria.
A major concern regarding the importance of the data: there are considerable technical challenges in generating Ca2+ depleted media. This is clear in that M. tuberculosis seems to be unaffected by Ca2+ in the medium - similar growth seems in Ca2+-free media to media with up to 10mM Ca2+ (Fig. S1). This raises a concern about the physiological relevance of the data (mammalian cells have intracellular Ca2+ of 0.01-0.1mM, extracellular free Ca2+ is around 1mM). The role of PE15/PPE20 in Ca2+ acquisition may be clearer if the authors ensure that the PDIM layer is intact. Specifically, there is a technical issue: The authors use Tween80 as a detergent. Tween-80 partially strips the outer cell wall of M. tuberculosis resulting in shedding of PDIM and PE/PPE proteins. Tyloxapol is a somewhat milder detergent. Some of the experiments would possibly show clearer phenotypes by use of Tyloxapol. In experiments where clumping is not a concern (ATP measurement), the cells can be pre-grown as indicated but then transferred to the multiwell plates in detergent-free media. At the time of processing of the cells for readout of, for example ATP, detergent can be used as needed. The authors could increase the impact of their work be exploring the role of PE15/PPE20 during pathogenesis of resting versus activated bone marrow macrophages where Ca2+ fluxes of the host cell play a role in host responses.
Minor:
The authors should consider citing Sharma et al (2021): PGRS Domain of Rv0297 of Mycobacterium tuberculosis functions in A Calcium Dependent Manner
Are there Ca2+ binding motifs in PPE20?
RNAseq data may need to be deposited in a public database.
Significance
In its current state, this work is somewhat incremental: the authors have provided data that suggest that PE15/PP20 are involved in Ca2+ uptake (data could be strengthened as suggested above). The physiological relevance of the PE15/PPE20 system remains unclear - no data on its role in pathogenesis.
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Referee #1
Evidence, reproducibility and clarity
PE/PPE proteins build up 10% of genome of Mtb, but the function of these proteins is only currently investigated in more detail. Recent studies show the involvement of individual PE/PPE proteins in the transport of nutrients, and more data supporting this functional role are about to emerge. Using RNA-seq, Boradia et al identified the pe15/ppe20 genes to be downregulated in response to calcium exposure. Purified PPE20 (but not PE15) appear to bind calcium in a thermal stability assay (though this claim needs further experimental support). The authors generated a Mtb pe15/ppe20 knockout strain and convincingly show in three types of assays (ATP levels, biofilm formation and lower signal in FRET measurements corresponding to lower calcium concentrations compared to the wild type strain) that the PE15/PPE20 proteins are involved in cellular calcium import, but do not appear to import magnesium. All phenotypes could be restored to the behavior of wildtype Mtb by complementing the KO strain with pe15/ppe20.
The manuscript is clearly written and easy to follow. The authors combined molecular biology (RNA-seq), biochemistry (proteins purification), biophysics (FRET) and microbiology (knockout generation, in vivo measurements) to reach their conclusions. Overall, the study reports novel and interesting data and as such is of high interest for the mycobacterial research community. However, some of the claims have a rather experimental basis, and thus the study needs to be strengthened with further experiments (or statements have to be removed) as outlined below.
Major points:
- Cellular localization of PE15/PPE20.
The authors do not describe the cellular fractionation method they applied (no mentioning of cellular localization experiments in materials and methods). The same applies to the main text (very superficial description). The authors show some Western blot data in Fig. S3, though the legend is superficial (abbreviations not explained) and the controls with markers for cellular localization appear to be lacking. Further, the authors do not prove that the FLAG-tagged PPE20 is functional.
The authors should extend discussion part of the manuscript. Several proteomic studies did not identify PE15 or PPE20 in the cell wall - doi: 10.1021/pr1005873, doi: 10.1091/mbc.E04-04-0329. At the same time PE15 (but not PPE20) is membrane or membrane-associated protein according to this work: doi: 10.1186/1471-2180-10-132. Quite recent work (https://doi.org/10.1073/pnas.1523321113) showed that PE15/PPE20 are secreted substrates of ESX-3 and these proteins have been found in the culture filtrate. Did authors analyze culture filtrate fraction by Western blotting? 2. Is PE15/PPE20 a channel?
A major claim of the authors is that PE15/PPE20 forms a (specific) channel for Ca2+ and not a porin-like protein that is permeable to a large set of solutes. However, this claim has its main experimental backing that PPE20 (purified alone from the cytosol of E. coli?) binds to Calcium in a (rather weirdly looking) "thermal melting assay" (further comments on these assays, see below). The second experiment supporting this idea is a lack of difference between wt and KO strain of Mtb in an assay that should report Mg2+ transport deficiency (Fig. S3). But here, a positive control of a mutant that is indeed deficient in Mg2+ import (and thus showing a phenotype) is lacking. In conclusion, the experimental basis on the grounds of which the authors claim PE15/PPE20 to be a specific Mg2+ channel is weak. On the other hand, the functional data clearly show a link between PE15/PPE20 and calcium uptake: Hence the data are solid enough to claim that PE15/PPE20 facilitates Ca2+ transport across the mycomembrane. 3. Thermal melting assay.
It is surprising to see that the thermal melting assays was done for PPE20 and PE15 as separately purified proteins. How did you purify PPE20 alone for this assay? It is broadly accepted for PE/PPE proteins that they only can be purified as pairs, including for PE15/PPE20 (https://doi.org/10.1073/pnas.0602606103). As for the cellular localization, the method section falls short in providing relevant information on how PPE20 and PE15 were purified in separate forms (it states they were co-expressed using a pETDuet vector). Further, the thermal melting assay only seemed to give some results for PPE20 alone, and not for PE15. There is no mentioning of the PE15/PPE20 complex in this assay. Further, the results are counter-intuitive, as Ca2+ addition leads to more precipitation at higher temperatures (and it does seem to weaken the stability of PPE20 instead of stabilizing it). How can the authors be sure that the presence of Ca2+ does not simply lead to more protein precipitation (via rather unspecific interactions) at elevated temperatures? Some positive controls with bona fide calcium binding protein in the same thermal melting setup would have helped to clarify this.
If the authors want to stick to their claims regarding Ca2+ binding to PE15/PPE20, they have to perform additional assays (e.g. equilibrium dialysis or ITC) with the entire PE15/PPE20 complex. Further, they have to show that PE15/PPE20 forms a proper oligomeric protein that is membrane bound and reasonably behaved on size exclusion chromatography, when expressed in and purified from E. coli. As it is doubtful that the authors can meet such quality standards, I would recommend to remove all statements regarding Ca2+ binding to PPE20 from the manuscript, as the underlying experiments are of poor quality. 4. RNA-seq data
The authors should include a table with all other identified genes that are potentially involved in calcium homeostasis. This is of interest because the KO strain is still capable of calcium import, hence other Ca2+ transport systems likely exist.
Minor comments:
- FRET experiments What is the binding affinity of the sensor for calcium?
- Figure 4D: one would expect a drop in FRET signal after EGTA addition, because this reverts the Ca2+ gradient from out-to-in (thus facilitating calcium flow into the cells) to in-to-out (EGTA actually acting as a sink into which all Ca2+ (also the one from within the cell) would flow). Can the authors explain?
- The experiments showing outer membrane localization of PE15/PPE20 are very important, but results of these experiments (western-blot and FRET) are shown in supplementary figures. They should be transferred/integrated into the main Figures.
- Line 166: the authors claim that the assay did not work in 7H9 due to low Ca2+ concentration in this medium. Why did the authors not just add a bit more calcium to show whether this claim holds true?
- Line 183: more detailed description on cellular fractionation and subsequent anti-FLAG Western needed here.
Referees cross-commenting
We agree with the concerns of reviewer#2 that the role of PDIM and use of detergent should be looked at more closely.
Likewise, the paper would strongly benefit from some further insights into the potential physiological role of PPE20/PE15 in calcium homeostasis.
Significance
Slow-growing mycobacteria like Mtb lack porins. Therefore, it is not clear how nutrients can be transported through the outer membrane. More and more data hint on PE/PPE protein family that can fulfill this function (Wang et al., Science 367, 1147-1151 (2020)). In the current work, the authors show that PE15/PPE20 are involved in calcium transport in Mtb. Mtb is a difficult model organism to work with because of its pathogenicity and slow rate of growth. Therefore, any information on nutrients transport in Mtb is highly appreciable.
The RNA-seq experiments as well as the genetic/functional experiments clearly show that PE15/PPE20 facilitates calcium import in Mtb. The corresponding sections and figures are convincing.
The experimental data attempting to show PE15/PPE20's cellular localization and its interaction with Ca2+ are currently weak, and need to be strengthened.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This paper demonstrates a link between oxidative stress, lipid biosynthesis, and targeted histone acetylation in fission yeast. In mutant cells with defects in lipid synthesis (cbf11, mga2 lacking transcription factors, and cut6 lacking acetyl-CoA carboxylase), transcripts of a number of genes implicated in resistance to oxidative stress are increased. This is associated with higher levels of H3K9 acetylation and increased tolerance to oxidative stress. These effects are mediated through Sty1, a stress-activated MAP kinase and the transcription factor Atf1.
It is also shown that H3K9 acetylation levels in the promoter region and just downstream of the transcriptional start site are increased in cbf11 mutants (Fig. 5A).
By mutational analysis, the authors implicate the acetyl transferases Mst1 and Gcn5 in this transcriptional effect. Other related acetyl transferases, Hat1, Elp3, Mst2, Rtt109 have been ruled out as main contributors to the dysregulation in unstressed cbf11 mutants. That specific acetyl transferases have been shown to be required is a strength of the investigation.
Major comments:
The hypothesis is put forward in the manuscript that altered acetyl-CoA levels in cbf1 mutants would underlie the dysregulation of genes induced by oxidative stress. Histone acetyl transferases compete for acetyl-CoA with lipid biosynthesis, and so with increased demand for acetyl-CoA underacetylation in the concerned promoters would result - specifically at H3K9. These results do not directly support the hypothesis, on the other hand they are not sufficient to rule it out.
Actually, we view this phenomenon the other way round: We primarily focus on exponentially growing cells, which have substantial demand for fatty acid (FA) production (= high acetyl-CoA consumption). So the level of promoter histone acetylation under these conditions is our baseline, or “normal” state. When FA production is decreased (cbf11 or cut6 mutants; inhibition of FA synthase by cerulenin…), stress gene promoters get *hyper*acetylated. We do not have any data on (or claims about) histone underacetylation compared to the baseline. Nevertheless, we now show that overexpression of Cut6/ACC results in decreased resistance to oxidative stress (Fig. 5C), which is compatible with the notion that increased acetyl-CoA consumption would result in insufficient histone acetylation at stress gene promoters during stress.
Acetyl-CoA levels were measured only in undisturbed cells, and the possibility remains that under oxidative stress there would be changes in acetyl-CoA pools that could explain this apparent contradiction - why did not the authors examine that?
Under oxidative stress, the Sty1 stress MAPK is activated, leading to a massive Atf1-dependent transcription wave, which is also associated with increased SAGA-dependent H3K9 acetylation (PMID: 21515633). This well-studied cellular response, however, is not the main focus of our study. Rather, we found a novel connection between perturbed lipid metabolism and increased expression of stress genes in cells *not challenged* by oxidative stress (i.e. Sty1-Atf1 are not hyperactivated). This is why we only measured acetyl-CoA concentrations in untreated cells.
The authors argue that although the global acetyl-CoA levels are not increased, local concentrations might be altered in a way to permit higher H3K9 acetylation levels at selected promoters. Although a formal possibility, this is rather far-fetched as a small and freely diffusible molecule like acetyl-CoA should quickly equilibrate within one cellular compartment. I think that although the overall relationships that the authors have established between oxidative stress, H3K9 acetylation levels with increased expression, and lipid biosynthesis, are compelling, the role of acetyl-CoA concentrations is not clear and should be de-emphasized.
Interestingly, acetyl-CoA production in the nucleus has been published by several studies (reviewed in PMID: 29174173), suggesting that local acetyl-CoA concentrations (microgradients) within the cell are functionally relevant. We agree that acetyl-CoA is a small molecule which, in theory, should diffuse quickly throughout the nucleocytoplasmic space. However, empirical evidence shows that the lipid synthesis in the cytosol and histone acetylation in the nucleus may not access a uniform nuclear-cytosolic pool of acetyl-CoA (PMID: 28099844, PMID: 28552616). This is related to the fact that the acetyl-CoA sink is large and acetyl-CoA may react with many proteins (i.e. any extra amounts will be consumed rapidly).
Even though we provide strong evidence that HAT activity is critical for the crosstalk between FA synthesis and stress gene expression, we do agree that we have not conclusively established the role of acetyl-CoA in the process. However, we still feel that it is justified to point out acetyl-CoA is a “possible” mediator molecule for the crosstalk in the Results and Discussion sections.
Minor comments:
In many of the bar diagrams, only a borderline statistical significance is indicated (p ~ 0.05) despite seemingly large numerical differences between the means. In the legends it is stated that one-sided Mann-Whitney U tests were used. This is a non-parametric test with low power - would it not have been better to use a t test?
We do agree that the non-parametric Mann-Whitney U test is rather conservative and, therefore, less sensitive for small sample sizes, such as n = 3. Our reason for using this particular test instead of the parametric t-test is that qPCR fold-change values come from a log-normal distribution, which is incompatible with t-test (requires normal distribution of data). Importantly, using conservative statistical testing does not invalidate our conclusions.
What do the error bars in the diagram show, SEM? If a non-parametric test is used, a parametric measure of variability is irrelevant.
The error bars represent standard deviation (SD). We do not see an issue here as, in our opinion, the visual style of numeric data presentation is independent from any chosen statistical testing methods.
It would be helpful to the reader to indicate directly in the diagram panels what is actually shown, not just "fold change vs ..." In Fig. 1, 2, 4 D and 5 we see mRNA levels, in Fig. 3 chromatin IP.
Done
Reviewer #1 (Significance (Required)):
The paper represents conceptual advances for our understanding of how stress responses, metabolism and transcriptional regulation are linked, although one of the links (acetyl-CoA levels in this case) is tenuous.
This manuscript belongs in a rich literature on stress responses on the gene expression level, mostly from studies in yeast. Potentially, it adds entirely new information on how cellular stress may be mechanistially linked to stress responses.
These results are potentially general and of broad interest to the biological community.
This reviewer is familiar with yeast genetics, stress responses, and quantification of gene expression.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
As more and more metabolic intermediates are found to also serve as co-factors for epigenetic modifications, it has been widely accepted that regulating the levels of these key metabolites can be an effective way to control nutrient related gene expression. Acetyl-CoA is one of those early examples. Increased acetyl-CoA was shown to promote local acetylation at growth genes (Mol Cell 2011 PMID: 21596309), and ACC deletion funnels more Acetyl-CoA towards histone acetylation reactions and causes global hyperacetylation (Ref 17). However, whether those increased metabolite/co-factor can exert signal-specific effects remains elusive. For instance, although increased acetyl-CoA stimulates the SAGA complex enzymatic activity, it is not clear whether it also causes SAGA to be targeted to new sites without external cues to induce new transcription factor binding. Does increased acetyl-CoA cause broad hyperacetylation at all inducible genes which are the primary targets for those HAT complexes?
In this manuscript, Princová et al. found that deletion of fatty acid synthesis transcriptional factors Cbf11 and Mga2 increases cell survival under H2O2 induced oxidative stress in S. pombe. They further showed that several stress-related genes increased upon Cbf11 deletion, and H3K9 acetylation at their promotor regions were elevated. They argued that FA-TF deletion may indirectly regulate stress-related genes potentially through influencing Acetyl-CoA level, although they failed to detect significant changes of global Acetyl-CoA levels. While it's interesting to see yet another example of metabolite-mediated gene expression regulation, the current manuscript only made incremental advance towards mechanistic principles of how these co-factors finetune specific gene expression program.
Specific comments:
- This work showed convincingly that deletion of CBF11 or MGA2 leads to resistance to oxidative stress. However, it provides little mechanistic insight into how deletion of Cbf11 increased the expression of stress response genes and why some HATs are involved but others not (Figure EV5).
We respectfully disagree with the notion that we only provide “little mechanistic insight” into the process whereby FA metabolism affects stress gene expression.
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First, we show that not only deletion of cbf11, but also a very specific manipulation of the rate-limiting FA-producing enzyme (Cut6/ACC; Fig. 4D), or chemical inhibition of FA synthase by cerulenin (new Fig. 4F) all lead to increased stress gene expression. On the other hand, overproduction of Cut6/ACC results in decreased stress gene expression and lower resistance to ox. stress (new Fig. 5B-C). These findings clearly show the specific and tight mutual relationship between FA synthesis and expression of stress genes.
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Second, we show that the DNA-binding activity of Cbf11 is critical for affecting stress gene expression levels, yet Cbf11 does not act as a stress gene repressor.
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Third, we show that, compared to e.g. peroxide treatment, stress gene mRNA levels are only moderately increased upon downregulation of FA synthesis. So the situation can be called stress gene “derepression”. At the same time the major stress-response regulators (Sty1-Atf1, Fig. 2A-C; Pap1, new Fig. 2D-E) are required for the derepression, but, importantly, neither of them shows increased activation compared to unstressed WT cells (Fig. 3A-C). These data suggest a qualitative difference between the two phenomena (canonical stress response vs dysregulation of FA synthesis). Furthermore, they hint at an important role of the chromatin environment.
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Fourth, we show that Gcn5/SAGA and Mst1, but not 4 other HATs, mediate the connection between FA metabolism and stress gene expression (Fig. 5D-E), and we show clear and specific H3K9 hyperacetylation of stress gene promoters in FA metabolism mutants (Fig. 5A), arguing that this is not a general acetylome issue.
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Fifth, we show that the stress genes affected by changes in FA metabolism show unusually high nucleosome (H3) occupancy in their transcribed regions (even in unperturbed WT cells; Fig. 5A bottom panels), which could dictate the observed specificity in regulation.
While we agree that our understanding is not yet complete, we have already described many mechanistic aspects of the link between FA metabolism and stress gene expression.
- Although in Cbf11 deletion cells, increased resistance to H2O2 is relied upon the Sty1/Atf1 pathway, the authors did not establish a link between lipid synthesis and Atf1 activity because Cbf11 deletion does not affect the phosphorylation of Atf1.
Sty1 and/or Atf1 show non-zero activity even in normal, healthy, unstressed cells. Importantly, Atf1 is bound to many target promoters even in the absence of stress (Fig. 3B; PMID: 20661279, PMID: 28652406). Moreover, Sty1 is actually needed for orderly cell cycle progression (sty1KO cells are elongated, a result of postponed mitotic entry; e.g. PMID:7501024), which we now mention in the Introduction and Discussion. Our point is that Sty1-Atf1 are not hyperactivated under normal conditions - this only happens during major stress insults. Thus, in unstressed cbf11KO cells, stress gene promoters are hyperacetylated, which may facilitate their (Sty1-Atf1 and Pap1-dependent) transcription, without the need for hyperactivation of the stress response regulators. Such increased transcriptional competence of stress promoters is consistent with our findings that upon peroxide treatment stress gene mRNA levels in cbf11KO exceed those in WT (Fig. 1B). We have amended the corresponding section of the Discussion to more clearly explain our conclusions and hypotheses.
- Cbf11 deletion causes elevated H3K9 acetylation at the promotor regions of a number of stress respond genes, the author did not mention whether demonstrate how lipid synthesis defect causes the hyperacetylation at these promoters.
As discussed in our manuscript, we suggest that following downregulation of FA synthesis, the surplus acetyl-CoA is used by Gcn5 and Mst1 HATs to hyperacetylate stress gene promoters.
- As all lipid-metabolism mutants show increased stress response, it would helpful to examine whether H2O2 induction of WT cells influence lipid synthesis, thus establish physiological links between FA synthesis and stress response.
We now mention in the Discussion section that, curiously, cut6/ACC mRNA levels are downregulated upon peroxide treatment. However, the significance of this finding is unclear as FA metabolism is strongly regulated at the post-translational level (PMID: 12529438). Unfortunately, we are not in a position to measure changes in metabolic fluxes upon stress. In any case, we believe that such experiments would be outside the scope of the current study.
Beside, fatty acid may be beneficial to fight oxidative stress because they maintain the integrity of cell membrane. What is the potential effect of CBF11 deletion in this aspect? The author may want to discuss it.
The reviewer suggests that higher production of FA would result in higher resistance to oxidative stress. However, our data do not indicate this - we show that under low FA synthesis the stress resistance is actually higher. Nevertheless, we acknowledge in the Discussion that the scenario suggested by the reviewer can occur, for example, in cancer cells which become more resistant to oxidative stress following increased lipid biosynthesis/storage.
- Since H2O2 treatment also causes change in glucose metabolism including upregulation of glucose transporter Ght5 (PMID: 30782292), it would be enlightening to see if there is a crosstalk between the lipid and glucose metabolisms. Does Ght5 expression increase upon H2O2 treatment in CBF11 deletion strain?
While the topic is interesting, we strongly believe that the relationship between glucose metabolism and stress gene expression is outside the scope of this study.
According to our data used in Fig. 4A, ght5 expression in cbf11KO at 60 min after 0.74 mM H2O2 treatment is downregulated 3-fold.
5 Different H2O2 concentration causes different stress response in pombe: Pap1 and Sty1 mediate responses for low and high H2O2, respectively. For fully activated Sty1 response, the concentration of H2O2, needs to reach 1mM (PMID: 17043891). In this study, the H2O2 concentration ranges from 0.5-1.5mM and Pap1 regulated Ctt1 does show increase upon H2O2 treatment. To test if suppressed lipid synthesis facilitates Sty1 dependent activation, it would be helpful to examine the activation of Pap1 (its nuclear translocation) to eliminate other influences.
We agree with the reviewer. We have now included data on the role of Pap1 in the crosstalk between lipid metabolism and stress gene expression. We show that Pap1 is required for increased expression of gst2 and ctt1 in untreated cbf11KO cells (Fig. 2D). We note that ctt1 is coregulated by both Pap1 and Atf1 (Fig. 2B, D). Also, Pap1 is partially required for H2O2 resistance of cbf11KO cells (Fig. 2E). Importantly, similar to Sty1-Atf, Pap1 is not hyperactivated (no nuclear accumulation) by 10 or 60 min of cerulenin treatment (Fig. 3C), while stress gene expression is upregulated at 60 min in cerulenin (Fig. 4F) and keeps increasing after 120 min (data not shown). These data collectively support our hypothesis that upon decreased FA synthesis, stress gene promoters become more transcription-competent without the requirement for hyperactivation of the corresponding stress gene regulators.
Reviewer #2 (Significance (Required)):
see above
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
This study examines the intriguing phenomenon that perturbation of fatty acid biosynthesis induces expression of stress-response genes by increased intracellular levels of acetyl-CoA and hyperacetylation of histones at the promoters of these genes. Loss of the CSL transcription factor Cbf11 results in induced expression of a subset of stress-response genes in unperturbed conditions and resistance to H2O2. These stress-response genes are not direct targets of Cbf11, but their upregulation is dependent on the Sty1-Atf1 pathway. Similar effects in upregulation of stress-response genes were observed in the cut6 hypomorph and mga2 deletion strain, however no change in global levels of acetyl-Co-A in the former as well as in the cbf11 deletion was detected. The upregulated stress-response genes appear to be linked to increased H3K9 acetylation in their promoters and dependent on the Gcn5 and Mst1 HATs.
The authors present good supportive evidence linking fatty acid biosynthesis to epigenetic regulation of stress response genes potentially mediated by intracellular levels of acetyl-CoA. This is an exciting area and the fission yeast model system is ideal to elucidate the molecular mechanisms behind this process. This is a substantial body of work with state-of-the art functional genomics approaches and LC-MS analysis. The data is of high quality and the manuscript is well written and relatively easy to read. Below are my comments for the manuscript.
It was determined that increased expression of stress-response genes in the cbf11 deletion is dependent on the presence of Sty1, and partially dependent on Atf1. How about Pap1 (or Prr1) - would this transcription factor that is also regulated by Sty1 be involved in the upregulation of the stress-response genes in the cbf11 deletion? Activation of Sty1 and Atf1 by phosphorylation was not observed in unperturbed cbf11 deletion cells which would be expected in the proposed model. This discrepancy was not well explained. Could activation of Sty1/Atf1/Pap1 in unperturbed cbf11 cells be assayed in a different way such as nuclear localization?
As these concerns were also raised by Reviewer 2, to avoid duplication, we kindly ask you to read our detailed responses above. Briefly, we have now included new data clarifying the role of Pap1 in the increased expression of selected stress genes in cbf11KO cells (or when FA synthesis is chemically inhibited) - comment #5 of Reviewer 2 above. Also, we explain why Sty1-Atf1 and/or Pap1 hyperactivation (i.e. above their activity level in untreated WT) is actually not needed in order for decreased FA synthesis to trigger a mild/moderate increase in stress gene expression - comment #2 of Reviewer 2 above. We have now also clarified this issue in the Discussion section.
As for the use of alternative methods for measuring the activation status of Sty1-Atf, we have already provided data from multiple independent and very sensitive methods (western blot, ChIP-qPCR; Fig. 3A-B). Also, it is questionable whether microscopy would be more sensitive than our current methods. Moreover, our H2O2-sensitive reporter does not indicate an increasingly oxidative environment inside cbf11KO cells, quite on the contrary (Fig. 1D).
It would strengthen the model that perturbation of fatty biosynthesis induces expression of stress-response genes and H2O2 resistance if more mutant strains other than cut6 and two of its known regulators were tested. Does the proposed model apply to any deficiency in fatty acid synthesis in general or only those that result in increased levels of acetyl-CoA? For example, would deletion strains of fas1, fas2, lsd90, lcf1, lcf2 or the4 show the same stress response as cut6, mga2, and cbf11 mutants?
The roles of lsd90, lcf1, lcf2 and the4 have been only poorly characterized so far, making it potentially difficult to interpret any stress-related phenotypes of these mutants. However, the role of the fatty acid synthase Fas1/Fas2 complex in FA production is well established. We have therefore inhibited FAS using cerulenin and found that this treatment also leads to increased stress gene expression (Fig. 5F), without causing Pap1 hyperactivation (Fig. 3C). Importantly, fas1/fas2 are not Cbf11 target genes, and FAS inhibition by cerulenin represents an acute intervention, very different from the long-term effects in cbf11/mga2/cut6 mutants.
Also, does overexpression of cut6+ confer sensitivity to H2O2?
Yes, our new data show that ~2-fold overexpression of cut6 both partially abolished the derepression of stress genes in cbf11KO cells (Fig. 5B), and increased sensitivity to H2O2 of WT cells (new Fig. 5C).
The authors hypothesize that induced expression of stress-response genes in the cbf11 deletion and cut6 hypomorph is due to H3K9 hyperacetylation because of increased acetyl-CoA abundance in the cell. However, LC-MS analysis showed no change in global abundance of acetyl-CoA in the cbf11 deletion and cut6 hypomorph although differential levels of acetyl-CoA in the nucleus relative to the rest of the cell cannot be ruled out. The authors mentioned that ppc1-537 and ssp2 null are known to have lower abundance of acetyl-CoA and the latter could suppress the cbf11 deletion-induced gene expression for two of three genes tested by qPCR. Can ppc1-537 also suppress the cbf11 deletion-induced gene expression? Are ppc1-537 and the ssp2 null sensitive to H2O2?
The ppc1-537 mutant is sick and has a growth defect, making it difficult to interpret any findings regarding its survival/resistance phenotype (see a similar issue with the cut6-621 mutant in Fig. 4E). Ssp2/AMPK has a pleiotropic role in the cell and its activity is actually controlled by Sty1-Atf1 under some stress conditions (PMID: 28515144) and the ssp2KO is resistant to osmotic stress (PMID: 28600551). All this makes it potentially difficult to derive reliable conclusions about ppc1 and ssp2. However, our current data on cut6 (ts hypomorph, Pcut6MUT, overexpression) and FAS/cerulenin are derived from precisely targeted and specific interventions, and support the proposed connection between FA synthesis and stress gene expression, and are consistent with the suggested role of acetyl-CoA (and its microgradients) in mediating the connection.
I think Rtt109 is H3K56 specific.
Indeed, H3K56 is the characterized specificity of Rtt109, and we indicate this explicitly in the manuscript. We wanted to make our HAT screen comprehensive since we could not presume which histone or even non-histone acetylation target(s) is involved in lipid metabolism-mediated stress gene expression. Even though we have observed increased H3K9ac (Gcn5/SAGA target), other modifications are likely involved since Mst1 affects stress gene expression in lipid mutants, but Mst1 is not known to target H3K9.
Reviewer #3 (Significance (Required)):
The authors present good supportive evidence linking fatty acid biosynthesis to epigenetic regulation of stress response genes potentially mediated by intracellular levels of acetyl-CoA. This is an exciting area and not all the molecular details have been elucidated in this process. S. pombe is ideal to study this fundamental process and discoveries would be applicable to other eukaryotic study organisms.
My expertise is in eukaryotic gene regulation, molecular genetics and functional genomics, so I am quite qualified to critically review this paper.
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Referee #3
Evidence, reproducibility and clarity
This study examines the intriguing phenomenon that perturbation of fatty acid biosynthesis induces expression of stress-response genes by increased intracellular levels of acetyl-CoA and hyperacetylation of histones at the promoters of these genes. Loss of the CSL transcription factor Cbf11 results in induced expression of a subset of stress-response genes in unperturbed conditions and resistance to H2O2. These stress-response genes are not direct targets of Cbf11, but their upregulation is dependent on the Sty1-Atf1 pathway. Similar effects in upregulation of stress-response genes were observed in the cut6 hypomorph and mga2 deletion strain, however no change in global levels of acetyl-Co-A in the former as well as in the cbf11 deletion was detected. The upregulated stress-response genes appear to be linked to increased H3K9 acetylation in their promoters and dependent on the Gcn5 and Mst1 HATs.
The authors present good supportive evidence linking fatty acid biosynthesis to epigenetic regulation of stress response genes potentially mediated by intracellular levels of acetyl-CoA. This is an exciting area and the fission yeast model system is ideal to elucidate the molecular mechanisms behind this process. This is a substantial body of work with state-of-the art functional genomics approaches and LC-MS analysis. The data is of high quality and the manuscript is well written and relatively easy to read. Below are my comments for the manuscript.
It was determined that increased expression of stress-response genes in the cbf11 deletion is dependent on the presence of Sty1, and partially dependent on Atf1. How about Pap1 (or Prr1) - would this transcription factor that is also regulated by Sty1 be involved in the upregulation of the stress-response genes in the cbf11 deletion? Activation of Sty1 and Atf1 by phosphorylation was not observed in unperturbed cbf11 deletion cells which would be expected in the proposed model. This discrepancy was not well explained. Could activation of Sty1/Atf1/Pap1 in unperturbed cbf11 cells be assayed in a different way such as nuclear localization?
It would strengthen the model that perturbation of fatty biosynthesis induces expression of stress-response genes and H2O2 resistance if more mutant strains other than cut6 and two of its known regulators were tested. Does the proposed model apply to any deficiency in fatty acid synthesis in general or only those that result in increased levels of acetyl-CoA? For example, would deletion strains of fas1, fas2, lsd90, lcf1, lcf2 or the4 show the same stress response as cut6, mga2, and cbf11 mutants? Also, does overexpression of cut6+ confer sensitivity to H2O2?
The authors hypothesize that induced expression of stress-response genes in the cbf11 deletion and cut6 hypomorph is due to H3K9 hyperacetylation because of increased acetyl-CoA abundance in the cell. However, LC-MS analysis showed no change in global abundance of acetyl-CoA in the cbf11 deletion and cut6 hypomorph although differential levels of acetyl-CoA in the nucleus relative to the rest of the cell cannot be ruled out. The authors mentioned that ppc1-537 and ssp2 null are known to have lower abundance of acetyl-CoA and the latter could suppress the cbf11 deletion-induced gene expression for two of three genes tested by qPCR. Can ppc1-537 also suppress the cbf11 deletion-induced gene expression? Are ppc1-537 and the ssp2 null sensitive to H2O2?
I think Rtt109 is H3K56 specific.
Significance
The authors present good supportive evidence linking fatty acid biosynthesis to epigenetic regulation of stress response genes potentially mediated by intracellular levels of acetyl-CoA. This is an exciting area and not all the molecular details have been elucidated in this process. S. pombe is ideal to study this fundamental process and discoveries would be applicable to other eukaryotic study organisms.
My expertise is in eukaryotic gene regulation, molecular genetics and functional genomics, so I am quite qualified to critically review this paper.
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Referee #2
Evidence, reproducibility and clarity
As more and more metabolic intermediates are found to also serve as co-factors for epigenetic modifications, it has been widely accepted that regulating the levels of these key metabolites can be an effective way to control nutrient related gene expression. Acetyl-CoA is one of those early examples. Increased acetyl-CoA was shown to promote local acetylation at growth genes (Mol Cell 2011 PMID: 21596309), and ACC deletion funnels more Acetyl-CoA towards histone acetylation reactions and causes global hyperacetylation (Ref 17). However, whether those increased metabolite/co-factor can exert signal-specific effects remains elusive. For instance, although increased acetyl-CoA stimulates the SAGA complex enzymatic activity, it is not clear whether it also causes SAGA to be targeted to new sites without external cues to induce new transcription factor binding. Does increased acetyl-CoA cause broad hyperacetylation at all inducible genes which are the primary targets for those HAT complexes?
In this manuscript, Princová et al. found that deletion of fatty acid synthesis transcriptional factors Cbf11 and Mga2 increases cell survival under H2O2 induced oxidative stress in S. pombe. They further showed that several stress-related genes increased upon Cbf11 deletion, and H3K9 acetylation at their promotor regions were elevated. They argued that FA-TF deletion may indirectly regulate stress-related genes potentially through influencing Acetyl-CoA level, although they failed to detect significant changes of global Acetyl-CoA levels. While it's interesting to see yet another example of metabolite-mediated gene expression regulation, the current manuscript only made incremental advance towards mechanistic principles of how these co-factors finetune specific gene expression program.
Specific comments:
- This work showed convincingly that deletion of CBF11 or MGA2 leads to resistance to oxidative stress. However, it provides little mechanistic insight into how deletion of Cbf11 increased the expression of stress response genes and why some HATs are involved but others not (Figure EV5).
- Although in Cbf11 deletion cells, increased resistance to H2O2 is relied upon the Sty1/Atf1 pathway, the authors did not establish a link between lipid synthesis and Atf1 activity because Cbf11 deletion does not affect the phosphorylation of Atf1.
- Cbf11 deletion causes elevated H3K9 acetylation at the promotor regions of a number of stress respond genes, the author did not mention whether demonstrate how lipid synthesis defect causes the hyperacetylation at these promoters.
- As all lipid-metabolism mutants show increased stress response, it would helpful to examine whether H2O2 induction of WT cells influence lipid synthesis, thus establish physiological links between FA synthesis and stress response. Beside, fatty acid may be beneficial to fight oxidative stress because they maintain the integrity of cell membrane. What is the potential effect of CBF11 deletion in this aspect? The author may want to discuss it.
- Since H2O2 treatment also causes change in glucose metabolism including upregulation of glucose transporter Ght5 (PMID: 30782292), it would be enlightening to see if there is a crosstalk between the lipid and glucose metabolisms. Does Ght5 expression increase upon H2O2 treatment in CBF11 deletion strain? 5 Different H2O2 concentration causes different stress response in pombe: Pap1 and Sty1 mediate responses for low and high H2O2, respectively. For fully activated Sty1 response, the concentration of H2O2, needs to reach 1mM (PMID: 17043891). In this study, the H2O2 concentration ranges from 0.5-1.5mM and Pap1 regulated Ctt1 does show increase upon H2O2 treatment. To test if suppressed lipid synthesis facilitates Sty1 dependent activation, it would be helpful to examine the activation of Pap1 (its nuclear translocation) to eliminate other influences.
Significance
see above
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Referee #1
Evidence, reproducibility and clarity
This paper demonstrates a link between oxidative stress, lipid biosynthesis, and targeted histone acetylation in fission yeast. In mutant cells with defects in lipid synthesis (cbf11, mga2 lacking transcription factors, and cut6 lacking acetyl-CoA carboxylase), transcripts of a number of genes implicated in resistance to oxidative stress are increased. This is associated with higher levels of H3K9 acetylation and increased tolerance to oxidative stress. These effects are mediated through Sty1, a stress-activated MAP kinase and the transcription factor Atf1.
It is also shown that H3K9 acetylation levels in the promoter region and just downstream of the transcriptional start site are increased in cbf11 mutants (Fig. 5A).
By mutational analysis, the authors implicate the acetyl transferases Mst1 and Gcn5 in this transcriptional effect. Other related acetyl transferases, Hat1, Elp3, Mst2, Rtt109 have been ruled out as main contributors to the dysregulation in unstressed cbf11 mutants. That specific acetyl transferases have been shown to be required is a strength of the investigation.
Major comments:
The hypothesis is put forward in the manuscript that altered acetyl-CoA levels in cbf1 mutants would underlie the dysregulation of genes induced by oxidative stress. Histone acetyl transferases compete for acetyl-CoA with lipid biosynthesis, and so with increased demand for acetyl-CoA underacetylation in the concerned promoters would result - specifically at H3K9.
These results do not directly support the hypothesis, on the other hand they are not sufficient to rule it out. Acetyl-CoA levels were measured only in undisturbed cells, and the possibility remains that under oxidative stress there would be changes in acetyl-CoA pools that could explain this apparent contradiction - why did not the authors examine that?
The authors argue that although the global acetyl-CoA levels are not increased, local concentrations might be altered in a way to permit higher H3K9 acetylation levels at selected promoters. Although a formal possibility, this is rather far-fetched as a small and freely diffusible molecule like acetyl-CoA should quickly equilibrate within one cellular compartment. I think that although the overall relationships that the authors have established between oxidative stress, H3K9 acetylation levels with increased expression, and lipid biosynthesis, are compelling, the role of acetyl-CoA concentrations is not clear and should be de-emphasized.
Minor comments:
In many of the bar diagrams, only a borderline statistical significance is indicated (p ~ 0.05) despite seemingly large numerical differences between the means. In the legends it is stated that one-sided Mann-Whitney U tests were used. This is a non-parametric test with low power - would it not have been better to use a t test? What do the error bars in the diagram show, SEM? If a non-parametric test is used, a parametric measure of variability is irrelevant.
It would be helpful to the reader to indicate directly in the diagram panels what is actually shown, not just "fold change vs ..." In Fig. 1, 2, 4 D and 5 we see mRNA levels, in Fig. 3 chromatin IP.
Significance
The paper represents conceptual advances for our understanding of how stress responses, metabolism and transcriptional regulation are linked, although one of the links (acetyl-CoA levels in this case) is tenuous.
This manuscript belongs in a rich literature on stress responses on the gene expression level, mostly from studies in yeast. Potentially, it adds entirely new information on how cellular stress may be mechanistially linked to stress responses.
These results are potentially general and of broad interest to the biological community.
This reviewer is familiar with yeast genetics, stress responses, and quantification of gene expression.
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www.biorxiv.org www.biorxiv.org
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Reply to the reviewers
1. General Statements
We are grateful to the reviewers for their time and expertise, and we have addressed all points they raised as detailed in our point-to-point response and highlighted the changes in the main manuscript. We have addressed all points raised by the reviewers and elaborated how this was done in a point-by-point reply. There are two new tables and a new supplementary figure. The figures and the text have been reshaped, according to the suggestions.
We are looking forward to your reply. Best regards, Yannick Schwab and Anna M Steyer
2. Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Serra Lleti et al. report a new software (CLEMSite) for fully automated FIB-SEM imaging based on locations identified beforehand in LM. The authors have implemented routines for automatically identifying common reference patterns and an automated FIB-SEM quality control. This allows autonomous data acquisition of multiple locations distributed over the entire sample dish. CLEMSite has been developed as a powerful tool for fast and highly efficient screening of morphological variations.
**Major comments:**
The performance of CLEMSite has been demonstrated by the authors with two typical biological example applications. The stated performance parameters such as correlation precision and reproducibility are highly convincing and supported by the presented data. The authors give detailed information on their workflow and how on to use CLEMSite, which should allow other researchers to implement this for their own applications. The only comment I have in this regard, and I might have overlooked it, but how will CLEMSite be made available to the scientific community?
Reply 1.1
We would like to warmly thank Reviewer #1 for their very supportive feedback. It is important to us to share our work with the community. Our prime intention is to offer CLEMSite as a proof of concept that has been demonstrated on a specific instrument, thus linked to a company (Zeiss). Because we are convinced this code can be adapted to other APIs provided by other vendors, we made it fully available via a Github repository (https://github.com/josemiserra/CLEMSite).
To make this more visible in the manuscript, we have modified this sentence to the first paragraph of the Results section:
“To control the FIB-SEM microscope, CLEMSite-EM interfaces commercial software (SmartSEM and ZEISS Atlas 5 from Carl Zeiss Microscopy GmbH) via a specific Application programming interface (API) provided by Zeiss. CLEMSite code is openly accessible and free to download from a Github repository (https://github.com/josemiserra/CLEMSite ).”
**Minor comments:**
The author mention that decreasing the z-resolution to 200 nm steps was critical to achieve high throughput. For applications that require higher resolution: is the only disadvantage a longer data acquisition time or are there also other limitations?
Reply 1.2:
Reviewer #1 is right, we have designed CLEMSite as a screening tool, where we emphasize the number of cells versus the resolution at which each cell is acquired. By acquiring images every 200 nm, we are gaining speed, but also stability. We have indeed noticed that below 50 nm, on occasions the beginning of the acquisition is not stable enough (the milling has to hit the front of the cross-section at view precisely), and it requires manual intervention to retract/advance the milling. In addition, to gain time in our current workflow, we have opted to not cover the region of interest with a platinum protective layer, which has no consequences when imaging at larger z steps because the overall time spent on one cell is very short. At higher z resolution regimes though, a non-protected block surface is inevitably damaged during the successive numerous mill & view cycles. We have added one sentence in the Methods section to make this point clearer.
“Note that to gain time in the preparation process for a run, we have not covered the ROI with a platinum protective layer and alternatively we increased the thickness of the gold coating of the full sample. In such cases, only low z-resolution acquisition is possible, as acquiring at a higher resolution would require sputtering of the sample surface.”
Finally, we may argue that if an experiment requires high-resolution acquisition, the time overhead spent to switch from one cell to the next (a few minutes) is not significant anymore relative to the time spent to acquire one cell (from several days to weeks). In such cases, automation for multi-site acquisitions would lose its relevance.
I would assume that locating the finer structural details in a much larger data set might also introduce additional challenges in the data analysis pipeline.
Reply 1.3:
We fully agree with Reviewer #1. In this proof of concept study though, we are not addressing the image analysis part but assess ultrastructural phenotypes manually using established stereology protocols. At the image resolution that we are using, our analysis is restricted to features such as volumes, surfaces, number of rather large organelles. Finer details, such as microtubules or fine contact sites between organelles would require a higher resolution, and indeed very likely other means to extract the morphometric data. State-of-the-art image analysis of isotropic FIB-SEM datasets is based on computer vision/machine learning. With such tools, the analysis of fine details is indeed accessible with very high accuracy, but at the cost of the throughput, at least for now as already mentioned in the Discussion section of the paper.
In Table 1 in the supplements, the units are missing for the targeting positions. On page 4, 4th line from the bottom, there is a typo in "reaaching a global targeting...".
Reply 1.4
We thank Reviewer#1 for their thorough inspection of the paper. We have corrected it accordingly.
Reviewer #1 (Significance (Required)):
With CLEMSite, the authors present a powerful new software tool for the FIB-SEM imaging community. The high level of automation allows high throughput data acquisition with minimal user interaction. To my knowledge, this is the first software that fully automatically recognises reference features and is able to run fully autonomously after points of interest have been selected in FM. This high throughput screening tool for FIB-SEM imaging would make a substantial technical contribution to the field of cellular imaging. My own expertise lies in the field of technical developments for CLEM and super-resolution FM. I am not able to judge the biological content of the manuscript.
We would like to thank Reviewer #1 for their constructive and encouraging feedback.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Review on "CLEMsite, a software for automated phenotypic screens using light microscopy and FIB-SEM" by Serra Lleti et al. The manuscript describes a toolset to correlate LM data with automated FIB-SEM of selected regions of interest. This allows 3D correlative microscopy of multiple adherent cells from a single resin block. This allows much needed high throughput in CLEM analysis to become quantitative. Two applications on Golgi apparatus morphology are shown.
**Major questions:**
-The software has been developed in collaboration between Zeiss/ Fibics in collaboration with academic groups and will only function on Zeiss SEMs that have the proper software. Thus, if I understand correct, it will not be of generic use and a more appropriate title would be 'CLEMsite, a software for automated phenotypic screens using light microscopy and Zeiss FIB-SEM"
Reply 2.1.
Reviewer #2 is right about the fact that our work was done on a Zeiss microscope and in CLEMSite’s current version, it would only work with that model, including firmware and software. As already phrased in the manuscript, we would like to stress our work is a proof-of-concept. For example, we wrote in the introduction that CLEMSite is a “software prototype”. We’ve also made clearer the links to Zeiss in the first paragraph of the Results (see also answer to reviewer 1.1)
CLEMSite is by no means designed to become an integrated part of current or future Zeiss microscopes. On the contrary, we have designed the software as an independent unit. All the parts of the software that are sending commands to the Zeiss API are indeed customized to that brand, but other functions are stand-alone units. In particular, the correlation strategy is independent of the microscope type and can be used generically. Similarly, the principles that we developed for finding the FIB-SEM coincidence point, or for selecting features-rich regions to perform the AFAS function would be valid whichever microscope model would be used.
For these reasons, we would prefer to avoid mentioning Zeiss already in the title of the manuscript.
- How is the described approach using FIB-SEM advantageous compared to methods like Serial Block-face EM (SBEM) and array tomography using serial section where larger fields of multiple cells can be imaged? Especially because the axial resolution was set to 200 nm and discussed as essential for the throughput speed.
Reply 2.2.
This is a very important point that we tried to bring across in the introduction of the manuscript. Other volume EM methods, such as SBEM and AT, like conventional TEM, require an ultramicrotome to produce thin sections (AT and TEM) or to remove the top layers from the resin block (SBEM). This inevitably requires trimming large specimens in order to accommodate the dimensions of the diamond knife used in the associated microtomes. FIB-SEMs does not have such limitations and selected volumes can be imaged from samples of any size, providing they fit in the chamber of the microscope. In our case, we were screening cells growing on a 1 cm2 surface area, which is already beyond what standard diamond knives can process. We would even argue that larger surfaces are at CLEMSite reach, but we have not tested this.
- Is the data FAIR available?
Reply 2.3
It is one of EMBL’s ambitions to make all data as FAIR as possible. For this study, we saved all the raw images and their corresponding embedded metadata as they came from the original software (ATLAS 5, Fibics for the SEM images and LAS X, Leica microsystems for the confocal images). The images published in this manuscript will be deposited on the EMPIAR data repository upon acceptance. The raw data and unpublished data, due to their size, will be fully available upon request to the authors. Additionally, their data is specifically generated for the correlation workflow, which is stored together with the image information as separated files. To store the information of logs we used text files, for intercommunication between processes, JSON, and XML to store coordinates in a readable format. As far as we know, there is no standard FAIR protocol yet that describes CLEM workflows in microscopy. We made our best possible efforts to archive our data in an understandable folder architecture, with detailed information on how to navigate through it, such that we are confident that our data could be mined by others in the future, thus reaching the goals of the FAIR charter.
- How is CLEMsite available? Is the code public or for sale?
Reply 2.4
It is important to us that our proof-of-concept can be used or adapted by others in the future. For this reason, we are sharing the full code that was developed for CLEMSite - See Reply 1.1 for further details.
**Other comments:**
- Can you comment on the flexibility of this method? It is described as a flexible method, but only HeLa cells (quite flat cells) and Golgi apparatus targeting was used. What about different cell types and what about targets with a less obvious EM morphology?
Reply 2.5
It is correct that we have demonstrated our workflow only on Hela cells which present a more or less homogeneous topology. Yet our workflow is flexible when it comes to the dimensions of the region of interest and the acquisition field of view, and can accommodate a wide range of cell shapes, as long as they adhere to a culture substrate. Dimensions of ROI and FOV can be adapted in the CLEMSite interface as described in Supplementary Figure 4. Following reviewer 2 question, we realize that this feature may not appear clearly and we have modified the corresponding section of the Result:
“The dimensions of the image stack, as well as the z resolution are set when initializing the run, via the CLEMSite interface (Supplementary Figure 4). Whilst every cell of one run can be acquired with the same recipe (as defined in ZEISS Atlas 5: sample preparation, total volume to be acquired, slice thickness and FIB currents applied at each step), CLEMSite-EM also offers individual definition of recipes, allowing a per cell adaptation of the shape or volume (Supplementary Fig. 4a).”
Changing the ROI size would thus accommodate the surface occupancy of a cell (in the plane parallel to the culture substrate) and changing the FOV would accommodate the cell’s height.
The morphology of the cell as it appears in the EM (SESI) does not alter the targeting strategy, since we are solely relying on the correlation, which means that the position of the target cell is extracted from the light microscopy images and the coordinate system provided by the gridded coverslip. Even if the cells were invisible at the surface of the resin block when inspected in the SEM, CLEMSite would still navigate to the proper region and create an image stack by FIB-SEM imaging.
- For EM acquisition ZEISS smartSEM with ATLAS was used. LM was recorded with a microscope from a different vendor. Can the software be used regardless of microscope type?
Reply 2.6
Yes, the correlation is based on collecting the stage coordinates from the light microscope, and on analyzing the images from the various magnifications and channels. This information can be obtained by most microscope types, but it might involve minor adaptations regarding the specific brand of a microscope (e.g. changes in the coordinate system of the stage used or the naming of the channels).
- Create less variation in the size of scale bars.
Reply 2.7
We have modified all figures to take this comment into account and thank Reviewer #2 for a good suggestion.
- M&M: High-resolution light microscopy: Why call this 'high resolution'?
Reply 2.8
We used this term to differentiate, in the feedback microscopy setup, the first stage where images are acquired at low magnification from the images acquired at high magnification. We agree that the term is misleading, so we decided to update the manuscript and change the term high resolution by higher magnification (the second stage in feedback microscopy).
Specs given seem randomly chosen: For example objective magnification yes, NA not; excitation wavelength yes, emission not.
Reply 2.9
We thank Reviewer #2 for spotting these missing details. We have edited the method section to add the NA and the emission wavelengths.
Reviewer #2 (Significance (Required)): See above: This depends on the availability of code, as well as the usability in FIB-SEM that is not based on Zeiss.
Reply 2.10
We hope our answers have addressed these concerns. When the code is indeed fully available, we can not at this stage presume of the transferability of CLEMSite to microscope from other manufacturers. Yet we would like to stress once more that our main aim is to demonstrate a proof of concept.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Schwab and coworkers present an automation software for correlative light and electron microscopy (CLEM) to acquire high-resolution SEM volume datasets at room temperature. This automation enables large-scale data collection and morphometric analysis of cell phenotypes. The paper is overall well written, but often assumes a lot of prior knowledge of the workflow, which might not be present in a general audience or for newcomers to the technique. This is also seen in the insufficient labeling and explanation of the figures. They seem a bit like presentation slides, which could be well understood with the help of the presenter/narrator, but alone lack a lot of information (see more specific comments below).
**Major Comments (in no particular order):**
- Final accuracy of ~ 5 µm ... is this really sufficient? Given that the size of many mammalian cells is ~10-15 µm, this is still a HUGE error. Of course, there is a tradeoff between throughput and accuracy, the area covered and speed. Nonetheless, this means a serious limitation in terms of the kind of targets / biological questions that can be addressed with this technique! (Especially in the context of "rare events") This should be discussed in more detail. Reply 3.1
We thank reviewer #3 for their constructive criticism of the work. Indeed, our final accuracy is 5 µm at best, which may at first glance appear as a disappointing value. This accuracy is the consequence of a couple of strategic decisions that we have made in designing the workflow, which will be further explained below. We have chosen to constantly opt for a large field of view that would be larger than the average cell size, thus mitigating the potential 5 µm offset in targeting. In our hands, this yielded satisfying results, yet we agree that a higher targeting precision would allow narrower fields of view and potentially an even increased throughput.
Our correlation strategy fully relies on the coordinate system built from the gridded pattern embossed at the surface of the culture dishes. The precision of CLEMSite automated targeting thus relies on i) its ability to properly detect the grid edges, both at the LM and at the ME, and ii) on the mesh size of the grid. To ensure a wide range of applications, we decided to design CLEMSite on commercial culture dishes, of which the MatTek gridded culture dishes appeared the most convenient, for each grid square presented a unique alphanumeric ID together with a relatively large and flat surface area to accommodate a large number of cells away from the grid pattern. Whilst such dishes showed a topology that satisfied our first criteria, the grid spacing was 600 µm. A smaller mesh size would have undoubtedly resulted in higher precision in the targeting but at the expense of losing free areas. Other commercial dishes with denser meshes unfortunately would not have ID engraved directly inside every square or we experienced difficulties in reproducibility during the sample preparation process to detach the glass from the resin block.
We also have excluded the option to design our own grids, which would have created another dependency for potential users from other laboratories.
Another possibility for targeting would be to register the fluorescence maps to the shapes of the cell as visible in the resin block. Adherent cells can be detected in the SEM if high energies are used to scan the surface of the blocks, and also if the block is not coated with a too thick layer of gold. In our experience, switching between voltages for acquiring such overviews and low voltages for acquiring FIB-SEM stacks is another source of imprecision and doesn’t improve the targeting in very confluent areas. Another interesting idea, as shown in Hoffman et al 2020, would be to scan the embedded samples by X-ray prior the FIB-SEM targeting, but not only this would imply that high-end X-ray machines would be available for such tasks, but would still require landmarks to register the X-ray maps to the SEM overviews. This would potentially yield a higher accuracy, but we have opted for the gridded substrates, judged more accessible to a large number of laboratories.
We tried to explain such a choice in the discussion, by adding this sentence in the description :
“Detection of local landmarks imprinted in the culture substrate enables automated correlation and targeting with a 5 µm accuracy. We estimate that this number could still be improved by customizing a gridded substrate with a smaller mesh size, as landmarks would be much closer to the targets. The detection algorithm we developed could be extrapolated to other customized dishes or commercial substrates for cell culture in SEM samples41. An advantage of using local landmarks for the correlation is that they mitigate the impact of sample surface defects or optical aberration across long distances. Alternatively, targeting individual cells with a FIB-SEM has been achieved by mapping the resin embedded cells with microscopic X-ray computed tomography44. We speculate that such tools could be an alternative to a gridded substrate, yet cannot predict its adaptability to large resin blocks such as the ones we used in this study. “
- Given that the whole point of the paper is "large scale automation", I would have preferred a few more examples/higher n-count. A comment on which type of targets the authors envision/have validated would be nice (also in the context of the limitation in accuracy). Reply 3.2
To our best knowledge, no one has ever imaged multiple cells automatically. So even 5 in a row is a high number.
In addition to this, we added an extra paragraph in the discussion.
“We believe that other research questions could benefit from this type of screening. As an example, the 2021 Human Protein Atlas Image Classification competition61 managed to classify multiple organelles of individual cells in fluorescence microscopy. Such machine learning models could be used to find rare events or particularly interesting phenotypes. In another example, in host-pathogen interactions, early infected cells might start to display a recognizable phenotype in a small subpopulation of cells62. In both cases, those marked cells could be used to establish a FIB-SEM screening to discover new morphological differences at the micrometer level.
To expand the applicability of these screenings beyond the proof-of-concept here presented, we propose two directions of improvement. First, by acquiring smaller enclosed volumes with isotropic resolution, we could target area-delimited organelles, like centrioles21. In this case, the full cell volume is neglected in favor of a small portion of it, but with higher z resolution. At the software level, that would require improving targeting accuracy by using smaller grids and extending the maps to 3D coordinates. 3D registration against a light microscopy Z-stack would considerably help to constrain the field of view during acquisition, thus reducing the imaging time and keeping the field of view position during tracking. At the instrument level, this would require, first, stabilizing the ion beam before the critical region is acquired, to compensate for the change between high currents for milling and fine currents for sectioning. Second, to make sure that the fine current beam hits exactly the front face of the milled cross-section, and then prevent milling artifacts. Finally, the second direction is to increase the number of samples acquired per session. That would imply ion beams that automatically reheat the Gallium source when it is exhausted (like proposed in Xu et al. 60), with faster algorithms for autofocus and autostigmatism in SEM.”
- It should be mentioned somewhere that "commercial dishes or coverslips" contain an imprinted grid pattern with numbers and letters to locate specific squares. [Again: probably clear to "aficionados" of the technique but totally unclear to newcomers/outsiders] Reply 3.3
We have added an explanation of the layout of the coordinate system in the part of the correlation strategy and the methods section “gridded dish with numbers and letters' to explain the correlation and targeting strategy better.
-
"It is important to keep the initial number high in order to compensate for the loss of targets" - what % of targets is lost exactly in the final step (FIB-SEM imaging)? The 10 cells out of 35 (29%!) that were not "of sufficient quality for further downstream analysis", were they lost/discarded because of problems in the automation (e.g. autofocus/tracking failure) or for other reasons (e.g. preservation of the cells during fixation/embedding)? Reply 3.4 In the main text we decide to explain the process of filtering better. We have added a supplementary figure showing different causes for problems of coordinate system detection due to scratches, cracks, or dirt. None of the cells in the study were discarded due to bad preservation, but the system being a proof of concept, we dealt with multiple difficulties that forced us to filter the acquired stacks for getting the ones showing the best quality. We also added supplementary material (Sup. Tables 3 and 4 with explanation) about the possible causes of cell losses during different experiments.
-
"One essential paradigm shift for increasing the acquisition throughput is the decision to decrease the resolution in the z-dimension, thus prioritizing the speed of acquisition and ultimately the total number of cells acquired in one run.". Surely, reducing z-resolution is an obvious way to speed up acquisition times. But this is not tied to the use of this software and obviously comes at a price ... this has been discussed before and is nothing novel. Hence "paradigm shift" might be a bit too strong. I however fully agree with CLEMSite's potential as a screening tool. Could a "high resolution" (isotropic) mode not be implemented, too? [then it would be up to the user to decide what to prioritize - throughput or resolution] Reply 3.5
We have replaced the word “paradigm shift” with “original strategy”. It is indeed up to the user to decide if higher z resolution or higher speed should be achieved by setting up different recipes.
Additionally, we direct the reviewer to read Reply 1.2.
- There is no mentioning of why this specific hardware was used. Are there any limitations that currently restrict the approach to Zeiss machines? Any plans supporting other vendors? Of course, there are always certain benefits with certain instruments. Or just simply no others were available... A comment on which part was performed by/at Zeiss and which in the labs would be useful to understand specific contributions. (Since a conflict of interest statement seems missing). Reply 3.6
The original plan was to set up a proof-of-principle study developing a program that is fully open source. We created an interface, which could plug any control via proprietary API, by simply adapting commands from the API to our interface. The idea is very similar to what is done in light microscopy open-source controllers, like the Micropilot software (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086017/).
That interface would be the place where to modify the software and add the external API and requires only that such API can be used with a .NET framework in C#. The programmer would have to modify only the following file:
https://github.com/josemiserra/CLEMSite/blob/master/CLEMSiteServer/TestApp/AtlasCom.cs.
We expect that FIB-SEMs are very similar across companies, at least in basic functionality (get images, get positions, mill execution for trench digging with a recipe file, ...). We thus believe our software could be adapted to other vendors. As an example, we used the Fibics API, but we could also program the same functionality with the Zeiss API to achieve the same goal.
Zeiss’s contribution to the project was i) providing a system during the initial phase of the project, and allocating time with programmers from FIBICS to help to provide the control API to be used by CLEMSite.
All experiments were performed at the European Molecular Biology Laboratory or Max-Planck Institute of Experimental Medicine.
**Figures:**
Figures should be improved. They often contain too little information to understand the concepts/results discussed and there's lots of white space. The legends should be improved accordingly. In general, a more concise and structured figure design could go a long way of improving the quality of the manuscript. Please find a few suggestions (for the main figures) below (but the same should be applied to the supporting figures):
Reply 3.7
We thank the reviewer for the suggestions on the figures in general. We have revisited all the figures and made corresponding changes as highlighted below.
Fig. 1: While I believe it is clear to me what each scheme is supposed to represent, someone less immersed in this topic (or just entering the field) may have problems navigating the figure. For example: what are all the different letters and numbers? What's the blue box with the trapezoid ("EM targets" - it may become clear later, but here it is not), what are the blue and the red arrowhead, respectively (I suppose EM and focused ion beam?). This should be improved and labeled accordingly.
We have addressed the queries by explaining the figure more explicitly in the legend (e.g. blue box, blue and red arrowhead). We have added i, ii, iii to separate b into subsections and adjusted the text accordingly.
Fig. 2: Again, a lot of annotation is missing. E.g. what is the 3rd insert in b exactly (edge-detection? After CNN identification?)? For most of the figure, yellow squares are used to indicate the zoom-in region, why not for b) 1st row? With the "zoo" of scale bars, wouldn't it make sense to either always show the same bar (e.g. 200 µm), or scale the images so things become more comparable? In this regard: a) 2nd column and e) 1st column represent the same FOV. Why are they shown with different magnification/cropping?
Reply 3.8
The scale bars have been homogenized when possible, in the case of b the image was zoomed to match a (first image in both cases). A yellow box was added for the zoom in b as suggested. We added descriptions in the figure legend.
Fig. 3: a) The procedure is well described in the legend, but no motivation is given in the text, why this is necessary. c) There's some floating density in the white space. Is this due to thresholding?
Already explained in figure legend.
Reply 3.9
We have adapted the text in the main manuscript to explain better that the coincidence point is normally found manually and for a routine with as little as possible intervention by an operator this had to be automated. We have also explained figure 3c more in the figure legend.
“The following steps, usually performed by a trained human operator, are triggered autonomously: localization of the coincidence point, needed to bring the FIB and SEM beams to point at the same position (Fig. 3a); milling of the trench to expose the imaging surface, detection of the trench to ensure a well-positioned imaging field of view (FOV) (Fig. 3b); automated detection of image features in the imaged surface needed to find an optimal location for the initial autofocus and autostigmation (AFAS) (Fig. 3c); and finally the stack acquisition (Fig. 3d).”
Fig. 4) Again, description/labeling of the figure is poor. E.g. what are the red outlines present in c) row 1-3 (but missing in row 4; why?)? [presumably these are the siRNA spots?] Is there any reason this figure could not be further subdivided into d), e), f) etc? As it stands, a lot of additional descriptors ("second from the left", "two images on the right") are necessary while a simple call to a), b), c) would be much easier...
Reply 3.10
We have more precisely described the siRNA spots in the legend more explicitly and have added headings to divide part c into a grid rather than adding letters/numbers to subdivide to make the figure more clear.
Fig. 5) Additional labeling (a,b,c...) could be helpful here, too. While intuitively I would assume that blue = DAPI and green = GFP, these things should be labeled or described in the legend. Especially in the 3D rendering it is quite unclear what is being portrayed. Is this an overlay of a FIB/SEM segmentation with the confocal 3D-data?
Reply 3.11
We have added headings to subdivide the images in b and explanations in the legends to explain the color-dye relation (blue is DAPI).
**Minor:**
The first element in the filtered list can thus be stored for the subsequent application of autofocus and autostigmation procedures (AFAS) (Supplementary Fig. 3c). [technically this has been defined before]
Reply 3.12
All typos and grammar-related issues have been addressed in the following ways:
A transformation is computed to register together the LM list and EM landmarks list, ...
“A transformation is computed to register the positions from the LM and the EM landmarks list, ... “
“The FOV is changed from a 305 μm by 305 μm, used for the detection of the trench, to a 36.4 μm by 36.4 μm in the exposed cross-section and an image of that cross-section is taken for analysis (Fig. 3c).”
The sample is positioned at the target coordinates of the first cell, and the Multisite module performs the coincidence point alignment of both the electron and ion beams (Fig. 3a and Supplementary Fig. 2a).
Reviewer #3 (Significance (Required)):
**Significance:**
It is clear that the kind of automation outlined here is necessary to elevate correlative SEM volume imaging to a "high-throughput" technique, which could become valuable for many biological questions. CLEMSite offers a valid technical solution and appears to be a solid implementation of the traditional/manual workflow. However, its presentation needs to be improved before we can support publication.
Reply 3.13
We have worked on different aspects of the presentation, rearranged the figures, and extended figure legends and hope this meets the reviewer’s expectations.
-
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Referee #3
Evidence, reproducibility and clarity
Summary:
Schwab and coworkers present an automation software for correlative light and electron microscopy (CLEM) to acquire high-resolution SEM volume datasets at room temperature. This automation enables large-scale data collection and morphometric analysis of cell phenotypes.
The paper is overall well written, but often assumes a lot of prior knowledge of the workflow, which might not be present in a general audience or for newcomers to the technique. This is also seen in the insufficient labeling and explanation of the figures. They seem a bit like presentation slides, which could be well understood with the help of the presenter/narrator, but alone lack a lot of information (see more specific comments below).
Major Comments (in no particular order):
• Final accuracy of ~ 5 µm ... is this really sufficient? Given that the size of many mammalian cells is ~10-15 µm, this is still a HUGE error. Of course, there is a tradeoff between throughput and accuracy, the area covered and speed. Nonetheless, this means a serious limitation in terms of the kind of targets / biological questions that can be addressed with this technique! (Especially in the context of "rare events") This should be discussed in more detail.
• Given that the whole point of the paper is "large scale automation", I would have preferred a few more examples/higher n-count. A comment on which type of targets the authors envision/have validated would be nice (also in the context of the limitation in accuracy).
• It should be mentioned somewhere that "commercial dishes or coverslips" contain an imprinted grid pattern with numbers and letters to locate specific squares. [Again: probably clear to "aficionados" of the technique but totally unclear to newcomers/outsiders]
• "It is important to keep the initial number high in order to compensate for the loss of targets" - what % of targets is lost exactly in the final step (FIB-SEM imaging)? The 10 cells out of 35 (29%!) that were not "of sufficient quality for further downstream analysis", were they lost/discarded because of problems in the automation (e.g. autofocus/tracking failure) or for other reasons (e.g. preservation of the cells during fixation/embedding)?
• "One essential paradigm shift for increasing the acquisition throughput is the decision to decrease the resolution in the z-dimension, thus prioritizing the speed of acquisition and ultimately the total number of cells acquired in one run.". Surely, reducing z-resolution is an obvious way to speed up acquisition times. But this is not tied to the use of this software and obviously comes at a price ... this has been discussed before and is nothing novel. Hence "paradigm shift" might be a bit too strong. I however fully agree with CLEMSite's potential as a screening tool. Could a "high resolution" (isotropic) mode not be implemented, too? [then it would be up to the user to decide what to prioritize - throughput or resolution]
• There is no mentioning of why this specific hardware was used. Are there any limitations that currently restrict the approach to Zeiss machines? Any plans supporting other vendors? Of course, there are always certain benefits with certain instruments. Or just simply no others were available... A comment on which part was performed by/at Zeiss and which in the labs would be useful to understand specific contributions. (Since a conflict of interest statement seems missing).
Figures:
Figures should be improved. They often contain too little information to understand the concepts/results discussed and there's lots of white space. The legends should be improved accordingly. In general, a more concise and structured figure design could go a long way of improving the quality of the manuscript. Please find a few suggestions (for the main figures) below (but the same should be applied to the supporting figures):
Fig. 1: While I believe it is clear to me what each scheme is supposed to represent, someone less immersed in this topic (or just entering the field) may have problems navigating the figure. For example: what are all the different letters and numbers? What's the blue box with the trapezoid ("EM targets" - it may become clear later, but here it is not), what are the blue and the red arrowhead, respectively (I suppose EM and focused ion beam?). This should be improved and labeled accordingly.
Fig. 2: Again, a lot of annotation is missing. E.g. what is the 3rd insert in b exactly (edge-detection? After CNN identification?)? For most of the figure, yellow squares are used to indicate the zoom-in region, why not for b) 1st row? With the "zoo" of scale bars, wouldn't it make sense to either always show the same bar (e.g. 200 µm), or scale the images so things become more comparable? In this regard: a) 2nd column and e) 1st column represent the same FOV. Why are they shown with different magnification/cropping?
Fig. 3: a) The procedure is well described in the legend, but no motivation is given in the text, why this is necessary. c) There's some floating density in the white space. Is this due to thresholding?
Fig. 4) Again, description/labeling of the figure is poor. E.g. what are the red outlines present in c) row 1-3 (but missing in row 4; why?)? [presumably these are the siRNA spots?] Is there any reason this figure could not be further sub-divided into d), e), f) etc? As it stands, a lot of additional descriptors ("second from the left", "two images on the right") are necessary while a simple call to a), b), c) would be much easier...
Fig. 5) Additional labeling (a,b,c...) could be helpful here, too. While intuitively I would assume that blue = DAPI and green = GFP, these things should be labeled or described in the legend. Especially in the 3D rendering it is quite unclear what is being portrayed. Is this an overlay of a FIB/SEM segmentation with the confocal 3D-data?
Minor:
The first element in the filtered list can thus be stored for the subsequent application of autofocus and autostigmation procedures (AFAS) (Supplementary Fig. 3c). [technically this has been defined before]
Typos/grammar:
In our case, we had two of such experiments, reaaching a global targeting accuracy (RMSE) of 8 {plus minus} 5 μm. A transformation is computed to register together the LM list and EM landmarks list, ...
The FOV is magnified from a 305 μm by 305 μm to a 36.4 μm by 36.4 μm surface area and an image of the cross-section is taken (Fig. 3c).
The sample is positioned at the target coordinates of the first cell, and the Multisite module performs the coincidence point alignment of both the electron and ion beams (Fig. 3a and Supplementary Fig. 3a).
To preserve the target, the sample is drifted 50 μm in x. [shifted?]
Significance
Significance:
It is clear, that the kind of automation outlined here is necessary to elevate correlative SEM volume imaging to a "high-throughput" technique, which could become valuable for many biological questions. CLEMSite offers a valid technical solution and appears to be a solid implementation of the traditional/manual workflow. However, its presentation needs to be improved before we can support publication.
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Referee #2
Evidence, reproducibility and clarity
Review on "CLEMsite, a software for automated phenotypic screens using light microscopy and FIB-SEM" by Serra Lleti et al.
The manuscript describes a toolset to correlate LM data with automated FIB-SEM of selected regions of interest. This allows 3D correlative microscopy of multiple adherent cells ¬from a single resin block. This allows much needed high throughput in CLEM analysis to become quantitative. Two applications on Golgi apparatus morphology are shown.
Major questions:
- The software has been developed in collaboration between Zeiss/ Fibics in collaboration with academic groups and will only function on Zeiss SEMs that have the proper software. Thus, if I understand correct, it will not be of generic use and a more appropriate title would be 'CLEMsite, a software for automated phenotypic screens using light microscopy and Zeiss FIB-SEM"
- How is the described approach using FIB-SEM advantageous compared to methods like Serial Block-face EM (SBEM) and array tomography using serial section where larger fields of multiple cells can be imaged? Especially because the axial resolution was set to 200 nm and discussed as essential for the throughput speed.
- Is the data FAIR available?
- How is CLEMsite available? Is the code public or for sale?
Other comments:
- Can you comment on the flexibility of this method? It is described as a flexible method, but only HeLa cells (quite flat cells) and Golgi apparatus targeting was used. What about different cell types and what about targets with a less obvious EM morphology?
- For EM acquisition ZEISS smartSEM with ATLAS was used. LM was recorded with a microscope from a different vendor. Can the software be used regardless of microscope type?
- Create less variation in the size of scale bars.
- M&M: High resolution light microscopy: Why call this 'high resolution'? Specs given seem randomly chosen: For example objective magnification yes, NA not; excitation wavelength yes, emission not.
Significance
See above: This depends on the availability of code, as well as the usability in FIB-SEM that is not based on Zeiss.
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Referee #1
Evidence, reproducibility and clarity
Summary:
Serra Lleti et al. report a new software (CLEMSite) for fully automated FIB-SEM imaging based on locations identified beforehand in LM. The authors have implemented routines for automatically identifying common reference patterns and an automated FIB-SEM quality control. This allows autonomous data acquisition of multiple locations distributed over the entire sample dish. CLEMSite has been developed as a powerful tool for fast and highly efficient screening of morphological variations.
Major comments:
The performance of CLEMSite has been demonstrated by the authors with two typical biological example applications. The stated performance parameters such as correlation precision and reproducibility are highly convincing and supported by the presented data. The authors give detailed information on their workflow and how on to use CLEMSite, which should allow other researchers to implement this for their own applications. The only comment I have in this regard, and I might have overlooked it, but how will CLEMSite be made available to the scientific community?
Minor comments:
The author mention that decreasing the z-resolution to 200 nm steps was critical to achieve high throughput. For applications that require higher resolution: is the only disadvantage a longer data acquisition time or are there also other limitations? I would assume that locating the finer structural details in a much larger data set might also introduce additional challenges in the data analysis pipeline.
In Table 1 in the supplements, the units are missing for the targeting positions.
On page 4, 4th line from the bottom, there is a typo in "reaaching a global targeting...".
Significance
With CLEMSite, the authors present a powerful new software tool for the FIB-SEM imaging community. The high level of automation allows high throughput data acquisition with minimal user interaction. To my knowledge, this is the first software that fully automatically recognises reference features and is able to run fully autonomously after points of interest have been selected in FM. This high throughput screening tool for FIB-SEM imaging would make a substantial technical contribution to the field of cellular imaging.
My own expertise lies in the field of technical developments for CLEM and super-resolution FM. I am not able to judge the biological content of the manuscript.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This study presents a first structural insight on formin mDia bound to actin filaments in physiological conditions. Based mainly negative stain EM, the authors use 2D and 3D class averaging to describe two main configuration of the formin at the filament barbed end. The two configurations support the previously proposed stair-stepping model, which was based on crystal structures, with an open state where the formin binds two actin monomers and a closed state where three monomers are bound. Because the majority of the structures fall in the first, open state, this supports the existence of this intermediate. The authors also show that the orientation of the free FH2 in this open state is somewhat flexible, as several sub-classes with different angles can be distinguished. Finally, they identify, for the first time, formin densities bound along the length of the filament.
The data is well presented and I don't have any major issue. The only point is that the information that all the initial structural data comes from negative stain EM comes should be put upfront. One gets the feeling that cryoEM is used throughout until one reads the section on cryoEM. Given that the methodology is now also established for cryoEM, it is regrettable that data was not collected with a 300kV microscope, which may have revealed more details of the conformations, but I understand microscope time is hard to come by, and the authors did a remarkable job from negative-stain EM.
The finding of formin densities binding along the length of the actin filament is very interesting. Besides the previous cited finding, it also reminds of the observations made in yeast where Bni1 (in S. cerevisiae; PMID 17344480) and For3 (in S. pombe; PMID 16782006) where shown to exhibit retrograde movement with polymerizing actin cables in vivo. This would be interesting to consider in the discussion.
Reviewer #1 (Significance (Required)):
This study extends our understanding of the mechanism of formin-mediated actin assembly, by providing a first structural observation in physiological conditions. While confirmatory of previously proposed model, but also excludes an alternative model, and offers novel observations of flexibility and binding along the actin filament length. It will be of great interest to researchers on the actin cytoskeleton.
My expertise is in the actin cytoskeleton and formins, but I am no expert in EM structural analysis.
We thank reviewer 1 for the very positive comments and for pointing out the relevance of our study for the actin cytoskeleton field. As advised, we now specify upfront in the abstract and in the introduction that most of the presented results were obtained from negative stain electron microscopy. Following the reviewer’s advice, we have enriched the discussion to highlight the retrograde movements of formins in actin cables observed in vivo.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Maufront et al. have used EM to study the conformation of mDia1 at the barbed end and the core of actin filaments to explain the molecular mechanism of the FH2 dimer processivity at these sites. Based on modelled structural data they tried to describe how the conformational changes in FH2 dimer lead to its partial dissociation, and then association with filaments during the process of translocation coupled to subunit addition at actin filaments barbed ends. This supports a previous study (Otomo et al. 2005, Nature), in which using X-ray crystallography structural data were used to propose a stair-stepping model for Bni1p translocation at the barbed ends during actin polymerization. The model for mDia1 binding to core filaments is also given. Moreover, using EM structure and the previously reported structures of actin (PDB: 5OOE), and actin with formin FH2 dimer (PDB: 1Y64), authors explained the dynamic nature of FH2 dimer at barbed ends of the filaments using the flapping model. But due to the low resolution of their structures ~ 26-29A0, the finer details of actin and the FH2 dimer structure at barbed ends could not be resolved, leaving open questions about the orientation of actin helical twist at this end during elongation. The authors tried several conditions to get high density barbed-end filaments, but that did not collect adequate number of particles, resulting in low number of particles selected for structure modelling purposes. However, to attain more physiologically relevant structure they used cryo-EM, but were successful in capturing only the open conformation structure of FH2 dimer (at low resolution). Thus, due to low resolution of structures the key findings have not added much to what we already know about the mechanism of FH2 dimer translocation during actin polymerization, except that their studies support the stair-stepping model (Otomo et al. 2005, Nature) and not that of "stepping second" model ( Paul and Pollard. 2008, Curr. Bio.). Thus, this manuscript does not merit publication in this journal.
We thank reviewer 2 for taking the time to read and review our study. However, we respectfully disagree with the statement that our findings “have not added much to what we already know about the mechanism of FH2 dimer translocation during actin polymerization”. As mentioned in our report, collecting EM data for formins in physiological conditions (at the barbed ends of growing filaments), as we do here for the first time, entails limitations on the number of particles one can observe and on the resulting resolution. Despite this rather low resolution, our data allow us to discriminate between two proposed models accounting for the processivity of formin FH2 domains at filament barbed ends. Being able to determine which of two competing models is valid (as the reviewer says we do) does add a lot to what we already know.
Major comments:
- Present study does not provide any new insight about the conformation of the actin dimer at the barbed ends of actin filaments when FH2 domains of formin are bound. This study appears to be more like an extension of previous research (Otomo et al. 2005, Nature), in which the authors used X-ray crystallography data to propose a model for actin filaments elongation by formin bound at the barbed ends.
As mentioned above, we respectfully disagree with this remark. First, in Otomo et al. 2005, formins are arranged in a crystal into a non-physiological “daisy chain” arrangement around a non-canonical tetramethyl rhodamine-actin filament. Our observations were made in physiological conditions displaying a single formin dimer at the barbed end of a polymerizing filament. Second, the stair stepping model originating from Otomo et al. was only inferred and extrapolated from the crystal structure and not directly observed. Both the open and the closed conformations were speculations, that had never been observed up to now. In our current report we directly visualize these two conformations. Third, the observations of Otomo et al. were obtained using formin Bni1p from yeast, not the mammalian formin mDia1, for which there is little (PDB 1V9B) structural data available describing the structure of a truncated mDia1 in the absence of actin. Finally, in addition to validating the stair-stepping model experimentally, we make unexpected observations that are totally absent from the model derived from Otomo et al. and subsequent studies.
The low resolution of structures is a major concern.
As mentioned above, the limited resolution is the price we had to pay for being in physiological conditions, with formins interacting with the barbed ends of growing actin filaments. Nonetheless, this resolution is sufficient to discriminate between the two previously existing models, and to make new observations, beyond these models.
Given the low resolution of data, how can the authors decide on the number (4) of classes of FH2 domain (in open state) and present them as "continuum of conformations". They stated "details featured in class 4 do not appear as sharp as in class 2". What was the basis of deciding on the sharpness level?
We agree that this point was unclear, and we thank the reviewer for pointing it out. The choice of the number of sub-classes for the open state is a trade-off between the sharpness (ie signal-to-noise ratio) of the resulting image, which is a direct consequence of the number of particles within each sub-class, and the internal variability within each sub-class. Class 4 might appear more “blurry” because it gathers particles displaying a range of angles. When increasing the number of generated classes in the 2D processing, we observe angular variations of the FH2 domains intermediate to the ones displayed in Figure 3. However, because increasing the number of classes results in averaging less particles per class, the generated classes appeared more noisy or “blurry” and not as “sharp”, as mentioned in the manuscript. Hence, we chose the number of displayed classes so that the signal-to-noise would remain satisfactory and sufficient to be able to determine the relative angle between the two FH2 domains. To make things clearer, “do not appear as sharp” was replaced by “displayed a lower signal-to-noise ratio and thus looked noisier”. The expression “sharp” was replaced by “enough contrast”.
The authors showed 30Å structure of FH2 domain encircling actin filaments towards their pointed ends, but said nothing about the kind of decoration it could be, a "daisy-chain" or "concentric circle"? Also, they did not mention anything about the orientation of actin helical twist and specific sites of binding. These information would provide new in-depth understanding of how formins binds while diffusing along the filaments.
The quality is sufficient to distinguish isolated FH2 dimers along the core of actin.
Accordingly, the FH2 dimers we observed along the core of our actin filaments adopt a conformation similar to that observed at the barbed end, as mentioned in the text (‘concentric circle’). This observation differs from the reported for INF2 which accumulated along filaments and may interact in a ‘daisy-chain‘ manner (Gurel et al, 2014 ; Sharma et al, 2014). From our data, we can thus assume that formins interact with F-actin along the core of filaments similarly to the way they do at the barbed ends, and might translocate in a two-step manner alongside the actin filament. As stated in the manuscript, the actin helical twist could not be deciphered. For docking the crystal structures within our EM envelope, we used the formin-actin contacts described previously in Otomo et al.
The author stated - "The leading FH2 domain likely provides a first docking intermediate for actin monomers that would help their orientation relative to the barbed end, resulting in a higher actin monomer on-rate". This statement was made on the basis of observing 79% times FH2 in the open state in their data set. This seems like an overstatement because they don't have any direct structural data to support such claim.
We agree with the reviewer that our statement, taken from the discussion section, is speculative, and we apologize if this was unclear. Our purpose was to propose a plausible mechanism, based on our structural data, since the FH2 domain stands in front of the barbed end in the “open conformation” and since it likely interacts with actin monomers. We have now rephrased our sentence to state more clearly that is a hypothetical mechanism : “We propose that… could provide…”.
In the Discussion they mentioned "the FH2 dimer would then be "lagging" behind the elongating barbed end if actin twisting back to 180{degree sign} occurs before the addition of actin monomer and this explains the diffusing along the actin filaments". Did authors encounter filaments with two formins bounds to them in their negative stain images? What is their view on this? In current data, they showed structure in which only one FH2 dimer is bound to the pointed ends of actin filaments. Have they tried increasing the concentration of formins to obtain structures with more than one formin is bound towards the pointed ends of actin filaments?
Following the recommendations from reviewer 2, we have performed an additional analysis and we now show typical examples of filaments observed with a formin along their core, including cases where two formins are observed on the same filament (Supplementary Figure 12). As we now explain in the discussion section, five different mechanisms (including lagging) can be invoked to explain how a formin can be located along the core of the filament. These five mechanisms can all account for the possibility to have more than one formin on the same filament.
The lagging mechanism, however, is the only one where we would expect that the filaments with a formin along their core are less likely to also have a formin at their barbed end (because the formin at the core spontaneously departed the bare barbed, that was left bare and with a shorter time to load another formin before fixation of the sample). A simple statistical analysis of our data leads to the estimation that 48 ± 7% (n=50) of actin filaments with a formin within their core also display a formin at their barbed ends. This is significantly less than for the global filament population, where 77 ±0.4% (n=10,461) of barbed ends are decorated with formins. This supports the lagging scenario as a likely mechanism putting formins along the core of the filament.
Regarding the specific suggestion to increase the formin concentration: We did screen different formin concentrations, but with higher concentrations the level of noise due to unbound formins was significantly increased in the image background and impeded a proper analysis. This is why we consistently used 100 nM formins.
To increase the density of short filaments for sample preparation, the authors used additional actin binding proteins "shown in supplementary Figure 2.C". There is no supplementary Figure 2.C. Moreover, it would be nice if the concentrations of these proteins are mentioned in the text.
We apologize for this mistake. Supplementary Figure 2.C has now been added and the protein concentrations have been added in the main text.
Minor comments:
- Figure 1 legend needs editing. E is missing in the legend.
Thanks for noticing this. We have added the missing legend for 1.E. 2. There is no supplementary Figure 2.C.
We apologize for this mistake. We have now added supplementary Figure 2.C.
It is recommended that the authors report the number of particle used during 2D and RELION 3D classifications in the figures. This would help in better understanding of the probability of the conformations mentioned in the text.
It was mentioned in the text. We have now made this information clearer to the reader.
Reviewer #2 (Significance (Required)):
This is the first direct study showing the two (open and closed) conformations of mDia1 FH2 domain at the barbed ends of actin filaments using EM and cryoEM. The study supports the proposed molecular mechanism of FH2 processivity at the barbed ends during filaments elongation using stair-stepping model reported earlier (Otomo et al. 2005, Nature). For the first time, FH2 has been shown to fluctuate between various angles with respect to static actin filaments, and on this basis they propose a flapping model (Fig 5). They explained the whole mechanism using structural proof, but the low resolution of data raises a question about their quality sufficiency to propose this mechanism. The overall novelty of this manuscripts is insufficient for the publication in this journal. Audience having understanding of the actin and actin binding proteins will be interested in this study. Additionally, researcher from the field of structural biology (EM and CryoEM) will be interested. I have been working in the field of actin and actin binding proteins for past 4 years. Over 10 years' experience in protein biochemistry, structural biology and molecular biology.
We do not fully understand why, on one hand, reviewer 2 indicates that “for the first time, FH2 has been shown to fluctuate between various angles…” and that “Audience having understanding of the actin and actin binding proteins will be interested in this study. Additionally, researcher from the field of structural biology (EM and CryoEM) will be interested.”. On another hand, reviewer 2 states that “The overall novelty of this manuscripts is insufficient for the publication in this journal.”, which seems contradictory with the above statements and comments.
Regarding novelty, we insist on the fact that we have achieved for the first time the direct observation of FH2 formin domains at a resolution sufficient to discriminate between two distinct models at the barbed ends, as well as to observe the presence of formin mDia1 along the core of actin filaments in conditions where nobody has proposed that this could happen.
In addition, we have not specified any specific journal within the possible ones from “review commons”, up to now.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
In this manuscript, Julien et al. use negative stain electron microscopy and cryo-EM to show two conformations of the FH2 domain for the formin mDia1 bound to the barbed end of an actin filament. These conformations support the "stair-stepping" model of FH2 domain movement with an elongating actin filament, as previously postulated by Otomo et al. (reference 1). The two states observe correspond to the "open" (~79%) and closed (~21%). The authors also show the conformational variability of the open state suggesting flexibility in this state. Finally, the authors observe FH2 domains encircling the actin filament at a distance from the barbed end, and suggest that the FH2 can diffuse from the barbed end down the filament.
Major comments:
1) Novel insights into formin function derived from this structure would raise impact. Issues that could be addressed include the following. Simply adding some lines to the discussion would not really add impact, but additional experimental/modeling work would.
We agree that comparing the binding mode of different formins on actin filaments, testing the impact of profilin, and assaying FH2 domains in the absence of FH1, as proposed below, would provide a broad set of interesting additional data. However, without claiming that our results can be generalized to all formins in all conditions, we believe that our findings are novel and should be of interest to a large community. The proposed additional experiments/modeling represent an impressive amount of work, and will be carried out in future investigations. We answer these comments in more details below.
- Whether this model really holds true for all FH2 domains. Formin FH2 dimerization and processive filament barbed end elongation are widespread features of formins, which have been evidenced for many organisms from metazoan to plants. Since we could dock the FH2 from yeast formin Bni1p to account for mammalian mDia1, we think the FH2 domain conformations may be conserved enough among species to display similar translocation mechanisms at the barbed ends of actin filaments, using a two-state mechanism. We chose to use the crystal structure from Bni1 formin (PDB 1Y64) because this structure was obtained in the presence of an actin filaments and brings some insights about the formin-actin contacts.
In order to convince reviewer 3, we superimposed the existing crystal structure of the FH2 mDia1 domain (PDB: 1V9D) with our model and reconstruction and show (Supplementary Figure 12) that the differences are minor. The mDia1 FH2 domains (atomic structures in red, PDB : 1V9D) are aligned with Bni1p FH2 domains (atomic structures in green and blue, PDB : 1Y64) previously fitted into the electron microscopy envelope of a barbed end capped by a formin in the « open state ». The FH2 domains are well aligned with a slight discrepancy in the knob/actin contact regions (blue arrows). This discrepancy most likely results from the absence of actin partners in the crystals obtained with mDia1 FH2 domains. The Bni1p structure thereby most accurately represents the knob/actin contact region. In addition, the folding of the lasso domain around the post domain is resolved in the Bni1p structure. Note here that the Bni1p lasso domains wrap equally well around the Bni1p post domain and the mDia1 post domain (green arrows).
- Whether the % time spent in the open and closed states might dictate the vastly different elongation rates mediated by different formins. For example, mDia1 is considered one of the 'faster' elongators (equivalent to actin alone in the absence of profilin), while fission yeast Cdc12 essentially caps filaments in the absence of profilin. We have discussed this aspect thoroughly in the discussion section to conclude that:” Our direct assessment of the open state occupancy rate thus provides important information on the molecular nature of the formin-barbed end conformations which could not be directly inferred from kinetic measurements, with or without mechanical tension, so far. Considering a gating factor of 0.9 and considering that formin mDia1 spends 79% of the time in the open state, we can compute that the on-rate for monomers would be slightly higher (14% higher) for an mDia1-bearing barbed end in the open state, than for a bare barbed end.”
We agree that repeating our set of EM experiments and analysis with other formins, like fission yeast Cdc12, would be interesting. However, this would take a long time, and falls out of the scope of our paper.
- Whether the % time spent in the open and closed states varies if filaments are actively elongating in the presence or absence of profilin. We have chosen not to include profilin in our experiments, and to limit the concentration of G-actin, in order to reduce the background in our EM micrographs. Also, a rapid filament elongation would increase the amount of F-actin per barbed end, while a dense population of short filaments is key to obtain accurate data (as we explain in the discussion, paragraph 1, p9).
We speculate that, by providing a link between the FH1 domains and the filament barbed end, profilin might very well alter the percentage of time spent in the open state, and mitigate lagging as mentioned in the discussion section. Properly addressing the impact of profilin with our EM experiments is very challenging, for the reasons we have explained. It would require further investigations, beyond the scope of this study.
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How this model impacts the interactions of formins with other proteins at the barbed end. For example, capping proteins. We did not include capping proteins (or other additional proteins) because we wanted to avoid increasing the number of particles from diverse nature per field of view, as they constitute a background that is detrimental for the analysis of EM micrographs. We would have add to sort out additional populations in the course of image analysis. We thus only mixed actin and formin in our assays.
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Do these results relate to formin function in disease? Because formins regulate actin polymerization, their malfunction is linked to a variety of diseases. We therefore expect our findings to be useful to researchers in the medical field. However, our study remains in the scope of basic research and primarily aims at understanding the mechanisms of formin-assisted actin polymerization.
2) The observation that formin FH2 domains can bind filament sides has been made several times. In particular, a structural model of the FH2 domain of the INF2 formin along the side of an actin filament (Gurel et al 2014, PMID 24915113). This publication also references other papers showing other formins binding to filament sides. There are two points to this comment:
- The model in Gurel et al is that the FH2 domain does not slide down the filament from the barbed end. Rather, the FH2 dimer has an appreciable dissociation rate, enabling it to encircle the filament without having to slide. This FH2 dissociation has been observed for another formin that has been shown to bind filament sides, FMNL1 (called FRL1 in the listed publication), in Harris et al 2006 (PMID 16556604). The authors must explain their reasoning for thinking that mDia1's FH2 can slide down the filament from the barbed end. One possibility is to make observations of this FH2 population in filaments that were not sonicated. What is the average distance of FH2s from the barbed end? We thank the reviewer for pointing our attention to this report from Gurel et al. which we now cite. Following this comment, as well as point 6 of reviewer 2, we now discuss the different mechanisms that could lead to our observation of mDia1 along the core of the filament. We provide a new analysis of our data (discussion section), arguing in favor of the lagging mechanism (i.e. ‘sliding down’ from the barbed end), without excluding the competing scenarios. Briefly, we compute that 48 ± 7% (n=50) of actin filaments with a formin within their core also display a formin at their barbed ends. This is significantly less than for the global filament population, where 77 ±0.4% (n=10,461) of barbed ends are decorated with formins. This supports the lagging scenario, which is the only one where a filament with a formin along its core should be less likely to also have a formin at its barbed end.
The distance of FH2s from the barbed end would provide additional information. However, it is difficult to estimate, since we often to not see the entire filament, and since we do not know which end is the barbed end.
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Interestingly, in some of the works studying formin binding to filament sides, mDia1 was shown to be rather poor in this property. It would be useful to get an idea of what % of the observed FH2s are in the filament core, as opposed to at the barbed end. Along with the additional analysis mentioned in the previous point, we have now estimated that about 8% of actin filaments display a formin within their core. We have added this number in the manuscript (end of the Results section). As a comparison, in our assays, 77% of filament barbed ends bear a formin.
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The authors must reference the past works showing FH2 binding to filament sides, particularly the structural work. At present, no mention of prior work on FH2 side binding is mentioned. As advised, we have now added additional references and more particularly Gurel et al, 2014.
3) My major technical concern in this manuscript is that the authors use the FH1-FH2-DAD domain of mDia1 for the imaging, but use FH2 structure of Bni1p for 3D characterization (Otomo et al.). Even though Bni1p has been used for functional and structural analysis, mDia1 and Bni1p FH2 domains share low sequence homology. In addition, mDia1 only partially complements loss of Bni1 function in vivo (Moseley et al., 2004 PMID 14657240). Can the authors use the partial structural information of the mDia1 FH2 from Shimada et al 2004 (PDB 1V9D, PMID 14992721)? Alternately, the authors could have used FH2 domain of Bni1p for imaging. At the very least, the authors should explain clearly why they used different proteins for imaging and modeling.
As mentioned above (please see our response to point 1.a), we chose to use the crystal structure from formin Bni1 (PDB 1Y64) because this structure was obtained in the presence of an actin monomers, and it thus brings some insights about the formin-actin contacts. The existing structures obtained from formin mDia1 does not include actin (full length by EM: Maiti et al, 2012; crystal structure of subdomains (without FH1): Otomo et al., 2010 PLoS one). It thus seems relevant, in the context of our investigations, to use a structure where formin-actin contacts could be at least partially inferred.
Further, we superimposed the existing crystal structure of the FH2 mDia1 domain (PDB: 1V9D) with our model and reconstruction and show that the differences are minor (please see the figure in our response to point 1.a, above).
4) The open and closed states are observed from negative staining data. However, the authors can only find one of the states (open) by cryo-EM, which decreases the confidence level of the paper's conclusions. It would be useful for the authors do a little more to try to find the closed conformation by cryo-EM.
Using Cryo-EM we can already recover the most abundant open conformation.
Unfortunately, as pointed out here, the number of particles obtained was too low to enable high resolution and reveal the two observed conformations. Indeed, considering a density of ~ 5 barbed ends par micrograph, the collection of tens of thousands of images would have been necessary, which was not realistic regarding the access we have to latest generation microscopes.
5) It is unclear whether there are additional effects of using FH1-FH2-DAD protein (not FH2 only) for the imaging, as it shows long protrusion at the tip of actin barbed end. To avoid those concerns the authors could use only FH2 domain of mDia1. Also the authors have to note that they used Bni1p structure because there are no published structures of mDia1 so far.
We had indeed tried to use a construct deprived of the flexible FH1 domain but the lower purity of this construct and the presence of aggregates led to the collection of lower quality EM micrographs. As profilin was not included in our assay, FH1 domains were not involved in actin polymerization at the barbed end and thus remain very flexible and unstructured. Consistently, we did not detect any additional electronic density that could result from the FH1 domains.
We indeed point out (p5) that “We used the crystal structure from yeast Bni1p FH2 domains in interactions with an actin filament, rather than the existing one from mammalian mDia1 formin FH2 dimer in isolation (PDB 1V9D), because actin-formin contacts are described in the Bni1p structure.” Minor comments:
1) Figure 1: It would be interesting if imaging is provided for mDia1 bound to filaments which it has nucleated. Would it be possible that binding to pre-formed filaments is different to that for mDia1-nucleated filaments?
This is a good suggestion for further investigations but it extends beyond the scope of this study: as we explain, our attempts to nucleate filaments from mDia1 lead to lower quality micrographs, and the sonication of preformed filaments was our best option. However, we do not expect the translocation mechanism of FH2 to differ, as a function of the nucleation history of the filament, since the formin interacts with a filament whose elongation it has assisted over several subunits.
2) Supplementary figure 2: Numbers of things in the S2 is unclear and poorly described in both results and methods. In particular, figure S2A, the definitions of the black and gray lines (steady state actin) is not clear. Are they containing 5% pyrene actin? Is that actin in polymerization buffer or in monomer-actin buffer? Is that actin incubated with actin polymerization buffer for a certain time before measurement of fluorescent intensity? In figure S2B, how the authors calculate the monomer actin concentration? The authors should provide the information in either results or methods part.
We apologize for the lack of information. Since this is a standard assay, we have now added more details in the Methods section (rather than in the Results section).
All curves shown in figure S2 were obtained with 5% pyrene actin. The gray curve shows the pyrene fluorescence intensity baseline from 1 µM G-actin monomers, obtained in G-buffer. The black curve is the fluorescence intensity at steady-state of 1 µM actin in polymerizing conditions, (after 1 hour of incubation at room temperature, at 5 µM, the sample was diluted without sonication and left for another hour before measuring the fluorescence intensity).
The monomeric actin concentrations shown in figure S2B are derived from the intensity level of pyrene at any time point during the experiment, using the simple equations we now present in the Methods section.
3) Supplementary figure 2 C: The figure and legend are missing in the manuscript. Furthermore, the authors describe that they used Gc-globulin to sequester monomeric actin in solution. Is gc-globulin widely used for actin monomer sequestration?
Thank you for noticing the missing panel which is now back in place. Indeed, Gc globulin is known to sequester G-actin (Van Baelen, H., R. Bouillon, and P. DeMoor. 1980. “Vitamin D binding protein (Gc-globulin) binds actin”. J. Biol. Chem. 255:2270-2272). This is why we have attempted to use it. We could see a slight effect but we did not want to increase the noise within our images with additional proteins that would have made the analysis more complicated.
CROSS-CONSULTATION COMMENTS Reviewer #1 mentions that the authors identify formin densities bound along the actin filament for the first time. I agree that the imaging of the mDia1 along the actin filament using electron microscopy is novel, but the concept of formin binding has already been found and studied well with other formins (PMID 16556604, PMID 24915113) and even mDia1 has poor binding activity compared to other formins. It was really nice of the authors to show the mDia1 side filament binding, but I don't think it is a striking finding.
I have no comment for Reviewer #2.
Reviewer #3 (Significance (Required)):
If the EM refinements and 3D rendering techniques are conducted rigorously (which this reviewer is unable to judge), the data support an existing theory of how FH2 domains interact with the actin barbed end. Overall, the data will be of interest in formin field. However, as written the paper confirms an existing model, and does not represent new insight. Impact would be raised by providing insights from these findings that impact formin function or disease.
We have answered this concern above. The existing models were speculative and not based on direct observations. They relied on data obtained in non-physiological conditions.
Here, we directly observe two distinct conformations in our structural data, and clearly validate one model over the other. This provides a major advancement in our understanding of formin interaction with actin filaments. In addition, we uncovered an unexpected behavior of formin mDia1, which can readily be found along the core of the filament without the aid of additional proteins, and we propose a mechanism based on our data to account for this observation.
Another main point is that the observation of FH2 domains bound along an actin filament, while interesting, is not novel. Others have found this for other formins, but those papers are not referenced here.
The direct binding of formins to the sides of actin filaments is thought to be specific to some particular formins (we now cite additional references in our manuscript, to discuss this point). Formin mDia1, which is a ubiquitous and widely studied mammalian formin (perhaps the most studied), has only been described to diffuse along actin filaments when a capping protein dislodges it from the barbed end (Bombardier et al. Nat Com 2015). Here, we show that formin mDia1 can be found encircling the core of actin filaments, in the absence of any capping protein. This behavior is novel and unexpected. It should open new avenues for research on formin mDia1, as well as on other formins.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, Julien et al. use negative stain electron microscopy and cryo-EM to show two conformations of the FH2 domain for the formin mDia1 bound to the barbed end of an actin filament. These conformations support the "stair-stepping" model of FH2 domain movement with an elongating actin filament, as previously postulated by Otomo et al. (reference 1). The two states observe correspond to the "open" (~79%) and closed (~21%). The authors also show the conformational variability of the open state suggesting flexibility in this state. Finally, the authors observe FH2 domains encircling the actin filament at a distance from the barbed end, and suggest that the FH2 can diffuse from the barbed end down the filament.
Major comments:
- Novel insights into formin function derived from this structure would raise impact. Issues that could be addressed include the following. Simply adding some lines to the discussion would not really add impact, but additional experimental/modeling work would.
- a. Whether this model really holds true for all FH2 domains.
- b. Whether the % time spent in the open and closed states might dictate the vastly different elongation rates mediated by different formins. For example, mDia1 is considered one of the 'faster' elongators (equivalent to actin alone in the absence of profilin), while fission yeast Cdc12 essentially caps filaments in the absence of profilin.
- c. Whether the % time spent in the open and closed states varies if filaments are actively elongating in the presence or absence of profilin.
- d. How this model impacts the interactions of formins with other proteins at the barbed end. For example, capping proteins.
- e. Do these results relate to formin function in disease?
- The observation that formin FH2 domains can bind filament sides has been made several times. In particular, a structural model of the FH2 domain of the INF2 formin along the side of an actin filament (Gurel et al 2014, PMID 24915113). This publication also references other papers showing other formins binding to filament sides. There are two points to this comment:
- a. The model in Gurel et al is that the FH2 domain does not slide down the filament from the barbed end. Rather, the FH2 dimer has an appreciable dissociation rate, enabling it to encircle the filament without having to slide. This FH2 dissociation has been observed for another formin that has been shown to bind filament sides, FMNL1 (called FRL1 in the listed publication), in Harris et al 2006 (PMID 16556604). The authors must explain their reasoning for thinking that mDia1's FH2 can slide down the filament from the barbed end. One possibility is to make observations of this FH2 population in filaments that were not sonicated. What is the average distance of FH2s from the barbed end?
- b. Interestingly, in some of the works studying formin binding to filament sides, mDia1 was shown to be rather poor in this property. It would be useful to get an idea of what % of the observed FH2s are in the filament core, as opposed to at the barbed end.
- c. The authors must reference the past works showing FH2 binding to filament sides, particularly the structural work. At present, no mention of prior work on FH2 side binding is mentioned.
- My major technical concern in this manuscript is that the authors use the FH1-FH2-DAD domain of mDia1 for the imaging, but use FH2 structure of Bni1p for 3D characterization (Otomo et al.). Even though Bni1p has been used for functional and structural analysis, mDia1 and Bni1p FH2 domains share low sequence homology. In addition, mDia1 only partially complements loss of Bni1 function in vivo (Moseley et al., 2004 PMID 14657240). Can the authors use the partial structural information of the mDia1 FH2 from Shimada et al 2004 (PDB 1V9D, PMID 14992721)? Alternately, the authors could have used FH2 domain of Bni1p for imaging. At the very least, the authors should explain clearly why they used different proteins for imaging and modeling.
- The open and closed states are observed from negative staining data. However, the authors can only find one of the states (open) by cryo-EM, which decreases the confidence level of the paper's conclusions. It would be useful for the authors do a little more to try to find the closed conformation by cryo-EM.
- It is unclear whether there are additional effects of using FH1-FH2-DAD protein (not FH2 only) for the imaging, as it shows long protrusion at the tip of actin barbed end. To avoid those concerns the authors could use only FH2 domain of mDia1. Also the authors have to note that they used Bni1p structure because there are no published structures of mDia1 so far.
Minor comments:
- Figure 1: It would be interesting if imaging is provided for mDia1 bound to filaments which it has nucleated. Would it be possible that binding to pre-formed filaments is different to that for mDia1-nucleated filaments?
- Supplementary figure 2: Numbers of things in the S2 is unclear and poorly described in both results and methods. In particular, figure S2A, the definitions of the black and gray lines (steady state actin) is not clear. Are they containing 5% pyrene actin? Is that actin in polymerization buffer or in monomer-actin buffer? Is that actin incubated with actin polymerization buffer for a certain time before measurement of fluorescent intensity? In figure S2B, how the authors calculate the monomer actin concentration? The authors should provide the information in either results or methods part.
- Supplementary figure 2 C: The figure and legend are missing in the manuscript. Furthermore, the authors describe that they used Gc-globulin to sequester monomeric actin in solution. Is gc-globulin widely used for actin monomer sequestration?
Referees cross-commenting
Reviewer #1 mentions that the authors identify formin densities bound along the actin filament for the first time. I agree that the imaging of the mDia1 along the actin filament using electron microscopy is novel, but the concept of formin binding has already been found and studied well with other formins (PMID 16556604, PMID 24915113) and even mDia1 has poor binding activity compared to other formins. It was really nice of the authors to show the mDia1 side filament binding, but I don't think it is a striking finding.
I have no comment for Reviewer #2.
Significance
If the EM refinements and 3D rendering techniques are conducted rigorously (which this reviewer is unable to judge), the data support an existing theory of how FH2 domains interact with the actin barbed end. Overall, the data will be of interest in formin field. However, as written the paper confirms an existing model, and does not represent new insight. Impact would be raised by providing insights from these findings that impact formin function or disease.
Another main point is that the observation of FH2 domains bound along an actin filament, while interesting, is not novel. Others have found this for other formins, but those papers are not referenced here.
- Novel insights into formin function derived from this structure would raise impact. Issues that could be addressed include the following. Simply adding some lines to the discussion would not really add impact, but additional experimental/modeling work would.
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Referee #2
Evidence, reproducibility and clarity
Maufront et al. have used EM to study the conformation of mDia1 at the barbed end and the core of actin filaments to explain the molecular mechanism of the FH2 dimer processivity at these sites. Based on modelled structural data they tried to describe how the conformational changes in FH2 dimer lead to its partial dissociation, and then association with filaments during the process of translocation coupled to subunit addition at actin filaments barbed ends. This supports a previous study (Otomo et al. 2005, Nature), in which using X-ray crystallography structural data were used to propose a stair-stepping model for Bni1p translocation at the barbed ends during actin polymerization. The model for mDia1 binding to core filaments is also given. Moreover, using EM structure and the previously reported structures of actin (PDB: 5OOE), and actin with formin FH2 dimer (PDB: 1Y64), authors explained the dynamic nature of FH2 dimer at barbed ends of the filaments using the flapping model. But due to the low resolution of their structures ~ 26-29A0, the finer details of actin and the FH2 dimer structure at barbed ends could not be resolved, leaving open questions about the orientation of actin helical twist at this end during elongation.
The authors tried several conditions to get high density barbed-end filaments, but that did not collect adequate number of particles, resulting in low number of particles selected for structure modelling purposes. However, to attain more physiologically relevant structure they used cryo-EM, but were successful in capturing only the open conformation structure of FH2 dimer (at low resolution). Thus, due to low resolution of structures the key findings have not added much to what we already know about the mechanism of FH2 dimer translocation during actin polymerization, except that their studies support the stair-stepping model (Otomo et al. 2005, Nature) and not that of "stepping second" model ( Paul and Pollard. 2008, Curr. Bio.). Thus, this manuscript does not merit publication in this journal.
Major comments:
- Present study does not provide any new insight about the conformation of the actin dimer at the barbed ends of actin filaments when FH2 domains of formin are bound. This study appears to be more like an extension of previous research (Otomo et al. 2005, Nature), in which the authors used X-ray crystallography data to propose a model for actin filaments elongation by formin bound at the barbed ends.
- The low resolution of structures is a major concern.
- Given the low resolution of data, how can the authors decide on the number (4) of classes of FH2 domain (in open state) and present them as "continuum of conformations". They stated "details featured in class 4 do not appear as sharp as in class 2". What was the basis of deciding on the sharpness level?
- The authors showed 30Å structure of FH2 domain encircling actin filaments towards their pointed ends, but said nothing about the kind of decoration it could be, a "daisy-chain" or "concentric circle"? Also, they did not mention anything about the orientation of actin helical twist and specific sites of binding. These information would provide new in-depth understanding of how formins binds while diffusing along the filaments.
- The author stated - "The leading FH2 domain likely provides a first docking intermediate for actin monomers that would help their orientation relative to the barbed end, resulting in a higher actin monomer on-rate". This statement was made on the basis of observing 79% times FH2 in the open state in their data set. This seems like an overstatement because they don't have any direct structural data to support such claim.
- In the Discussion they mentioned "the FH2 dimer would then be "lagging" behind the elongating barbed end if actin twisting back to 180{degree sign} occurs before the addition of actin monomer and this explains the diffusing along the actin filaments". Did authors encounter filaments with two formins bounds to them in their negative stain images? What is their view on this? In current data, they showed structure in which only one FH2 dimer is bound to the pointed ends of actin filaments. Have they tried increasing the concentration of formins to obtain structures with more than one formin is bound towards the pointed ends of actin filaments?
- To increase the density of short filaments for sample preparation, the authors used additional actin binding proteins "shown in supplementary Figure 2.C". There is no supplementary Figure 2.C. Moreover, it would be nice if the concentrations of these proteins are mentioned in the text.
Minor comments:
- Figure 1 legend needs editing. E is missing in the legend.
- There is no supplementary Figure 2.C.
- It is recommended that the authors report the number of particle used during 2D and RELION 3D classifications in the figures. This would help in better understanding of the probability of the conformations mentioned in the text.
Significance
This is the first direct study showing the two (open and closed) conformations of mDia1 FH2 domain at the barbed ends of actin filaments using EM and cryoEM. The study supports the proposed molecular mechanism of FH2 processivity at the barbed ends during filaments elongation using stair-stepping model reported earlier (Otomo et al. 2005, Nature). For the first time, FH2 has been shown to fluctuate between various angles with respect to static actin filaments, and on this basis they propose a flapping model (Fig 5). They explained the whole mechanism using structural proof, but the low resolution of data raises a question about their quality sufficiency to propose this mechanism. The overall novelty of this manuscripts is insufficient for the publication in this journal.
Audience having understanding of the actin and actin binding proteins will be interested in this study. Additionally, researcher from the field of structural biology (EM and CryoEM) will be interested. I have been working in the field of actin and actin binding proteins for past 4 years. Over 10 years' experience in protein biochemistry, structural biology and molecular biology.
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Referee #1
Evidence, reproducibility and clarity
This study presents a first structural insight on formin mDia bound to actin filaments in physiological conditions. Based mainly negative stain EM, the authors use 2D and 3D class averaging to describe two main configuration of the formin at the filament barbed end. The two configurations support the previously proposed stair-stepping model, which was based on crystal structures, with an open state where the formin binds two actin monomers and a closed state where three monomers are bound. Because the majority of the structures fall in the first, open state, this supports the existence of this intermediate. The authors also show that the orientation of the free FH2 in this open state is somewhat flexible, as several sub-classes with different angles can be distinguished. Finally, they identify, for the first time, formin densities bound along the length of the filament.
The data is well presented and I don't have any major issue. The only point is that the information that all the initial structural data comes from negative stain EM comes should be put upfront. One gets the feeling that cryoEM is used throughout until one reads the section on cryoEM. Given that the methodology is now also established for cryoEM, it is regrettable that data was not collected with a 300kV microscope, which may have revealed more details of the conformations, but I understand microscope time is hard to come by, and the authors did a remarkable job from negative-stain EM.
The finding of formin densities binding along the length of the actin filament is very interesting. Besides the previous cited finding, it also reminds of the observations made in yeast where Bni1 (in S. cerevisiae; PMID 17344480) and For3 (in S. pombe; PMID 16782006) where shown to exhibit retrograde movement with polymerizing actin cables in vivo. This would be interesting to consider in the discussion.
Significance
This study extends our understanding of the mechanism of formin-mediated actin assembly, by providing a first structural observation in physiological conditions. While confirmatory of previously proposed model, but also excludes an alternative model, and offers novel observations of flexibility and binding along the actin filament length. It will be of great interest to researchers on the actin cytoskeleton.
My expertise is in the actin cytoskeleton and formins, but I am no expert in EM structural analysis.
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Reply to the reviewers
In this section we list all the comments done by the three referees and our corresponding action.
Regarding Reviewer #1:
- On "mechanical control": The authors show changes in circadian power fraction with changes in YAP and with cytoskeletal inhibitors, but there are no properly-controlled experiments that directly perturb mechanics. The authors show a correlation between YAP nuclear/cytoplasmic ratio and circadian power, but YAP N/C alone is not a readout of mechanotrasndcution, per se. The authors have shown two different experiments where cells are cultured on a stiff (30kPa) substrate and soft substrate (300Pa), but they do not shown a direct comparison of YAP nuclear localization and circadian power under these two conditions in the same experiment. Direct, controlled perturbation of mechanical cues is necessary to support the title's use of the phrase "mechanical control."
We agree with the referee that further mechanical perturbations could strengthen our conclusions. In our original manuscript we directly controlled the mechanical environment by culturing cells on substrates of 300Pa and 30kPa in stiffness. These differences in stiffness were not sufficient to drive changes in circadian power fraction and YAP localisation, as depicted in Fig. 3C (we note that the direct comparison requested by the referee is shown in that figure). We hypothesise that this negative result is due to a very low “rigidity threshold” or to secretion of extracellular matrix that stiffens the initially soft substrate. In any case, we plan to strengthen the “mechanical control” message of our paper with one or more of the below experiments:
A) We will measure circadian power fraction and YAP localisation in even extremer stiffness/adhesion conditions, using 300 Pa and 30 kPa polyacrylamide gels with a different fibronectin coating protocol, as described in Elósegui-Artola et al., 2017. This allows a much finer control of the concentration of fibronectin coated, so we can reach low enough levels to compromise the cell adhesion to the substrate and cross down the threshold that would lead to cytosolic localisation of YAP. We will perform this experiment in presence of the FUD peptide, which inhibits matrix deposition (Tomasini-Johansson et al., 2001; this peptide has already been tested in our lab).
B) We will use the approach described in Fig. 2E to compare the circadian power fraction in cells spread in stadium-shaped islands of 2400 um2 and 1200 um2. Oakes et al., 2014 already showed that traction forces exerted by 3T3 fibroblasts depend on the size of the spread area of the cells, so we expect differences in mechanotransduction that should affect YAP localisation and, if our hypothesis is correct, the RevVNP circadian oscillations.
C) We will abolish the physical connection between the actin cytoskeleton and the nucleus by disrupting the LINC complex via the overexpression of a dominant negative (DN) nesprin-1 KASH domain (Lombardi et al., 2011). The plasmid designed for the inducible overexpression of the DN KASH domain, originally tested in NIH3T3 cells (Mayer et al., 2019), is available in our lab and has been used to prove that uncoupling cytoskeleton and nucleus leads to nuclear YAP decrease in single cells (Kechagia at al., 2022). We will aim to increase the circadian power fraction in low density cells upon the overexpression of the DN KASH domain.
Elosegui-Artola A, Andreu I, Beedle AEM, Lezamiz A, Uroz M, Kosmalska AJ, Oria R, Kechagia JZ, Rico-Lastres P, Le Roux AL, et al (2017) Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores. Cell 171: 1397-1410.e14
Kechagia Z, Sáez P, Gómez-González M, Zamarbide M, Andreu I, Koorman T, Beedle AEM, Derksen PWB, Trepat X, Arroyo M, et al (2022) The laminin-keratin link shields the nucleus from mechanical deformation and signalling Cell Biology
Lombardi ML, Jaalouk DE, Shanahan CM, Burke B, Roux KJ & Lammerding J (2011) The Interaction between Nesprins and Sun Proteins at the Nuclear Envelope Is Critical for Force Transmission between the Nucleus and Cytoskeleton*. Journal of Biological Chemistry 286: 26743–26753
Mayer CR, Arsenovic PT, Bathula K, Denis KB & Conway DE (2019) Characterization of 3D Printed Stretching Devices for Imaging Force Transmission in Live-Cells. Cel Mol Bioeng 12: 289–300
Oakes PW, Banerjee S, Marchetti MC & Gardel ML (2014) Geometry regulates traction stresses in adherent cells. Biophysical Journal 107: 825–833
Tomasini-Johansson BR, Kaufman NR, Ensenberger MG, Ozeri V, Hanski E & Mosher DF (2001) A 49-Residue Peptide from Adhesin F1 of Streptococcus pyogenes Inhibits Fibronectin Matrix Assembly*. Journal of Biological Chemistry 276: 23430–23439
2. On "via YAP/TAZ": In addition to above, it is necessary to show that the changes in Circadian power fraction induced by mechanical cues in fact require YAP/TAZ signaling. Thus, an experiment comparing soft (300Pa) substrate with Stiff (30kPa) substrate in the presence or absence of YAP/TAZ is necessary to state that YAP and TAZ are the mechanistic mediators of mechanical cues on the clock.
We are currently generating via CRISPR-KO and shRNA silencing a YAP1/TAZ double mutant. We plan to use this cell line in those conditions where YAP is prominently nuclear (low density in stiff substrates) with the purpose of rescuing the RevVNP circadian power fraction.
- While the TEAD-binding domain mutant experiment is elegant, to claim that TEAD is the transcriptional mediator, it must be demonstrated that this mutant indeed fails to induce TEAD-mediated transcription. This could be simply executed by demonstrating that the CCD mutant expresses reduced CTGF and Cyr61 (for example), compared to the 5SA, under these conditions. Further, endogenous YAP is still active and available to bind to TEAD in this system, which should be discussed.
We plan to carry out quantitative real-time PCR of CTGF and Cyr61 in all the YAP mutants and the control. Regarding the presence of endogenous YAP, we will clarify in the text that a) the overexpression of the different YAP mutants was done in high-density conditions, where endogenous YAP is significantly less localised in the nucleus, and that b) the levels of the exogenous YAP are much higher (we already have western blots showing this).
- In Figure 3a: The cell perimeter needs to be shown either by actin staining or by brightfield images. The manually marking of cell boundaries is insufficient, specifically because the drugs used in this experiment affect the cytoskeleton. It would be very helpful to see this via actin staining or in the least with brightfield images.
The cell perimeter was drawn based on the cytosolic YAP immunostaining, whose levels are high enough to infer the cell shape (higher resolution images can be attached if necessary). As stated in the manuscript, the YAP nuclear-to-cytosolic ratio is calculated using two adjacent areas of identical size, one inside the nucleus and the other one just outside (see Materials and Methods/Immunostainings), so the exact cell shape is irrelevant for this particular quantification.
Regarding Reviewer #2:
Effects on the circadian clock
- The authors use the fluorescent reporter created by Nagoshi from sections of the Rev-erbα gene. This reporter is widely used to estimate relative circadian timing in individual cells but it does not provide direct information on the circadian clock activity. In other words, while Reverb rhythmic expression is driven by the clock, it is not known whether less-rhythmic or non-rhythmic expression or change in expression level of Rev-erbα is affecting the core clock. For example, it has been shown that Rev-erbα knock-down cells are rhythmic as long as Rev-erb-beta is present. Thus, one major shortcoming of the current version of the manuscript is the missing dissection between Rev-erbα rhythmicity/expression and the circadian clock. More concretely, it remains unclear whether the change in Rev-erbα expression is a direct effect or caused by a defect clock. Since the authors presume a direct effect of YAP/TAP on Rev-erb expression, the former is likely. If that is the case, the data could be interpreted as that (missing) mechanic stimuli can lead to nuclear YAP/TAZ, which rises the level of Rev-erbα (and maybe interfere with its rhythmic accumulation). Beyond Rev-erbα expression, there may or may not be an effect on the circadian clock (core clock, CCGs). With the current version we do not know since the authors do not look beyond Rev-erbα expression. Thus, the claims on circadian clock or circadian rhythms in their cells is not studied in this version of the manuscript. The current version is still very interesting and provides insights into the Rev-erbα modulation, but additional work would be needed to show links with the core clock machinery. For this the authors could show influence (or at least correlation) of the YAP/TAZ/REVERBA phenotype on the oscillations of core clock genes or clock-controlled genes. Either through the use of alternative (ideally constitutive) reporters (e.g. PER2, BMAL1, fluorescent or LUC), or/and by analyzing RNA/Protein of core clock genes or output genes. This would not be necessary for all experiments, but at least for some were its possible (e.g. experiments with drugs perturbations). Otherwise, any claim like "YAP/TAZ perturbs the circadian clock ..." or "the circadian clock deregulation in nuclear YAP-enriched cells" is potentially flawed and has to be removed/reformulated.
We agree with the reviewer. In order to understand if the core clock is affected, beyond REV-ERBA, by YAP/TAZ expression and localisation, we plan to perform the two experimental approaches explained below. For both of them we will use high-density cells with and without YAP-5SA overexpression since the other conditions (drugs, micropatterned cells, low density) may not render enough cells for analytical approaches that are not based on fluorescent microscopy (real-time qPCR or luminescence recordings). Also, the potential results obtained with YAP-5SA overexpression will be more informative regarding causality YAP-circadian clock than those using the other conditions described in the manuscript.
- We will use NIH3T3 bmal1::luc cells (already generated in our lab with the pABpuro-BluF plasmid; https://www.addgene.org/46824/) and an adapted microscopy-based system to track bioluminescence. We will need to give our cells a synchronisation shock since the single-cell signal with this reporter is too low and noisy to perform single-cell tracking.
- We will check during 48 hours, every 4 hours, the mRNA levels of Bmal1, Clock, Cry1, Per2, Yap1 and Rev-erbα via quantitative real-time PCR. As in A), we will need to synchronise our cells prior RNA collection. In case the expression of the other components of the clock are not affected by YAP-5SA overexpression, we will modify the message of our manuscript to emphasize the role of REV-ERBA. As the referee mentions (and we thank them for that comment), finding that the modulation of Rev-erbα is mechano-sensitive and dependent on YAP/TAZ signalling would be still very relevant, given the role of this factor in metabolism, inflammation, mitochondrial activity, or Alzheimer’s disease, as discussed in lines 231-235 in the manuscript.
- The authors aim to discard the possibility of paracrine signals by showing no increase in circadian power fraction of cells growing in low density with conditioned medium (Figure 2D). A paracrine signal coming from an oscillatory system is likely to oscillate and in that case, I do not see how growing cells in constant conditional medium can discard the effects of an oscillatory paracrine signal. I believe the elegant experiment shown in Figure 2E more precisely address this issue.
The reviewer is right in the sense that paracrine coupling of circadian oscillators would require a circadian paracrine signal, like shown in Finger et al., 2021, and that we provide sufficient experimental evidence of a mechanics- rather than paracrine-driven control of the RevVNP circadian oscillations. Specifically, by using micropatterning (Fig. 2E) and gap closure (Fig. 2A) we show that cells under the same paracrine medium are able to display acute differences in RevVNP expression. The experiment with conditioned medium, which is a traditional technique used in some papers in the field like in Noguchi et al., 2013, was performed to rule out the possibility that secreted factors, even if not circadian, could ultimately impact the low-density cells’ circadian clock. We will rephrase the manuscript to stress out this reasoning.
Finger AM, Jäschke S, del Olmo M, Hurwitz R, Granada AE, Herzel H & Kramer A (2021) Intercellular coupling between peripheral circadian oscillators by TGF-β signaling. Science Advances 7
Noguchi T, Wang LL & Welsh DK (2013) Fibroblast PER2 circadian rhythmicity depends on cell density. Journal of Biological Rhythms 28: 183–192
Data analysis methodology:
- Single-cell circadian recordings like the ones analyzed here are characterized by noisy amplitude and non-sinusoidal waveforms with fluctuating period (Bieler et al., 2014; Feillet et al., 2014). The authors interpolate, smooth, detrend and normalize their data; operations that are known to introduce spectral artifacts that can mislead the interpretation of the power spectrum. Moreover, the time-series pre-processing operations described by the authors in the methods sections is incomplete and the authors should more explicitly describe all their operations with exact methods applied, filter parameters and time-windows sizes (if applicable). To validate their pre-processing steps the authors could provide their time-series analysis pipeline code and/or provide a few examples of raw versus pre-processed data together with their respective spectrums before and after pre-processing. In addition, the authors could provide their raw trace signal data together with the corresponding post-processed signal data as plain text files.
In our response to the reviewers, we will address this point exactly as requested by the reviewer. We will rewrite our methods section to explain better our analysis pipeline, clarifying that we do not apply detrending, that we resort rarely to interpolation of missing points, and stating the specifics of the standard low-pass filter we apply. We will then strengthen Supplementary Figure 1 with more examples of raw-data and processed data, and will provide raw trace signal data and the corresponding processed data to illustrate our approach.
- The authors rely on Fourier analysis and a reasonable self-made definition of circadian strength named as "circadian power fraction". Using a stationary-based method for noisy non-stationary data can lead to inaccurate spectrum power estimations. As the current version of the manuscript does not provide any alternative/complementary analysis method nor we have any available raw signal data it is unclear if their analysis appropriately represents the circadian power. The authors could consider implementing complementary data-analysis strategies to validate their conclusions. Fortunately, there are multiple suitable data analysis strategies already available that are exactly designed for this kind of data (eg. (Price et al., 2008; Leise et al., 2012; Leise, 2013; Bieler et al., 2014; Mönke et al., 2020). This time-series analysis methods is a crucial step as all main results on this manuscript rely on the authors self-made definition of circadian power. This is particularly important as there is no standardized method in the circadian field to estimate circadian rhythmicity and/or circadian power of single-cell traces.
We will take this point into consideration by running a complementary analysis of our data with one of the methods recommended by the reviewer. Our choice is pyBOAT, as presented in Mönke et al. (2020), because on first inspection its implementation of the wavelet method appears to be the most suitable for our dataset type. If we find that our time-series are too short for these methods we will use the RAIN algorithm (Thaben and Westermark, 2014) instead.
Mönke G, Sorgenfrei FA, Schmal C & Granada AE (2020) Optimal time frequency analysis for biological data - pyBOAT Systems Biology
Thaben PF & Westermark PO (2014) Detecting Rhythms in Time Series with RAIN. J Biol Rhythms 29: 391–400
- The authors mainly show circadian power fraction and analyze rhythmicity scores/powers. Is there the a chance that a rise in the basal expression level of Rev-erbα is reducing the rhythmicity score? Or to phrase it otherwise, the absolute amplitude may remain the same, but the relative amplitude may be reduced? Would that affect the FT analysis power scored? To clarify this the authors could provide an analysis of the relative amplitude in addition to the circadian intensity (as in Fig.1C).
Our analysis pipeline subtracts the mean signal from each cell’s intensity-time trace, and then divides each trace by its standard deviation. This procedure eliminates any bias due to basal expression of Rev-erbα. We will address this point by clarifying the methods section and providing examples in Supplementary Figure 1 of raw data with high-basal levels and low basal levels, showing their pre- and post-processed spectra.
Minor points by text-line:
YAP and TAZ should be introduced to the reader during introduction. by set a of proteins. Here the authors probably meant that cells were not reset nor entrained during the experiment. "..expression depends on..". This is a correlation, not proof of causation is shown until this point. This is an overstatement. Using the term "provoked" suggests a causal relationship not shown. Similarly last sentence "This result established.... is caused..". Again, this is an overstatement as only correlation is shown. According to their description the authors are not using any image-preprocessing steps, eg background subtraction or other filters. Is this correct? It is not clear what image metric for the single-cell signals are the authors using, eg. integrated nuclear intensity or mean/median nuclear intensity. I am not familiar with TrackMate but it might be possible to export and share with the readers the image-analysis pipeline used which would clarify any questions about image processing and signal extraction.
We thank the reviewer for pointing out all these minor points. We will address each one of them to make the paper clearer.
Regarding Reviewer #3:
The authors state in lines 163-165: 'This striking anticorrelation reveals that the robustness of the Rev-erbα circadian expression depends on the nucleocytoplasmic transport of YAP and its mechanosensitive regulation'. Although interesting, the data in figure 3 to which this statement refers is, as the authors identify, correlative, rather than causative. I would strongly suggest altering this statement to better reflect the data.
We will modify the text to eliminate this overstatement.
It looks to me as though all experiments were carried out in the same clonal reporter 3T3 line. To avoid possible issues with founder effects, I would ask that the authors repeat the initial experiment in figure 1B, and the associated analysis as in 1C-E with a different clonal 3T3 line. Hopefully this will not be very arduous, as the methods suggest that multiple clonal 3T3 reporter lines were made initially. With time to defrost, plate, record and analyse the data, I would hope that this would not take more than six weeks maximum.
We will perform the experiments regarding the cell density effect on the RevVNP oscillations (Fig. 1) in another clonal 3T3 line as the reviewer suggests. We have already initiated the experimental repeats with the alternative clone.
I would note that the custom software used for analysis does not appear to be generally available. I would assume that the authors would make this available upon request.
We will extend the explanation of our method as suggested by Reviewer #2 and make the code available to the community.
Experiments appear to have been adequately replicated in terms of n. However, the robustness of these findings would be supported though use of a different clonal reporter line, as discussed above.
We will solve this problem as stated above.
Statistical analysis is generally appropriate. I would suggest including statistical analysis in figures 3B and S4B to demonstrate that the pharmacological treatments are indeed having a statistically significant effect on the MAL and YAP nuclear/cytoplasmic ratio.
We will perform the corresponding statistical analysis on those data.
For Figure 4, it is not stated which statistical tests have been used, with only P values given in table S1. Please state which test has been used.
We will specify the statistical test used in the figure legend.
Furthermore, it would be valuable to see if it is possible to perform statistical analysis looking at the populations should in Figure 4A, to either support or refute the statement made in Line 189-90 that 'we overexpressed 5SA-S94A-YAP, a mutant version of YAP unable to interact with TEAD and observed that the cells recovered, to a large extent, both the RevVNP circadian power fraction and the REV-ERBα basal levels displayed by the wild-type high-density population'
The p-values corresponding to that dataset are represented in Table S1, but we will move them to the figure legend so the extent of the differences between the YAP mutants and the control becomes more noticeable. This applies too to the next comment of the reviewer.
Additionally, it is a little unclear to me why exact p values are reported in table S1. It seems that they might be better placed in the relevant figure legend.
Minor comments:
Although the authors took good care to try to ensure that there was minimal phase synchrony between cells, it would be good to see some analysis to confirm that these efforts were successful. This is of particular concern, given that many things that commonly happen during cell handling, such as temperature change and media change, even with conditioned media, can act to synchronise cells. Hopefully, this information should be available from your existing analysis.
All our experiments, except for the gap closure ones (which imply an unavoidable medium shock after the removal of the gasket where the cells are cultured to achieve high density) are carried out in a similar way (see Materials and Methods). This approach does not involve the typical shock of serum, dexamethasone, or other hormones, because we want to avoid biochemical signalling that could mask the “pure” effect of mechanics on the pathways that affect the circadian clock. In any case, a certain level of synchrony should not affect the analysis we perform, since this is single cell-based and does not consider the phase but the strength of its circadian frequency. But as requested by the reviewer we will analyze the phase signal and report the results if relevant to the project.
It would be informative to see both phase and period analysis for the data shown in figure 2C. Do cells at the edge show differences in relative synchrony following the removal of the PDMS barrier and Rev-erba induction? Is there a period difference between cells at the edge and those that remain confluent?
We agree with the referee that the “shock” received by the cells at the edge should work as a reset of their circadian phase and we have tried to analyse this effect. However, there are technical limitations that make this analysis difficult, mainly the short duration of the experiment and the fact that these cells transition very fast, upon gap closure, from a non-circadian to a circadian behaviour. We will attempt to better report this interesting effect by using the WAVECLOCK (Price et al., 2008) or the pyBOAT method (Mönke et al., 2020), suggested by Reviewer #2, which are designed to analyse non-stationary data.
Mönke G, Sorgenfrei FA, Schmal C & Granada AE (2020) Optimal time frequency analysis for biological data - pyBOAT Systems Biology
Price TS, Baggs JE, Curtis AM, Fitzgerald GA & Hogenesch JB (2008) WAVECLOCK: wavelet analysis of circadian oscillation. Bioinformatics 24: 2794–2795
Figure 2B - the text states that those cells far from the edge oscillate robustly thoughout the experiment, but this is not easy to see from this kymograph due to the dynamic range. Is there another way of presenting this that might make it easier to confirm?
We will calculate the circadian power fraction of the “bulk” cells as we do for the other conditions described in the manuscript. We can also show examples of individual traces if the average shown in Fig. 2C or the kymograph in Fig. 2B are not clear enough.
Figure 1D-E - the text provides periodicity for the high-density cells, but not the low density ones. Could you provide periodicity for both populations - do they differ?
We will represent in more detail the results of the frequency analysis on the low-density cells so the diversity of periods (frequencies) at this condition gets more evident.
Figure S3 - it is interesting to note the difference in population rhythmicity between the bulk and edge data here, which is not seen so clearly in cells without thymidine. Could the authors comment on this?
We agree with the referee that there is an obvious difference regarding RevVNP expression (mainly on the edge cells but also in the bulk) between the experiments with and without thymidine. We hypothesise this is due to the pronounced decrease in cell divisions in the presence of thymidine, which considerably slows down the gap closure and impacts the density of the entire cell population. We will comment this effect in the manuscript.
Line 148 - it is unclear here what is meant by 'the onset of circadian oscillations'. Could you rephrase this for clarity?
We will change that sentence.
Line 173 - a few words to highlight that Lats is a kinase and the function of YAP phosphorylation by Lats would aid clarity here. Similarly, explanation of the functional difference between the protein with 4 Serine to alanine mutations and 5 mutations and why both of these mutants were used would be helpful.
We will clarify this point following the reviewer’s suggestion.
Line 174 - for accuracy, this should perhaps read 'fibroblast circadian clock', as this work is only in 3T3 cells, and therefore may not apply more generally.
We will implement this change.
Line 202 - could you expand to explain the existing limitations of studying cell signalling cascades in synchronised cells? This is not clear to me. Thanks.
We will discuss the signalling effects caused by 50% serum shocks and other traditional ways to synchronise the cells as requested by the reviewer.
Figures 1D and 4B - the choice of colour range used in these kymographs is skewed towards the warmer colours, making it quite hard to discern differences between the groups. I would suggest using the cooler colour range for a greater proportion of the data set, to make rhythmicity, or lack of it, clearer to see.
We will invest further efforts to finding the optimal colour map and range for our datasets.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this study, the authors employ the NIH 3T3 fibroblast cell line to study the effect of cell density and the associated mechanical cues on cellular circadian rhythmicity. For this, they generate a Rev-erbα:VENUS line and, combined with constitutive nuclear mCherry expression, are able to track Rev-erbα: expression in single cells within populations of differing densities. Using overexpression of the transcriptional co-regulator YAP and specific mutants thereof, they suggest a role for YAP and its associated transcription factor family, TEAD, in the regulation of Rev-erbα expression under conditions of differing cell density.
Major comments:
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Are the key conclusions convincing? Yes, the major conclusions are supported by the data shown.
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Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
The authors state in lines 163-165: 'This striking anticorrelation reveals that the robustness of the Rev-erbα circadian expression depends on the nucleocytoplasmic transport of YAP and its mechanosensitive regulation'. Although interesting, the data in figure 3 to which this statement refers is, as the authors identify, correlative, rather than causative. I would strongly suggest altering this statement to better reflect the data.
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Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
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Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
It looks to me as though all experiments were carried out in the same clonal reporter 3T3 line. To avoid possible issues with founder effects, I would ask that the authors repeat the initial experiment in figure 1B, and the associated analysis as in 1C-E with a different clonal 3T3 line. Hopefully this will not be very arduous, as the methods suggest that multiple clonal 3T3 reporter lines were made initially. With time to defrost, plate, record and analyse the data, I would hope that this would not take more than six weeks maximum.
- Are the data and the methods presented in such a way that they can be reproduced?
Yes. I would note that the custom software used for analysis does not appear to be generally available. I would assume that the authors would make this available upon request.
- Are the experiments adequately replicated and statistical analysis adequate?
Experiments appear to have been adequately replicated in terms of n. However, the robustness of these findings would be supported though use of a different clonal reporter line, as discussed above.
Statistical analysis is generally appropriate. I would suggest including statistical analysis in figures 3B and S4B to demonstrate that the pharmacological treatments are indeed having a statistically significant effect on the MAL and YAP nuclear/cytoplasmic ratio.
For Figure 4, it is not stated which statistical tests have been used, with only P values given in table S1. Please state which test has been used.
Furthermore, it would be valuable to see if it is possible to perform statistical analysis looking at the populations should in Figure 4A, to either support or refute the statement made in Line 189-90 that 'we overexpressed 5SA-S94A-YAP, a mutant version of YAP unable to interact with TEAD and observed that the cells recovered, to a large extent, both the RevVNP circadian power fraction and the REV-ERBα basal levels displayed by the wild-type high-density population'
Additionally, it is a little unclear to me why exact p values are reported in table S1. It seems that they might be better placed in the relevant figure legend.
Minor comments:
- Specific experimental issues that are easily addressable. Although the authors took good care to try to ensure that there was minimal phase synchrony between cells, it would be good to see some analysis to confirm that these efforts were successful. This is of particular concern, given that many things that commonly happen during cell handling, such as temperature change and media change, even with conditioned media, can act to synchronise cells. Hopefully, this information should be available from your existing analysis.
It would be informative to see both phase and period analysis for the data shown in figure 2C. Do cells at the edge show differences in relative synchrony following the removal of the PDMS barrier and Rev-erba induction? Is there a period difference between cells at the edge and those that remain confluent?
- Are prior studies referenced appropriately?
To the best of my knowledge, yes.
- Are the text and figures clear and accurate?
Figure 2B - the text states that those cells far from the edge oscillate robustly thoughout the experiment, but this is not easy to see from this kymograph due to the dynamic range. Is there another way of presenting this that might make it easier to confirm?
Figure 1D-E - the text provides periodicity for the high-density cells, but not the low density ones. Could you provide periodicity for both populations - do they differ?
Figure S3 - it is interesting to note the difference in population rhythmicity between the bulk and edge data here, which is not seen so clearly in cells without thymidine. Could the authors comment on this?
Line 148 - it is unclear here what is meant by 'the onset of circadian oscillations'. Could you rephrase this for clarity?
Line 173 - a few words to highlight that Lats is a kinase and the function of YAP phosphorylation by Lats would aid clarity here. Similarly, explanation of the functional difference between the protein with 4 Serine to alanine mutations and 5 mutations and why both of these mutants were used would be helpful.
Line 174 - for accuracy, this should perhaps read 'fibroblast circadian clock', as this work is only in 3T3 cells, and therefore may not apply more generally.
Line 202 - could you expand to explain the existing limitations of studying cell signalling cascades in synchronised cells? This is not clear to me. Thanks.
- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
Figures 1D and 4B - the choice of colour range used in these kymographs is skewed towards the warmer colours, making it quite hard to discern differences between the groups. I would suggest using the cooler colour range for a greater proportion of the data set, to make rhythmicity, or lack of it, clearer to see.
Significance
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
This work provides a potential mechanism for the modulation of cellular rhythmicity under conditions of varying cell density, a currently relatively understudied area to which the contribution made here will be valuable. However, the work is limited by its use of only one cell type (NIH 3T3) and one reporter (Rev-erb:VENUS), which makes the work difficult to generalise in the context of all cell types and environments that exist in a mammalian context.
- Place the work in the context of the existing literature (provide references, where appropriate).
Previous work has identified YAP activity as a mechanism of signalling growth substrate stiffness (Halder et al. 2012, Panciera et al. 2017). It has also been speculated, but not demonstrated, that YAP might influence circadian rhythmicity (Streuli and Meng, 2019). This work provides some initial evidence to support this speculation.
- State what audience might be interested in and influenced by the reported findings.
This work would be of genera; interest to those working on mammalian cellular circadian rhythmicity. Additionally, given YAP's status as an oncogene, this work would also be relevant to those considering circadian disruption in cancer.
- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
Mammalian circadian cell biology and biochemistry.
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Referee #2
Evidence, reproducibility and clarity
Summary:
Abenza et al. investigate an important question of how the physical environment affects the properties of the individual circadian clocks. The authors utilize a set of clever experiments, pharmacological manipulations and data analysis techniques to unveil a potential role of YAP/TAZ in the circadian clock.
Major comments:
Effects on the circadian clock
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The authors use the fluorescent reporter created by Nagoshi from sections of the Rev-erbα gene. This reporter is widely used to estimate relative circadian timing in individual cells but it does not provide direct information on the circadian clock activity. In other words, while Reverb rhythmic expression is driven by the clock, it is not known whether less-rhythmic or non-rhythmic expression or change in expression level of Rev-erbα is affecting the core clock. For example, it has been shown that Rev-erbα knock-down cells are rhythmic as long as Rev-erb-beta is present. Thus, one major shortcoming of the current version of the manuscript is the missing dissection between Rev-erbα rhythmicity/expression and the circadian clock. More concretely, it remains unclear whether the change in Rev-erbα expression is a direct effect or caused by a defect clock. Since the authors presume a direct effect of YAP/TAP on Rev-erb expression, the former is likely. If that is the case, the data could be interpreted as that (missing) mechanic stimuli can lead to nuclear YAP/TAZ, which rises the level of Rev-erbα (and maybe interfere with its rhythmic accumulation). Beyond Rev-erbα expression, there may or may not be an effect on the circadian clock (core clock, CCGs). With the current version we do not know since the authors do not look beyond Rev-erbα expression. Thus, the claims on circadian clock or circadian rhythms in their cells is not studied in this version of the manuscript. The current version is still very interesting and provides insights into the Rev-erbα modulation, but additional work would be needed to show links with the core clock machinery. For this the authors could show influence (or at least correlation) of the YAP/TAZ/REVERBA phenotype on the oscillations of core clock genes or clock-controlled genes. Either through the use of alternative (ideally constitutive) reporters (e.g. PER2, BMAL1, fluorescent or LUC), or/and by analyzing RNA/Protein of core clock genes or output genes. This would not be necessary for all experiments, but at least for some were its possible (e.g. experiments with drugs perturbations). Otherwise, any claim like "YAP/TAZ perturbs the circadian clock ..." or "the circadian clock deregulation in nuclear YAP-enriched cells" is potentially flawed and has to be removed/reformulated.
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The authors aim to discard the possibility of paracrine signals by showing no increase in circadian power fraction of cells growing in low density with conditioned medium (Figure 2D). A paracrine signal coming from an oscillatory system is likely to oscillate and in that case, I do not see how growing cells in constant conditional medium can discard the effects of an oscillatory paracrine signal. I believe the elegant experiment shown in Figure 2E more precisely address this issue.
Data analysis methodology:
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Single-cell circadian recordings like the ones analyzed here are characterized by noisy amplitude and non-sinusoidal waveforms with fluctuating period (Bieler et al., 2014; Feillet et al., 2014). The authors interpolate, smooth, detrend and normalize their data; operations that are known to introduce spectral artifacts that can mislead the interpretation of the power spectrum. Moreover, the time-series pre-processing operations described by the authors in the methods sections is incomplete and the authors should more explicitly describe all their operations with exact methods applied, filter parameters and time-windows sizes (if applicable). To validate their pre-processing steps the authors could provide their time-series analysis pipeline code and/or provide a few examples of raw versus pre-processed data together with their respective spectrums before and after pre-processing. In addition, the authors could provide their raw trace signal data together with the corresponding post-processed signal data as plain text files.
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The authors rely on Fourier analysis and a reasonable self-made definition of circadian strength named as "circadian power fraction". Using a stationary-based method for noisy non-stationary data can lead to inaccurate spectrum power estimations. As the current version of the manuscript does not provide any alternative/complementary analysis method nor we have any available raw signal data it is unclear if their analysis appropriately represents the circadian power. The authors could consider implementing complementary data-analysis strategies to validate their conclusions. Fortunately, there are multiple suitable data analysis strategies already available that are exactly designed for this kind of data (eg. (Price et al., 2008; Leise et al., 2012; Leise, 2013; Bieler et al., 2014; Mönke et al., 2020). This time-series analysis methods is a crucial step as all main results on this manuscript rely on the authors self-made definition of circadian power. This is particularly important as there is no standardized method in the circadian field to estimate circadian rhythmicity and/or circadian power of single-cell traces.
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The authors mainly show circadian power fraction and analyze rhythmicity scores/powers. Is there the a chance that a rise in the basal expression level of Rev-erbα is reducing the rhythmicity score? Or to phrase it otherwise, the absolute amplitude may remain the same, but the relative amplitude may be reduced? Would that affect the FT analysis power scored? To clarify this the authors could provide an analysis of the relative amplitude in addition to the circadian intensity (as in Fig.1C).
Minor points by text-line:
- YAP and TAZ should be introduced to the reader during introduction.
- by set a of proteins.
- Here the authors probably meant that cells were not reset nor entrained during the experiment.
- "..expression depends on..". This is a correlation, not proof of causation is shown until this point. This is an overstatement.
- Using the term "provoked" suggests a causal relationship not shown.
- Similarly last sentence "This result established.... is caused..". Again, this is an overstatement as only correlation is shown.
- According to their description the authors are not using any image-preprocessing steps, eg background subtraction or other filters. Is this correct?
- It is not clear what image metric for the single-cell signals are the authors using, eg. integrated nuclear intensity or mean/median nuclear intensity. I am not familiar with TrackMate but it might be possible to export and share with the readers the image-analysis pipeline used which would clarify any questions about image processing and signal extraction.
References:
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Bieler, J, Cannavo, R, Gustafson, K, Gobet, C, Gatfield, D, and Naef, F (2014). Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells. Mol Syst Biol 10, 739.
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Leise, TL (2013). Wavelet analysis of circadian and ultradian behavioral rhythms. J Circadian Rhythms 11, 5.
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Leise, TL, Wang, CW, Gitis, PJ, and Welsh, DK (2012). Persistent Cell-Autonomous Circadian Oscillations in Fibroblasts Revealed by Six-Week Single-Cell Imaging of PER2::LUC Bioluminescence. PLoS One 7, 1-10.
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Mönke, G, Sorgenfrei, F, Schmal, C, and Granada, A (2020). Optimal time frequency analysis for biological data - pyBOAT. BioRxiv 179, 985-986.
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Price, TS, Baggs, JE, Curtis, AM, FitzGerald, GA, and Hogenesch, JB (2008). WAVECLOCK: wavelet analysis of circadian oscillation. Bioinformatics 24, 2794-2795.
Significance
I believe this manuscript is of high significant both for the circadian as well as the mechanobiology fields. Readers from single-cell signalling studies will also be very interested in this work.
To my knowledge the discussed link has not been studied before at single cell level, which as the authors show can provide multiple new insights.
I do work with similar single-cell signals, have broad expertise in microscopy, image analysis methods, time series analysis, and the circadian clock mechanisms but very little experience in mechanobiology.
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Referee #1
Evidence, reproducibility and clarity
Here Abenza & Rossetti et al. show that in 3T3 fibroblasts, the circadian clock depends on cell density, correlates with YAP activity, and further demonstrate that circadian power fraction is suppressed by genetic YAP activation (5SA), but is rescued by expression of 5SA YAP without the tead-binding domain. This is a striking study on an important question; however, the data do not directly support the conclusions and the title of the paper. These conclusions/title should be altered, or supported with additional experiments, as detailed below:
Major Critiques:
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On "mechanical control": The authors show changes in circadian power fraction with changes in YAP and with cytoskeletal inhibitors, but there are no properly-controlled experiments that directly perturb mechanics. The authors show a correlation between YAP nuclear/cytoplasmic ratio and circadian power, but YAP N/C alone is not a readout of mechanotrasndcution, per se. The authors have shown two different experiments where cells are cultured on a stiff (30kPa) substrate and soft substrate (300Pa), but they do not shown a direct comparison of YAP nuclear localization and circadian power under these two conditions in the same experiment. Direct, controlled perturbation of mechanical cues is necessary to support the title's use of the phrase "mechanical control."
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On "via YAP/TAZ": In addition to above, it is necessary to show that the changes in Circadian power fraction induced by mechanical cues in fact require YAP/TAZ signaling. Thus, an experiment comparing soft (300Pa) substrate with Stiff (30kPa) substrate in the presence or absence of YAP/TAZ is necessary to state that YAP and TAZ are the mechanistic mediators of mechanical cues on the clock.
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While the TEAD-binding domain mutant experiment is elegant, to claim that TEAD is the transcriptional mediator, it must be demonstrated that this mutant indeed fails to induce TEAD-mediated transcription. This could be simply executed by demonstrating that the CCD mutant expresses reduced CTGF and Cyr61 (for example), compared to the 5SA, under these conditions. Further, endogenous YAP is still active and available to bind to TEAD in this system, which should be discussed.
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In Figure 3a: The cell perimeter needs to be shown either by actin staining or by brightfield images. The manually marking of cell boundaries is insufficient, specifically because the drugs used in this experiment affect the cytoskeleton. It would be very helpful to see this via actin staining or in the least with brightfield images.
Significance
This is an exciting paper that potentially links mechanotransduction to the circadian clock. While my group is not focused on circadian rhythms, and I don't have the background to comment on the measurements or robustness of circadian power, the idea is striking and significant.
I strongly recommend inclusion of loss-of-function approaches (either genetic or pharmacologic) in addition to the gain-of-function methods employed here to support the necessity of YAP/TAZ signaling. Also, appropriately controlled experiments to show true mechanical effects on the circadian clock are necessary.
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www.biorxiv.org www.biorxiv.org
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)): ____ *A significant criticism of the paper is an assumption that readers will be familiar with all of the findings in the author's previous 2016 paper and the PGL-1 papers by Aoki et al. Minimal context is given for each approach. *
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
*Some conclusions are not well supported and require further analysis, proper controls, and more extensive descriptions of the experiments performed. *
We have addressed the reviewer’s concerns as detailed below.
Most importantly, the central conclusion and title of the paper is that composition can buffer the dynamics of individual proteins within liquid-like condensates. In other words, in vitro condensation assays often do not recapitulate LLPS behavior in vivo. That said, the findings in this study would be significantly strengthened and complemented by observing endogenously tagged PGL-3 and PGL-3 mutants in living worms, considering the efficiency of using CRISPR in C. elegans to insert tags and make precise mutations.
The original manuscript already contained data where we microinjected wild-type PGL-3 and mutant PGL-3 proteins (recombinantly purified) into adult C. elegans gonads to assay how the P granule phase supports diffusion of these proteins.
In the revised version, we now include additional data which shows “dynamics buffering” in transgenic worms generated using CRISPR/Cas9 technology. Briefly, we used CRISPR/Cas9 to generate transgenic C. elegans which expresses PGL-3-mEGFP or PGL-3(D425-452)-mEGFP from the native pgl-3 locus. In vitro, wild-type PGL-3-mEGFP protein generates liquid-like condensates. On the other hand, the recombinantly purified PGL-3(D425-452)-mEGFP protein generates condensates that are non-dynamic. In contrast to these observations in vitro, both wild-type PGL-3-mEGFP and PGL-3(D425-452)-mEGFP show similar dynamics (half-time of FRAP recovery) within P granules in vivo.
*To improve readability, the introduction to P granules should be expanded, and include the reasons for looking at the nematode-specific PGL-3 protein among all the other known P granule proteins. A recap of previous findings on PGL-3 phase separation, in vivo and in vitro, is warranted, starting with the significant results of Saha et al 2016. Setting up the investigative questions in the context of recent work on PGL-1 (Aoki, et al) is also necessary. *
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
The physiological concentration of PGL-3 should be more transparent, including why some experiments in this study are done at physiological concentrations while others are not. Describing why salt concentrations, crowding agents, and protein abundance are similar or different for each experiment is necessary and relevant. For example, after showing in Figure 1 that PGL-3 protein phase separates, the paragraph starting on line 161 says that it was previously shown that PGL-3 doesn't phase separate at physiological concentrations without RNA. One has to go back to Figure 1 to realize it was done differently than Figure 2 and Saha 2016.
The concentrations of PGL-3 protein and use of crowding agents (if any) have already been specified within figures or figure legends. Salt concentrations used are specified within figure legends or materials and methods section.
We have added the following paragraph to the materials and methods section of the revised manuscript.
“Saha et al. 2016 showed that at physiological concentrations (approx. 1 mM), the PGL-3 protein is unable to phase separate into condensates. At these concentrations, mRNA promotes phase separation of PGL-3. To assay for mRNA-dependence of condensate assembly, it is therefore essential to use physiological concentrations of the PGL-3 protein or mutants (e.g. Figure 2). However, these condensates are generally too small to assay rate of internal rearrangement of PGL-3 molecules within condensates using fluorescence recovery after photobleaching experiments. Therefore, to generate large condensates for measuring internal rearrangement of PGL-3 or mutant molecules, we primarily used higher concentrations of these proteins where binding to RNA is not essential for phase separation. However, to mimic the in vivo P granule phase as closely as possible, we generally added constituent proteins in proportion to their in vivo abundance estimated in Saha et al. 2016.”
The added paragraph in the Introduction section of the revised manuscript may be helpful to the readers. * *
*Statements in the same paragraph like "in contrast to full-length PGL-3, mRNA does not support phase separation..." should be qualified by stating the concentration observed, with or without salts or other crowding agents. Similarly, line 230 "suggests that interactions involving the disordered C-terminal region of PGL-3 are not essential for the fast dynamics" and should be qualified with "at non-physiological concentrations and with XX crowding agents or salt concentration." It would be more consistent if physiological concentrations were consistent from figure to figure, as extra variables weaken some of the stated conclusions. *
We thank the reviewer for this suggestion. However, we feel the statements (without full experimental details within main text) help convey the conceptual essence of the findings better. Of course, all these statements contain reference to figures or prior publications which provide relevant details about experimental conditions.
*The 2010 review reference stating that there are 40 P granule enriched proteins is outdated. More recent reviews put the number much higher. This is relevant because the approach to put PGL-3 in a more physiological environment by including just PGL-1, GLH-1 and mRNA with the condensate assays, out of ~100 P granule enriched proteins, may not be sufficient to conclude "that the influence of complex composition on dynamics is modest" (line 223), or imply that the multicomponent nature of the P granule is reconstituted by adding these components (line 355). *
We revised the text to indicate that P granules contain approx. 70 proteins and added appropriate references.
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Based on current information of constitutive P granule components (PGL-1, PGL-3, GLH-1, GLH-2, GLH-3, GLH-4, DEPS-1, MIP-1 and mRNA), (Kawasaki et al, 1998, 2004; Spike et al, 2008a, 2008b; Price et al, 2021; Cipriani et al, 2021; Phillips & Updike, 2022) we reconstituted P granule-like phase in vitro with mRNA, PGL- and GLH- proteins that likely constitute the most abundant components within P granules in vivo (based on concentration estimates in Saha et al. 2016).
We do appreciate the reviewer’s comment that more components can be added to our in vitro reconstitution in addition to the limited set of components used in our study. However, we feel it is interesting to observe that a limited set of components can support dynamics buffering (the main message of the paper). Further, the complementary in vivo experiments show that the P granule phase can also support dynamics buffering.
*Figure 1C needs to include PGL-3(370-693) in the analysis. Figure 1E is also incomplete without a comparison of FRAP recovery between PGL-3(1-452) and full PGL-3 as the control.
*
Fig. 1c already includes data with PGL-3 (370-693) [top row, central panel]. FRAP recovery data with full-length PGL-3 is already available in Supplementary Fig. 2c, g.
*Figure 4C is missing an essential control where PGL-3 and S1 FRAP is performed without PGL-1, GLH-1, and mRNA. *
In the revised version, we have added Supplementary Fig. 5f, where FRAP recovery of the following condensates are plotted together: 1) PGL-3 alone, 2) S1 alone, 3) PGL-3 + PGL-1, GLH-1 and mRNA, 4) S1 + PGL-1, GLH-1 and mRNA.
*It would also help show sup Fig4A in the main figure to show concentration dependence. *
We revised Fig. 4 to address the reviewer’s suggestion.
Consider adding subtitles to supplementary figures.
We considered the suggestion but felt it may not be essential.
*M&M should include an explanation for statistical analysis *
We added a paragraph describing statistical analysis within the Materials and Methods section.
*CROSS-CONSULTATION COMMENTS I am also in agreement with the comments and critiques of reviewers 2 and 3.
* Reviewer #1 (Significance (Required)): The paper by Saha and colleagues investigate the in vitro liquid-liquid phase separation propensity of a P granule protein PGL-3 and its structural domains. The findings largely replicate and support the phase-separation properties of a paralogous protein called PGL-1, as recently described by Aoki et al. 2021. Furthermore, they show that the dynamics demonstrated by recombinant PGL-3 may be maintained or buffered by the complex composition of P granules.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
*Jelenic et al. describe the effect of partner proteins on the FRAP dynamics of recombinant PGL-3 protein and variants in in vitro condensates and C elegans p-granules. The study shows that the N terminal a-helical dimerization domains is required for condensate formation and modulate of it alters aggregation and the FRAP dynamics of its condensates. Interestingly, a construct including the entire IDR region (370-693) by itself does not phase separate on its own at these conditions. The K126E K129E mutant (known previously to disrupt dimerization) and the deletion mutant abrogate llps. A mutant construct that shuffles the sequence in the region 423-453 called S1 here reduces the helicity and the condensate FRAP dynamics but recovered in the presence of a few P granule components. Also, the reduced dynamics of partially unfolded PGL-3 condensates are also rescued by the p-granule components to a certain degree of the unfolded PGL3 concentrations. This threshold concentration for recovering the condensate dynamics is further reduced in the helix reducing S1 mutant, which is also dependent on the number and the nature of P granule components.
Overall, the study aims to probe how "composition can buffer protein dynamics within liquid-like condensates" - yet several underlying aspects of the study do not fully support that conclusion. The introduction does not sufficiently introduce the known structural information of the two dimerization domains in C elegans PGL proteins for which structures are known. The region is discussed as "alpha helical" but really there are two evolutionarily conserved independently folding dimerization domains (referring to the mutants as "reduced alpha helicity" is not helpful - these are mutations that destabilize a folded domain).*
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
*Additionally, the abstract and introduction ignore the aspects of aggregation (touched on in discussion) - this is likely what the disruption to the helical region in residue 450 region is doing (the helix is not on the dimer interface based on homology / sequence identity to the crystal structure of PGL-1 central dimerization domain. *
We think elucidating the molecular mechanism of apparent aggregation of PGL-3 (D425-452) could be an interesting direction for future investigation. Here, we focused our analysis predominantly on the mutant S1 since it generates liquid-like condensates with ~20- fold slower dynamics (compared to wild-type) in contrast to non-dynamic condensates/aggregates. Therefore, influence of other P granule components on the dynamics of PGL-3 in liquid-like condensates is easier to address using the mutant S1 rather than PGL-3 (D425-452). We didn’t find evidence that S1 aggregates as we did not detect aggregates of S1 molecules using fluorescence confocal microscopy and the slow dynamics in condensates of S1 does not change significantly over 24 h (Supplementary Fig. 3f).
However, in the revised version, we now include additional in vivo data with C. elegans expressing the aggregation-prone PGL-3 (D425-452)-mEGFP. Briefly, we used CRISPR/Cas9 to generate transgenic C. elegans which expresses PGL-3-mEGFP or PGL-3(D425-452)-mEGFP from the native pgl-3 locus. In vitro, wild-type PGL-3-mEGFP protein generates liquid-like condensates. On the other hand, the recombinantly purified PGL-3(D425-452)-mEGFP protein generates condensates that are non-dynamic. In contrast to these observations in vitro, both wild-type PGL-3-mEGFP and PGL-3(D425-452)-mEGFP show similar dynamics (half-time of FRAP recovery) within P granules in vivo.
Finally, the "dynamics buffering" is not really clearly established and could also be explained as small concentrations of aggregated proteins act like clients while increasing the concentration results in aggregation and "cross linking" in the entire droplet - and this concentration is never achieved in the in worm experiments so it is not clear. In other words, the change in FRAP dynamics not observed in worms is perhaps not surprising if small amount of recombinant proteins are incorporated into the granules. *
*
Data with the S1 mutant establishes that dynamics buffering can be observed in condensates with different sets of additives both in vitro (Fig. 5a, b) and in vivo (Fig. 4a, b). Further, data with condensates of S1 containing the additives PGL-3 (K126E K129E) or S1 (K126E K129E) demonstrate that dynamics (half-time of FRAP recovery) within S1 condensates, and in turn “dynamics buffering” depend on inter-molecular interactions. With respect to the hypothesis proposed by the reviewer, we did not detect aggregates within S1 condensates using confocal fluorescence microscopy.
In contrast to S1 condensates, condensates containing partially unfolded PGL-3-mEGFP together with PGL-1, GLH-1 and mRNA showed spatial inhomogeneities in fluorescence signal throughout the condensate (Fig. 4g). We have not tested if areas with higher fluorescence signal represent aggregates. It is a possibility that the partially unfolded PGL-3-mEGFP fluorescence signal becomes more homogeneous if higher concentrations of additives (PGL-1, GLH-1 and mRNA) are used. However, the presented data demonstrate the significant effect of the P granule components (PGL-1, GLH-1 and mRNA) on the FRAP recovery rate of partially unfolded PGL-3-mEGFP in condensates (compare figures Fig. 3e and Fig. 4g).
However, consistent with dynamics buffering, the P granule phase in vivo supports wild-type dynamics of different PGL-3 constructs over a range of concentrations - PGL-3(D425-452)-mEGFP at physiological concentration (CRISPR transgenic strain, Fig. 4e) or at higher concentrations (microinjected S1 and partially unfolded PGL-3-mEGFP, Fig. 4b).
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*It is also not clear what the mechanism of the changes is - is the protein driven to fold more properly (despite S1 disruption of its conserved sequence) inside the condensate? Does it still self interact and act as a dimerization domain? Does this change disrupt interactions? *
We agree with the reviewer that identifying the precise structural changes of the S1 protein within the condensate vs. dilute phase could be an interesting direction for future investigation. However, we have already discussed the issues raised by the reviewer in the original manuscript.
“Our data is consistent with the model that other regions of S1 molecules cooperate with residues 425-452 (shuffled) to generate stronger inter-molecular interactions. For instance, addition of the mutant S1 (K126E K129E) enhances dynamics of S1 within condensates in contrast to maintaining the slower dynamics observed within condensates of S1 alone. This suggests that the interactions disrupted by the mutations K126E and K129E also contribute to slow S1 dynamics. One possibility is that interactions involving the residues K126 and K129 favor S1 conformations that enhance 425-452 (shuffled)-dependent interactions. Indeed, the mutations K126E K129E have been reported to interfere with interactions among N-termini of PGL-3 molecules (Aoki et al, 2021). While two self-association domains within the α-helical N-terminus of PGL-3 have been mapped (Aoki et al, 2021, 2016), structural insights into those associations are limited. However, PGL-3 shares significant sequence similarity with another protein PGL-1. Crystal structures are available for fragments of the PGL-1 protein that show the two self-association domains at the N-terminus are predominantly α-helical and globular in nature (Aoki et al, 2016, 2021). Therefore, one possibility is that shuffling the sequence 425-452 of PGL-3 or heat-induced unfolding of PGL-3 exposes hydrophobic residues that become available to participate in inter-molecular interactions.”
What is the real mechanism by which PGL-3 phase separates if not via the disordered domains? *
*
We agree with the reviewer that elucidating the detailed mechanism of phase separation of PGL-3 is an interesting direction for future investigation. However, we feel this is not required to support the main message of this manuscript.
Throughout the manuscript, the term "dynamics" is used to indicate FRAP, but it would be better to define what is meant (diffusion of PGL-3 in condensates) instead of using dynamics a term that could mean many things. Secondly, FRAP cannot directly measure liquidity etc (see recent critiques by McSwiggen elife 2019, etc) so it is better to be cautious in the claims. Finally, discussing "dyanmics buffering" adds more terminology where it is not needed - perhaps say "changes to diffusion of PGL-3 in condensates".
We feel it is useful to introduce a term that describes our observation. To our knowledge, our observation is novel and therefore requires a new term to describe it.
However, we do appreciate the concern raised by the reviewer. We used a more generic term “dynamics buffering” in contrast to the more specific “diffusion buffering” since we did not directly estimate diffusion behavior at the ‘single-molecule’ level. However, we already described what we mean by “dynamics buffering” in the text as follows.
“We used condensates of similar size for our analysis (average ± 1 SD of diameter of condensates are 6.4 ± 1.7 mm (Fig. 5a) and 5.9 ± 0.4 mm (Fig. 5b)). Therefore, dynamics buffering here is likely to represent similar diffusion rates of S1 within condensates.”
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*The "N-terminus" is not 65% of the protein. One could define this as the N-terminal domain, but again there are two clear folded domains in the first 65% of the protein and this needs to be described better. *
We revised the text to replace the terms “N-terminus” and “N-terminal domain” to “N-terminal fragment”.
*The description of "stickers" and the references to tau and hnRNPA1 are confusing as this is a predominantly ordered domain while those are IDRs. *
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We feel this is important as it aids discussing our work in the context of current literature describing the mechanisms of macromolecular phase separation.
The suggestion in the discussion that "P granule components support dynamics by participating in intermolecular interactions wth PGL-3-mEGFP molecules" is not well supported because no interaction assays are performed and no mutaitons are made that disrupt these interactions to test this.
Indeed, we have not conducted interaction assays or mutational analysis to directly test this. However, our detailed analysis with the S1 mutant supports this suggestion.
While partially unfolded PGL-3-mEGFP molecules lose 30% of a-helicity, the a-helicity of the S1 mutant is reduced by 15% compared to wild-type PGL-3. Data with S1 and partially unfolded PGL-3-mEGFP molecules show that loss of a-helicity correlates with slower diffusion of protein molecules within condensates. Using the mutants PGL-3 (K126E K129E) and S1 (K126E K129E), we show that diffusion rate of S1 molecules within condensates depend on inter-molecular interactions, and presence of other P granule components support faster diffusion rate of S1 molecules within condensates. Therefore, we feel it is safe to speculate that intermolecular interactions with P granule components can support dynamics of a “more unfolded” (compared to S1) version of PGL-3 molecule. * *
*More detailed analysis of some of the claims: Claim 1: An a-helical region mediates the phase separation of PGL-3, and the C-terminal disordered region by itself does not phase separate. The N-terminal dimerization is essential for LLPS. The C-terminal IDR interactions with mRNA facilitate the LLPS. Comments: The authors show sufficient experimental data using microscopy and FRAP on truncated constructs with the N-terminal and C-terminal regions - but see above regarding how these are described - a proper domain structure with the folded domains shown and the RGG motifs highlighted should be added and integrated throughout the discussion. *
In the revised version of the manuscript, we described the predicted PGL-3 domains within a paragraph in the introduction: “The interactions that support phase separation of the PGL-3 protein remains unclear. Structural studies on the orthologous PGL-1 protein revealed two dimerization domains. This raises the possibility that PGL-3 also contains similar dimerization domains, and phase separation depends on interactions involving these domains.”
Our Fig. 1a already includes the schematic representation of PGL-3 with predicted N-terminal and Central Dimerization domains and RGG repeats.
*They show that the N-terminus is necessary and adequate for LLPS, and the C-terminus by itself does not phase separate. But, how does the N-terminal domains phase separate? This is not explained - what are the interactions? *
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Also, a di-mutant (K126E K129E) that is known, and also authors use SEC-MALS to show their N-terminal construct is consistent with the published results. Disrupting the n-terminal dimerization prevents phase separation, suggesting the importance of these residues in the N-terminus for self-assembly and LLPS. The Microscopy data backs the claim that the mRNA-mediated LLPS is facilitated by binding with C-terminus. However, the m-RNA binding to IDR is not sufficient for LLPS. Yet, the authors do not explain how higher salt prevents phase separation - again the mechanism of phase separation is unclear. Is it multivalent interaction of the two dimerization domains? A basic model (that is tested) would be important.
We agree with the reviewer that elucidating the detailed mechanism of phase separation of PGL-3 is an interesting direction for future investigation. However, we feel this is not required to support the main message of this manuscript.
However, our manuscript already provides some relevant insights as follows.
“To investigate the underlying mechanism further, we began by testing if the N-terminal α-helical region of PGL-3 can self-associate. Our analysis using size exclusion chromatography followed by multi-angle light scattering (SEC-MALS) showed that this PGL-3 fragment 1-452 forms a dimer (Supplementary Fig. 2f). Mutation of two residues (K126E K129E) have been shown to interfere with interactions among the N-termini of PGL-3 molecules (Aoki et al, 2021). We mutated these two residues within the full-length PGL-3 protein (K126E K129E) (Fig. 1a) and found that this mutant PGL-3 (K126E K129E) protein cannot phase separate even at high protein concentrations up to ~130 µM (Fig. 1b, c). Addition of mRNA does not trigger phase separation of this protein at physiological concentrations either (Fig. 2a, b). Taken together, our data is consistent with a model where association among folded N-termini of PGL-3 molecules is essential for phase separation.”
A likely possibility is that phase separation of PGL-3 depends on electrostatic inter-molecular interactions among the folded N-terminal fragment of PGL-3 molecules. Therefore, high salt prevents phase separation.
Are the tags removed to ensure that phase separation is not caused by tags or remaining linker regions? Is the protein purified to be without nucleic acid contamination or other purity metrics?
Most of the experiments were done with only 5% of total protein tagged with 6x-His-mEGFP. No additional tags were present on the constructs. For recombinant expression and purification, proteins were cloned such that it is possible to remove the 6xHis-mEGFP tag following treatment with TEV protease. Following removal of the 6xHis-mEGFP tag, the residual linker is just two amino acid residues long. We used 100% tagged-protein for our experiments only in very few cases (indicated in the figure legends).
To demonstrate purity of recombinant proteins, SDS-PAGE gels with all protein constructs used in this study are shown in Supplementary Fig. 1.
To minimize contamination of nucleic acids, we treated samples with Benzonase during the course of purification.
To assess the extent of nucleic acid contamination, the ratio of absorbance at 260 nm and 280 nm (A260/A280) was monitored. In exceptional cases with high A260/A280 values, we analyzed samples further by purifying RNA from the sample using RNA purification kit (Qiagen) and found that RNA represented 1% or less of the sample mass.* *
Claim2: The N-terminal a-helical region modulates the dynamics within condensates. The IDR region has minimal effect on the fast dynamics of PGL-3. Comments: The authors show that the full-length PGL-3 condensates have modest influence of components by comparing the FRAP half times with or without the P granule components, including mRNA. However, have the authors tried this in the presence of mRNAs for the constructs lacking the IDRs as they have several RGG domains and bind with mRNA and are likely to change the dynamics.
We thank the reviewer for this suggestion. However, this experiment is not essential to support the claim made in the context of homotypic condensates of PGL-3 : “The N-terminal a-helical region modulates the dynamics within condensates. The IDR region has minimal effect on the fast dynamics of PGL-3.”
*The authors report the importance of the N-terminal a-helical region by making a construct that lacks/disrupts a part of the helices lowers the thermal stability and significantly lowers the dynamics of the condensates. Also unfolding of helices is shown to reduce the dynamics. One primary concern is whether these "rescued" protein dynamics imply protein functionality. *
An assay of “functionality” e.g. an enzymatic activity of the PGL-3 protein is not available.
However, we compared the fecundity of C. elegans worms expressing from the native pgl-3 locus, PGL-3-mEGFP or the mutant protein PGL-3(D425-452)-mEGFP, to assay the functionality of P granules in these strains. We found that worms of both genotypes produced similar number of offspring (Fig. 4d). This suggests that deletion of residues 425-452 of PGL-3 does not result in significant loss of function of P granules.
Are these semi denatured proteins refolded in the presence of P-granule components?
We feel that identifying the precise structural changes of the semi-denatured PGL-3 proteins within the condensate vs. dilute phase could be an interesting direction for future investigation.
Finally, it is not clear why the authors chose to disrupt folding of the central dimerization domain?
The manuscript included a paragraph to describe the rationale.
“This suggests that interactions involving the disordered C-terminal region of PGL-3 are not essential for the fast dynamics within condensates. Therefore, we addressed the role of the N-terminal α-helical region (1-452) in driving dynamics. In order to avoid engineering mutations that result in significant misfolding of PGL-3 and concomitant loss of its ability to phase separate, we focused our mutational analysis close to the junction of the folded N-terminus and the disordered C-terminus of PGL-3. Surprisingly, we found that a full-length PGL-3 construct (D425-452) that lacks only 27 residues phase separates into condensates that are non-dynamic (Fig. 3a, c). Sequence analysis of the PGL-3 protein predicts that this region 425-452 spans two α-helices (one complete helix and fraction of a second helix) (Supplementary Fig. 3d). We generated a PGL-3 construct (hereafter called ‘S1’) (Fig. 3a) in which the sequence in the region, 425-452, is shuffled while keeping the overall amino acid composition unchanged. We found that S1 phase separates into condensates that are 20- fold less dynamic than with wild-type PGL-3 (Fig. 3d, Supplementary Fig. 3c).”
Saying that "reduced alpha-helicity of PGL-3 correlates with slower dynamics in condensates" may be factual in these assays but "correlation" should be expanded upon to include mechanism and to me it seems that the statement should read "aggregation of PGL-3 causes slower dynamics in condensates" (both the partially destabilized mutant and the fully unfolded WT show similar effects perhaps to different degrees).
We feel that identifying the precise structural changes of the semi-denatured PGL-3 proteins within the condensate vs. dilute phase could be an interesting direction for future investigation.
We did not use the term "aggregation" since we did not detect aggregates of S1 molecules using fluorescence confocal microscopy.
*CROSS-CONSULTATION COMMENTS I agree with the other reviewer's comments and critiques, I have concerns about the biological relevance and also the biophysical mechanisms. Reflecting on the other reviewers' comments, the papers could provide more depth in one or both of these areas to come to firm conclusions that are either revealing about PGL biology or elucidate a (possible) general biophysical mechanism. *
In the revised version, we now include additional data which shows “dynamics buffering” in transgenic worms generated using CRISPR/Cas9 technology. Briefly, we used CRISPR/Cas9 to generate transgenic C. elegans which expresses PGL-3-mEGFP or PGL-3(D425-452)-mEGFP from the native pgl-3 locus. In vitro, wild-type PGL-3-mEGFP protein generates liquid-like condensates. On the other hand, the recombinantly purified PGL-3(D425-452)-mEGFP protein generates condensates that are non-dynamic. In contrast to these observations in vitro, both wild-type PGL-3-mEGFP and PGL-3(D425-452)-mEGFP show similar dynamics (half-time of FRAP recovery) within P granules in vivo.
Reviewer #2 (Significance (Required)): *Hence, although the authors shows how inclusion of other components can alter the one protein component phase separation, this is done with entirely artificial means of destabilizing the fold of one of the domains which likely leads to aggregation. So the true impact of the work is hard to understand because the mutations impact on the basic biophysical properties of the domain (stability, interaction) are not completely characterized and the reason for disrupting this folding is not clear. *
A major impact of our work is elucidation of a novel “dynamics buffering” property within biomolecular condensates in vitro. Our in vivo data is consistent with this finding.
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We have chosen two orthogonal ways of perturbing the PGL-3 protein (i.e. mutations and temperature-dependent unfolding) to assay the effect on diffusion rate against different levels of perturbation (e.g. 30% loss of a-helicity in heat-denatured PGL-3-mEGFP vs. 15% loss of a-helicity in the S1 mutant, compared to wild-type PGL-3). Studying the phase separation behavior of these “artificially-generated” constructs provided the understanding that dynamics of PGL-3 in condensates depends on inter-molecular interactions, and slower dynamics generally correlate with stronger inter-molecular interactions. Further, interactions among two or more P granule components can buffer against large change in dynamics / aggregation within the P granule phase. These insights may lay the groundwork for addressing how more “natural” modifications (e.g., post-translational modifications, high local concentration of “sticky” molecules) may influence dynamics within biomolecular condensates in vivo.
Based on current knowledge of P granule composition, chaperone proteins (e.g. heat-shock family proteins) do not show abundant concentration within P granules. However, it is unclear if chaperone proteins are completely excluded from the P granule phase. Therefore, we speculate that weak interactions among two or more non-chaperone proteins contribute significantly to “dynamics buffering” within the P granule phase in vivo.
In the discussion section of the manuscript, we had speculated that “dynamics buffering” may potentially explain observations reported in the nucleolus: “Similarly, interactions among components could be a potential mechanism of storage of misfolding-prone proteins in non-aggregated state within the liquid-like nucleolus under stress in vivo (Frottin et al, 2019).”
Our finding is also relevant in the context of synthetic biology with applications that require steady diffusion rate of macromolecules during biochemical reactions within biomolecular condensates.
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My field of expertise is protein phase separation and protein structure. * *
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary: P granules are liquid condensates found in the developing germlines and embryos of C. elegans. Prior work by the authors and others have established P granules as a tractable model to investigate the basic biophysical properties of liquid condensates. Much of the prior published work focused on specific P granule scaffold proteins, PGL-1 and PGL-3. How attributes of these PGL proteins and the effect of other P granule components affect condensate properties is not fully understood. Here, Jelenic, et al. probe the biophysical properties of PGL-3. Using recombinant protein, they show that an N-terminal, alpha-helical region of PGL-3 is sufficient for liquid condensate formation and that N-terminal assembly is required for this formation. Creation of a scrambled alpha-helical region in PGL-3 and heat treatment affects PGL-3 fluidity. This fluidity can be "rescued" in vivo and in vitro with the inclusion of other P granule factors, including wildtype PGL-3, PGL-1, GLH-1 and mRNA. The authors note an inverse correlation between fluidity and mutant PGL-3 fluorescent intensity. They propose a model that heterotypic compositions of condensates can buffer their fluidity against components with stronger multivalent interactions. *
MAJOR: 1. PGL-3 is a fantastic model to study the biophysical properties of a liquid condensate. But as the authors address in their discussion, the S1 mutant will likely affect the central domain folding, at its minimum causing exposure of a hydrophobic surface not typically exposed in biology. These helices are found at the terminal portion of the domain determined in the crystal structure and as depicted in the authors' Figure 1A. While the cause of S1's enhanced molecular interactions does not affect the in vitro work presented in this manuscript, it does affect how the conclusions connect to the biological nature of P granules and liquid condensates more generally. *
We have chosen two orthogonal ways of perturbing the PGL-3 protein (i.e. mutations and temperature-dependent unfolding) to assay the effect on diffusion rate against different levels of perturbation (e.g. 30% loss of a-helicity in heat-denatured PGL-3-mEGFP vs. 15% loss of a-helicity in the S1 mutant, compared to wild-type PGL-3). Studying the phase separation behavior of these “artificial” constructs provided the understanding that dynamics of PGL-3 in condensates depends on inter-molecular interactions, and slower dynamics generally correlate with stronger inter-molecular interactions. Further, interactions among two or more P granule components can buffer against large change in dynamics / aggregation within the P granule phase. These insights may lay the groundwork for addressing how more “natural” modifications (e.g., post-translational modifications, high local concentration of “sticky” molecules) may influence dynamics within biomolecular condensates in vivo.
Based on current knowledge of P granule composition, chaperone proteins (e.g. heat-shock family proteins) do not show abundant concentration within P granules. However, it is unclear if chaperone proteins are completely excluded from the P granule phase. Therefore, we speculate that weak interactions among two or more non-chaperone proteins contribute significantly to “dynamics buffering” within the P granule phase in vivo.
In the discussion section of the manuscript, we had speculated that “dynamics buffering” may potentially explain observations reported in the nucleolus: “Similarly, interactions among components could be a potential mechanism of storage of misfolding-prone proteins in non-aggregated state within the liquid-like nucleolus under stress in vivo (Frottin et al, 2019).”
Our finding is also relevant in the context of synthetic biology with applications that require steady diffusion rate of macromolecules during biochemical reactions within biomolecular condensates.
- Recombinant PGL-3 experiments added PGL-1, GLH-1 and mRNA simultaneously and measured fluidity. It will be interesting to know which components contribute to fluidity and whether fluidity enhancement of each component is dependent on one another. Addition experiments with each component should be included and/or at least discussed in the main text. *
Our data with S1-mEGFP or PGL-3-mEGFP (pre-heated at 50°C) proteins microinjected into C. elegans gonads, and the transgenic strain expressing PGL-3(D425-452)-mEGFP from the pgl-3 locus showed that the P granule phase can support fast dynamics of these mutant PGL-3 constructs. Since P granules have a complex composition, one possibility is that fast dynamics of these constructs is supported by interactions involving many P granule components. We found that using only a limited set of P granule components (PGL-1, GLH-1 and mRNA) can buffer dynamics of S1 in condensates in vitro.
In absence of a systematic analysis investigating the individual role of approx. 70 P granule proteins in buffering S1 dynamics in condensates in vitro, we have claimed in the text that dynamics-buffering of S1 in condensates is supported by interactions among two or more components. However, we do appreciate the reviewer’s comment and feel it would be interesting to investigate the contribution of individual P granule components towards fluidity in future studies. We have discussed this in the ‘Discussion’ section of the manuscript.
- The biological relevance of PGL-1, GLH-1, and mRNA were not discussed in the main text. How these factors contribute to P granule assembly and function should be mentioned in the Introduction or Results. *
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
*MINOR: 1. Line 20, "most non-membrane-bound compartments...have complex composition": Are there examples of condensates that do not have complex composition? *
Not all non-membrane-bound compartments may have been characterized. To accommodate this possibility, we refrained from making a more general statement, but stated “most non-membrane-bound compartments…”.
- Lines 40-43, RNA interactions driving LLPS: Please include citations from the Parker Lab (e.g. Van Treeck and Parker, Cell. 2018 doi: 10.1016/j.cell.2018.07.023) *
We added the reference suggested by the reviewer.
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*
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Line 60, condensates contain hundreds of different proteins and RNA: Please cite at least a few examples of condensates with their components identified. *
We added some references following suggestion by the reviewer.
- Lines 82-84, PGL-3 drives assembly: Please cite Kawasaki, et al. Genetics 2004 for the discovery of PGL-3. *
We added the reference suggested by the reviewer.
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Lines 88-89, PGL-3 N-terminal fragment predominantly alpha-helical: The PGL domain structures should be cited here as supporting evidence that these regions are composed primarily of alpha helices (Aoki, et al 2016, 2021) *
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*
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
- Lines 158-159, driving forces for phase separation: This statement should be removed or expanded. The authors point regarding the protein concentrations is not clear here but clarified in the Discussion (Lines 691-693). Recommend removing due to its speculative nature. *
We retained the speculative comment in the results section. We feel that this prepares the readers for the discussion later in the manuscript.
- Lines 210: Add commas before and after "PGL-1 and GLH-1"*
We addressed the reviewer’s suggestion.
- Lines 218-219: add "and" instead of comma between PGL-1 and GLH-1 *
We addressed the reviewer’s suggestion.
- Lines 238-239, alpha-helices: The PGL CDD structure should also be referenced here (Aoki, et al 2016). *
To address this concern, we have added a paragraph in the Introduction section of the revised manuscript.
- Lines 680-682, MEG proteins: Please cite accordingly. *
We added the reference suggested by the reviewer.
- Lines 694-695, heterotypic interactions: Please cite Saha, et al. 2016. *
We added the reference suggested by the reviewer.
- Figure 1: Add space between 1 and mM DTT *
We addressed the reviewer’s suggestion.
- Figure 2b: Please provide statistics between condensate numbers. *
We provide statistics between condensate numbers in Fig. 2b.
- Figure 4A: The region of the germline imaged and analyzed should be mentioned in the caption or the main text. *
We revised the Figure legend of Fig. 4a to address this issue.
- Figure 4B,C: Please include statistics between the FRAP curves. *
We have included statistics comparing FRAP curves in Supplementary Fig. 4a-c.
- Figure 4D: It will be helpful to compare this curve to Figure S4A in the same graph. Please also include graph statistics. *
We have revised Fig. 4 to address the reviewer’s suggestion.
- Figure 5: The data points are difficult to resolve. Recommend use of color.*
We considered the suggestion, but felt it works better in the original form.
- Figure 6: This is a very general model that does not highlight the extensive experimental work performed by the authors. Recommend incorporating PGL-3, mutants and P granule factors into this model. *
We thank the reviewer for appreciating our extensive work. However, we retained the original Fig. 6 for the sake of simplicity.
- Methods, Line 939, C. elegans section: What worms were used? TH623? Please describe the genotype. *
We have included a table listing the strains used in the study and their genotype. * CROSS-CONSULTATION COMMENTS While my review was arguably the more favorable of the three, I agree with the other reviewers' comments and evaluation, particularly with Reviewer #1. As written in my review, my primary concern was the biological relevance of the work.*
Reviewer #3 (Significance (Required)):
Overall, the in vitro work presented investigating the biophysical properties of this minimal P granule system was thorough and well-analyzed, and the manuscript was clearly written. Additional citations and statistics will improve the manuscript and the strength of the conclusions, respectively. The biological relevance of this study to P granule form and function in vivo, and to condensates in vivo, is debatable. This work will interest those who study condensate biology, the biophysics of protein-protein and protein-RNA interactions, and RNA biochemists more generally.
A major impact of our work is elucidation of a novel “dynamics buffering” property within biomolecular condensates in vitro. Our in vivo data is consistent with this finding.
We have chosen two orthogonal ways of perturbing the PGL-3 protein (i.e. mutations and temperature-dependent unfolding) to assay the effect on diffusion rate against different levels of perturbation (e.g. 30% loss of a-helicity in heat-denatured PGL-3-mEGFP vs. 15% loss of a-helicity in the S1 mutant, compared to wild-type PGL-3). Studying the phase separation behavior of these “artificially-generated” constructs provided the understanding that dynamics of PGL-3 in condensates depends on inter-molecular interactions, and slower dynamics generally correlate with stronger inter-molecular interactions. Further, interactions among two or more P granule components can buffer against large change in dynamics / aggregation within the P granule phase. These insights may lay the groundwork for addressing how more “natural” modifications (e.g., post-translational modifications, high local concentration of “sticky” molecules) may influence dynamics within biomolecular condensates in vivo.
- *
Based on current knowledge of P granule composition, chaperone proteins (e.g. heat-shock family proteins) do not show abundant concentration within P granules. However, it is unclear if chaperone proteins are completely excluded from the P granule phase. Therefore, we speculate that weak interactions among two or more non-chaperone proteins contribute significantly to “dynamics buffering” within the P granule phase in vivo.
In the discussion section of the manuscript, we had speculated that “dynamics buffering” may potentially explain observations reported in the nucleolus: “Similarly, interactions among components could be a potential mechanism of storage of misfolding-prone proteins in non-aggregated state within the liquid-like nucleolus under stress in vivo (Frottin et al, 2019).”
Our finding is also relevant in the context of synthetic biology with applications that require steady diffusion rate of macromolecules during biochemical reactions within biomolecular condensates.
*I have expertise in P granules, protein/RNA biochemistry, condensate assembly, and C. elegans. *
References
Aoki ST, Kershner AM, Bingman CA, Wickens M & Kimble J (2016) PGL germ granule assembly protein is a base-specific, single-stranded RNase. Proceedings of the National Academy of Sciences of the United States of America
Aoki ST, Lynch TR, Crittenden SL, Bingman CA, Wickens M & Kimble J (2021) C. elegans germ granules require both assembly and localized regulators for mRNA repression. Nat Commun 12: 996
Cipriani PG, Bay O, Zinno J, Gutwein M, Gan HH, Mayya VK, Chung G, Chen J-X, Fahs H, Guan Y, et al (2021) Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules. Elife 10: e60833
Frottin F, Schueder F, Tiwary S, Gupta R, Körner R, Schlichthaerle T, Cox J, Jungmann R, Hartl FU & Hipp MS (2019) The nucleolus functions as a phase-separated protein quality control compartment. Science 365: 342–347
Kawasaki I, Amiri A, Fan Y, Meyer N, Dunkelbarger S, Motohashi T, Karashima T, Bossinger O & Strome S (2004) The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development. Genetics 167: 645–661
Kawasaki I, Shim YH, Kirchner J, Kaminker J, Wood WB & Strome S (1998) PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell 94: 635–645
Phillips CM & Updike DL (2022) Germ granules and gene regulation in the Caenorhabditis elegans germline. Genetics 220: iyab195
Price IF, Hertz HL, Pastore B, Wagner J & Tang W (2021) Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans. Elife 10: e72276
Saha S, Weber CA, Nousch M, Adame-Arana O, Hoege C, Hein MY, Osborne Nishimura E, Mahamid J, Jahnel M, Jawerth L, et al (2016) Polar Positioning of Phase-Separated Liquid Compartments in Cells Regulated by an mRNA Competition Mechanism. Cell 166: 1572-1584.e16
Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, Yee C, Bennett K & Strome S (2008a) Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins. Genetics 178: 1973–1987
Spike CA, Bader J, Reinke V & Strome S (2008b) DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells. Development (Cambridge, England) 135: 983–993
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
Summary:
P granules are liquid condensates found in the developing germlines and embryos of C. elegans. Prior work by the authors and others have established P granules as a tractable model to investigate the basic biophysical properties of liquid condensates. Much of the prior published work focused on specific P granule scaffold proteins, PGL-1 and PGL-3. How attributes of these PGL proteins and the effect of other P granule components affect condensate properties is not fully understood. Here, Jelenic, et al. probe the biophysical properties of PGL-3. Using recombinant protein, they show that an N-terminal, alpha-helical region of PGL-3 is sufficient for liquid condensate formation and that N-terminal assembly is required for this formation. Creation of a scrambled alpha-helical region in PGL-3 and heat treatment affects PGL-3 fluidity. This fluidity can be "rescued" in vivo and in vitro with the inclusion of other P granule factors, including wildtype PGL-3, PGL-1, GLH-1 and mRNA. The authors note an inverse correlation between fluidity and mutant PGL-3 fluorescent intensity. They propose a model that heterotypic compositions of condensates can buffer their fluidity against components with stronger multivalent interactions.
MAJOR:
- PGL-3 is a fantastic model to study the biophysical properties of a liquid condensate. But as the authors address in their discussion, the S1 mutant will likely affect the central domain folding, at its minimum causing exposure of a hydrophobic surface not typically exposed in biology. These helices are found at the terminal portion of the domain determined in the crystal structure and as depicted in the authors' Figure 1A. While the cause of S1's enhanced molecular interactions does not affect the in vitro work presented in this manuscript, it does affect how the conclusions connect to the biological nature of P granules and liquid condensates more generally.
- Recombinant PGL-3 experiments added PGL-1, GLH-1 and mRNA simultaneously and measured fluidity. It will be interesting to know which components contribute to fluidity and whether fluidity enhancement of each component is dependent on one another. Addition experiments with each component should be included and/or at least discussed in the main text.
- The biological relevance of PGL-1, GLH-1, and mRNA were not discussed in the main text. How these factors contribute to P granule assembly and function should be mentioned in the Introduction or Results.
MINOR:
- Line 20, "most non-membrane-bound compartments...have complex composition": Are there examples of condensates that do not have complex composition?
- Lines 40-43, RNA interactions driving LLPS: Please include citations from the Parker Lab (e.g. Van Treeck and Parker, Cell. 2018 doi: 10.1016/j.cell.2018.07.023)
- Line 60, condensates contain hundreds of different proteins and RNA: Please cite at least a few examples of condensates with their components identified.
- Lines 82-84, PGL-3 drives assembly: Please cite Kawasaki, et al. Genetics 2004 for the discovery of PGL-3.
- Lines 88-89, PGL-3 N-terminal fragment predominantly alpha-helical: The PGL domain structures should be cited here as supporting evidence that these regions are composed primarily of alpha helices (Aoki, et al 2016, 2021)
- Lines 158-159, driving forces for phase separation: This statement should be removed or expanded. The authors point regarding the protein concentrations is not clear here but clarified in the Discussion (Lines 691-693). Recommend removing due to its speculative nature.
- Lines 210: Add commas before and after "PGL-1 and GLH-1"
- Lines 218-219: add "and" instead of comma between PGL-1 and GLH-1
- Lines 238-239, alpha-helices: The PGL CDD structure should also be referenced here (Aoki, et al 2016).
- Lines 680-682, MEG proteins: Please cite accordingly.
- Lines 694-695, heterotypic interactions: Please cite Saha, et al. 2016.
- Figure 1: Add space between 1 and mM DTT
- Figure 2b: Please provide statistics between condensate numbers.
- Figure 4A: The region of the germline imaged and analyzed should be mentioned in the caption or the main text.
- Figure 4B,C: Please include statistics between the FRAP curves.
- Figure 4D: It will be helpful to compare this curve to Figure S4A in the same graph. Please also include graph statistics.
- Figure 5: The data points are difficult to resolve. Recommend use of color.
- Figure 6: This is a very general model that does not highlight the extensive experimental work performed by the authors. Recommend incorporating PGL-3, mutants and P granule factors into this model.
- Methods, Line 939, C. elegans section: What worms were used? TH623? Please describe the genotype.
CROSS-CONSULTATION COMMENTS
While my review was arguably the more favorable of the three, I agree with the other reviewers' comments and evaluation, particularly with Reviewer #1. As written in my review, my primary concern was the biological relevance of the work.
Significance
Overall, the in vitro work presented investigating the biophysical properties of this minimal P granule system was thorough and well-analyzed, and the manuscript was clearly written. Additional citations and statistics will improve the manuscript and the strength of the conclusions, respectively. The biological relevance of this study to P granule form and function in vivo, and to condensates in vivo, is debatable. This work will interest those who study condensate biology, the biophysics of protein-protein and protein-RNA interactions, and RNA biochemists more generally.
I have expertise in P granules, protein/RNA biochemistry, condensate assembly, and C. elegans.
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