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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Redhardt and colleagues describe a structure of the voltage and Ca-activated Slo1 channel in complex with an auxiliary subunit, γ1. In complex with γ1, Slo1 adopts an open state that closely resembles previous open state structures. Of γ1, only the single membrane-spanning helix, which binds to the periphery of the Slo1 VSD, is resolved. There, it establishes several interactions with Slo1 that authors propose may favor adoption of the open state, potentially explaining how γ1 can shift I-V profile of Slo1 to be activated at more negative membrane potentials. The interactions described fit well with existing mutagenesis analyses.

While this report provides a first glimpse of how γ1 can bind to Slo1, its impact will be minimal. It describes a single structural snapshot and there are no functional analyses presented. Additional analyses would be helpful in understanding of how γ1 can regulate Slo1 channels.

We thank the reviewer for their honest judgment. We agree that validating the structure by biochemical and/or functional data would have significantly strengthened the manuscript. However, we are convinced that our structural data alone already provides significant novel understanding of the assembly of the Slo1-γ1 complex and regulation of Slo1 by γ1. Thus, we feel that publication of this manuscript is justified by the high importance of Slo channels and our data will have an impact in the field.

__Major comments: __ 1. The authors propose several models for how γ1 regulates Slo1, yet none of them are experimentally evaluated. For example, on page 8, it is written that "we propose that the combination of three different principles, namely shape complementarity, covalent anchoring and lowering the resting state potential by a positively charged intracellular stretch, act in concert to stabilize an active VSD conformation in the Slo1-γ1 complex." This is a testable hypothesis and one that should be experimentally evaluated to better understand regulation by γ1.

We agree with the reviewer that experimental validation of this hypothesis would have been an asset. Nevertheless, we think that our structural data in context of previous functional data e.g. from Li et al. 2015,2016) and also in comparison with the other two manuscripts on the same topic which have been published while this manuscript was under review, allows us to draw conclusions about the mechanism of γ1-mediated activation of Slo1. We have now, however, toned down some of the earlier statements and changed parts of our interpretations in light of the novel findings by Yamanouchi et al. and Kallure et al.

The authors analysis of the extracellular domain of γ1 is incomplete. The only presented structure was performed with C4 symmetry imposed, in which extracellular domains were largely lost. The authors propose that these domains are dynamic and that their dynamism would enable simultaneous binding of both γ and b subunits, as occurs in cells. A more thorough analysis of the dynamics and well as potential asymmetric conformations should be performed to better understand how these domains interact with Slo1.

We completely agree with the reviewer that a thorough analysis of the extracellular domain is important and thank the reviewer for their valuable suggestions. We had attempted such analysis already from the beginning, but were not successful. More specifically, we have attempted reconstructions with lower symmetry (C2 and C1) from the beginning or by symmetry relaxation after initial C4 reconstruction. Also, we tested different masking and signal subtraction strategies in combination with different global and local refinements, as well as symmetry expansion and 3D classification. Unfortunately, none of these strategies led to a better resolved LRR module.

We now think that in comparison with Kallure et al. and Yamanouchi et al., the ice in our sample was thinner, which allowed us to reach higher resolution in the core particle (Slo1 and γ1 TM helix), but at the cost of the γ1 LRRs being denatured or at least distorted by the air-water interface.

The refinement statistics suggest that the model was incompletely refined. This reviewer was not provided with the map or models, but the validation report lists a clashscore of 9 and 5.7% of the rotamers as being outliers, both of which are high for the reported resolution of the structure. It is also strange that the Q-score varied between different γ1 protomers. Why are the four protomers not identical when the map is 4-fold symmetric? The authors should carefully inspect their model to insure that it is as correct as possible.

We thank the reviewer for pointing this out, and while the values for clashscores and rotamers were not outside the range of values typically found in many other cryo-EM structures, we agree that there was still some room for improvement. We have worked on this and could lower the values to a clashscore of 7.0 and 1.8 % rotamer outliers.

The difference in Q-score is also something not too uncommon since, while the map is indeed C4-symmetric, during model refinement the NCS restraints are not completely preventing small deviations between the protomers. We have now also successfully attempted to minimize these differences further.

Reviewer #1 (Significance (Required)):

The impact of this report is limited. Functional analyses will be necessary to uncover precisely how gamma subunits regulate Slo1 channels.

We thank the reviewer for this honest statement, but respectfully disagree. While additional functional analyses would have certainly boosted the impact, we are certain that our structural data and their interpretation will be very valuable for the field, because they provide (as stated by Reviewer 3) new insights into the regulation of Slo channel activity by the γ subunits and suggest (as stated by reviewer 2) a novel mechanism of activation of voltage-gated ion channels..

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary This study presents a high resolution cryo-EM study of a voltage-gated Ca++-dependent K+ channel in the presence of a gamma1 subunit. Analysis of the structure and sequence alignments suggest a novel mechanism of activation of voltage-gated ion channels.

__Major comments __ The major issue in this paper is that it is only a structural biology paper. There is no structure-function relationship study, no functional studies of mutants that could validate -or not- the inferred underlying mechanism. Even though the authors have identified good candidates for mutations (e.g. p. 6) they have not attempted to validate their importance experimentally. As a result, reading their discussion is somewhat frustrating and full of assumptions, as indicated by sentences (p.7) like

"a possible mechanism... might be... which would make... more likely".

"... which might act ... seems important... might indicate... might lower... likely most pronounced... could be responsible..."

"... might play an important role... does not allow a certain conclusion..."

We completely agree with the reviewer that the paper would have been much stronger if we would have been able to perform biochemical or functional assays testing mutations in the binding interface. However, this would have unfortunately been beyond the scope of the project. We are nevertheless confident that our structural data will be of value for the field, also in context of the two structure-function papers that have been published since which confirm and validate our data and provide the link to function.

__Minor comments which could be confidently addressed __ The Introduction contains no description of the state-of-the-art in the field concerning the available structures in the same system or similar ones. Hence, it is difficult to judge for people outside the field if the novelty. is incremental or significant.

We have adjusted the introduction to explicitly mention previously published structural data on the Slo channels.

References 10 and 42 (eLife) lacj some details.

We have adjusted said references accordingly.

__Reviewer #2 (Significance (Required)): ______

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Significance general assessment As it turns out, at least two papers in exactly the same field just appeared: -one in Molecular Cell by a Japanese group, which is much more developed and contains functional tests and structure-function relationships, in addition to beautiful structures (available on-line early December) https://www.sciencedirect.com/science/article/pii/S1097276523009218

-one in biorxiv, deposited yesterday https://www.biorxiv.org/content/biorxiv/early/2023/12/20/2023.12.20.572542.full.pdf

Advances wrt known results See above. As a result of these new papers in Mol Cell and biorxiv, I think the authors should reconsider submitting their article elsewhere, perhaps for a more specialized audience.

We agree with the reviewer that in light of the other two publications which both were published a while after we deposited our preprint on biorxiv and while the manuscript was under review, the uniqueness of our data is somewhat lowered. However, since our data is overall in large agreement with these two other publications, but we report a structure at significantly higher resolution and from a different species (indeed the first Slo1 structure from rabbit, a model organism of BK channel characterization in the last decades), we are confident that our data are still very valuable for the field and qualify for publication in one of the affiliate journals of Review Commons. After all, the fact that three papers reporting very similar data were published within a few weeks (plus another preprint reporting structures of a Slo channel, but unrelated to γ subunits) illustrates the importance for understanding the regulation of this essential ion channel and the impact of all structural data enhancing this understanding, and independent confirmation by three different labs is something very valuable to the community.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

"This manuscript by Redhardt et al. presents the cryo-EM structure of the Slo K+ channel from rabbits in conjunction with its auxiliary subunit, γ1, and proposes a mechanistic model for regulating channel activation. "This manuscript by Redhardt et al. presents the cryo-EM structure of the Slo K+ channel from rabbits in conjunction with its auxiliary subunit, γ1, and proposes a mechanistic model for regulating channel activation. The Slo channel, also known as the large-conductance calcium-activated potassium channel or BK channel, is an ion channel type found in various cell membranes, including neurons, muscle cells, and other tissue types. Its key features encompass Ca2+ activation, voltage dependence, and regulation by auxiliary subunits. Different auxiliary subunits have been shown to modulate channel functions distinctly; notably, the γ1 subunit enables channel activation at lower voltages compared to the wild-type channel. This manuscript offers a structural-functional framework that enhances our comprehension of how Slo channels are regulated by auxiliary subunits, such as gamma and beta subunits. While the structure of Slo channels in complex with the beta subunit is understood, the binding and interaction of the gamma subunit with the channels remain elusive due to the absence of corresponding structures. Along these lines, the presented structure here indeed provides new insights into the regulation of Slo channel activity by the gamma subunit. However, there are some important questions below that should be addressed."

1. In Figure 1D panel, the calcium ions appear to be indistinct, likely due to the figure's low resolution. The authors are recommended to enhance the figure quality and consider a better positioning to effectively illustrate the ions.

We have adjusted the coloring of calcium ions Fig. 1D to increase their visibility.

It would be beneficial for the readers if the authors provided detailed methodology explaining how they arrived at the 7% and 11% coexpression, aiding in the complex formation. Additionally, it would be informative to know the observed shift in the size exclusion chromatography (SEC) profile of Slo1-Y1 compared to apo Slo1.

We have arrived at these concentrations of the respective viruses by empirically testing ranges between 3 % and 15 %. We have now added a sentence to the manuscript to explain this.

Is there any rationale behind initially purifying using strep affinity followed by His affinity?

The idea behind using a dual-affinity protocol is to ensure that all purified complexes contain at least one copy of Slo1 and one copy of γ1. Using the Strep tag first allows to remove most contaminants already in the first step, due to its higher specificity compared to the His tag. We have added a sentence to the methods section to explain this.

Regarding the Slo1 tetramer with gamma subunit binding, are there other classes where one, two, or three gamma subunits are bound to Slo1? Or is there only one class where all protomers of Slo1 are occupied by the gamma subunit? How do these classes appear when refined in C1 symmetry? Are there classes displaying C1 or C2 symmetry, or is the four-fold symmetry preserved across all refined classes?"

We exclusively observe complexes with four γ1 subunits. This is also in agreement with the other two recent publications reporting Slo1-γ1 complex structures, but could in principle be an artifact of artificial overexpression. Also when we refine the particles in C1, we retain C4 symmetry and do not observe any classes with C2 or C1 symmetry.

The authors utilized nearly 1.9 million particles to reconstruct the final class, resulting in a high resolution. Is such a large number of particles truly necessary to achieve high resolution in this context?

The large number of particles is not strictly necessary, i.e. we could obtain similar quality by using fewer particles. In the end, we have now further classified down to ~827k particles, which very slightly improved the resolution and quality of the map.

Authros mentioned that F273 of γ1 forms pi-stacking interactions, it remains unclear with which components of the channel these interactions occur.

F273 forms (slightly distorted) T stacking interactions with F164 in S2 and F187 in S3. We now changed the sentence in the manuscript to mention the residues that line the hydrophobic pocket to make it more clear which elements contribute to the interaction with F273.

The authors propose that the disulfide bond between the γ subunit and Slo1 could play a crucial role in their interaction. Was there any observation of a covalent linkage in SDS page analysis? Furthermore, how would this interaction be affected if either cysteine C253 of gamma1 or C141 on the channel were mutated or neutralized?"

We have run all our SDS-PAGE experiments under reducing conditions, thus destroying any disulfide bridges that might have been present in the complex. We have now, however obtained a slightly better defined reconstruction (as pointed out in our answer to point 5 raised by this reviewer) where we do not see as clear continuous density anymore between the two cysteine side chains. Thus, we have removed the cystine bond from the final model and have adjusted text and figures accordingly. We still think that it might be more than coincidence that those two side chains come into such close proximity, though, and still discuss the possibility of a cystine bridge in the manuscript.

Author's state that "The presence of several immobile positive charges on the intracellular side in close proximity to the VSD as in the case of the Slo1-γ1 complex is likely to locally lower the resting state potential and repulse the gating charges, thereby reducing the energy to overcome for the VSD to transition to the active conformation." Authors need to be little more elaborative here as it is not clear what authors mean repulse of gating charges.

We have expanded our description of the proposed repulsive effect of the positive charges in the manuscript and in addition also discuss the additional role of the charges in stabilizing the Ca2+-bound conformation of the gating ring as proposed by Yamanouchi et al.

Probably beyond this study but I was wondering whether it is possible that Beta and gamma subunit can together assemble as heteromers to form a cage-like structure with contribution from both.

We agree with the reviewer that this is an interesting question which we have also thought about and one which should be tested, but as the reviewer already mentioned, this would go beyond the present study and should be subject to an independent follow-up investigation.

Are there any specific lipids observed within the structure that could potentially contribute to the functional conformation or stability of the complex?"

Given the high resolution of our structure, we observe a number of ordered lipid and detergent molecules, most of which were located at similar positions as in previous structures of Slo channels. Besides those molecules clustering in the deep cleft between neighboring voltage-sensor domains, we also observe lipid densities close to the binding site of γ1 on the distal side of the VSD. However, as their relevance for γ1 binding is unclear, we don’t discuss them in the manuscript. In general, of course, we agree with the reviewer that lipids can have a large impact on the function of membrane proteins.

It would be interesting to see if the kink in the gamma subunit is entirely neutralized through mutations of proline and glycine, how these alteration might impact the assembly of the mutated gamma subunit with the channel. The authors should provide insights into whether this mutated form of the gamma subunit assembles effectively with the channel and whether there are functional consequences associated with this alteration.

As shown by Kallure et al., substituting P270 in the kink by serine (the native residue at this position in γ3) strongly diminished the ability of γ1 to associate with Slo1 in vitro, demonstrating the importance of the kink and providing a rationale for the observed differences in the potency of the TM helices of γ1 and γ3 in Slo1 activation.

It would be generally beneficial for the authors to provide functional insights that can support the physiological relevance of this kink in the gamma subunit. Understanding the potential consequences of this mutation and its implications for the assembly and function of the channel complex will offer valuable insights into the physiological role of the kink.

We absolutely agree with the reviewer that functional insights on the relevance of the kink would be very valuable, but we think that the available experimental data together with the natural sequence differences in γ1-γ4 and the correlation with their physiological activity are very clear indications that the kink is relevant. However, future follow-up studies that prove this beyond any doubt would be valuable.

Is it known that binding of beta or gamma subunit can impact the subsequent binding of beta and gamma to channels. If it is, it need to be discussed briefly in the discussion part.

This is, to the best of our knowledge, not known. The only existing data that suggests co-presence of beta and gamma subunits on Slo1, reported in Gonzalez-Perez et al., 2015, stems from electrophysiological experiments and does not reveal anything about hierarchy and temporal order of binding events.

Reviewer #3 (Significance (Required)):

The Slo channel, also known as the large-conductance calcium-activated potassium channel or BK channel, is an ion channel type found in various cell membranes, including neurons, muscle cells, and other tissue types. Its key features encompass Ca2+ activation, voltage dependence, and regulation by auxiliary subunits. Different auxiliary subunits have been shown to modulate channel functions distinctly; notably, the γ1 subunit enables channel activation at lower voltages compared to the wild-type channel. This manuscript offers a structural-functional framework that enhances our comprehension of how Slo channels are regulated by auxiliary subunits, such as gamma and beta subunits. While the structure of Slo channels in complex with the beta subunit is understood, the binding and interaction of the gamma subunit with the channels remain elusive due to the absence of corresponding structures. Along these lines, the presented structure here indeed provides new insights into the regulation of Slo channel activity by the gamma subunit.

We thank the reviewer for this positive assessment of the data and agree that our structural data, also when regarded together with the complementary manuscripts by Kallure et al. and Yamanouchi et al., provides significant new insight into the assembly and activity of γ subunits.

You need role when there is not a built in tag for the behaviour you would like to have

That's easily done.

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Author Response

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

The authors of this study seek to visualize NS1 purified from dengue virus infected cells. They infect vero cells with DV2-WT and DV2 NS1-T164S (a mutant virus previously characterized by the authors). The authors utilize an anti-NS1 antibody to immunoprecipitate NS1 from cell supernatants and then elute the antibody/NS1 complex with acid. The authors evaluate the eluted NS1 by SDS-PAGE, Native Page, mass spec, negative-stain EM, and eventually Cryo-EM. SDS-PAGE, mas spec, and native page reveal a >250 Kd species containing both NS1 and the proteinaceous component of HDL (ApoA1). The authors produce evidence to suggest that this population is predominantly NS1 in complex with ApoA1. This contrasts with recombinantly produced NS1 (obtained from a collaborator) which did not appear to be in complex with or contain ApoA1 (Figure 1C). The authors then visualize their NS1 stock in complex with their monoclonal antibody by CryoEM. For NS1-WT, the major species visualized by the authors was a ternary complex of an HDL particle in complex with an NS1 dimer bound to their mAB. For their mutant NS1-T164S, they find similar structures, but in contrast to NS1-WT, they visualize free NS1 dimers in complex with 2 Fabs (similar to what's been reported previously) as one of the major species. This highlights that different NS1 species have markedly divergent structural dynamics. It's important to note that the electron density maps for their structures do appear to be a bit overfitted since there are many regions with electron density that do not have a predicted fit and their HDL structure does not appear to have any predicted secondary structure for ApoA1. The authors then map the interaction between NS1 and ApoA1 using cross-linking mass spectrometry revealing numerous NS1-ApoA1 contact sites in the beta-roll and wing domain. The authors find that NS1 isolated from DENV infected mice is also present as a >250 kD species containing ApoA1. They further determine that immunoprecipitation of ApoA1 out of the sera from a single dengue patient correlates with levels of NS1 (presumably COIPed by ApoA1) in a dose-dependent manner.

In the end, the authors make some useful observations for the NS1 field (mostly confirmatory) providing additional insight into the propensity of NS1 to interact with HDL and ApoA1. The study does not provide any functional assays to demonstrate activity of their proteins or conduct mutagenesis (or any other assays) to support their interaction predications. The authors assertion that higher-order NS1 exists primarily as a NS1 dimer in complex with HDL is not well supported as their purification methodology of NS1 likely introduces bias as to what NS1 complexes are isolated. While their results clearly reveal NS1 in complex with ApoA1, the lack of other NS1 homo-oligomers may be explained by how they purify NS1 from virally infected supernatant. Because NS1 produced during viral infection is not tagged, the authors use an anti-NS1 monoclonal antibody to purify NS1. This introduces a source of bias since only NS1 oligomers with their mAb epitope exposed will be purified. Further, the use of acid to elute NS1 may denature or alter NS1 structure and the authors do not include controls to test functionality of their NS1 stocks (capacity to trigger endothelial dysfunction or immune cell activation). The acid elution may force NS1 homo-oligomers into dimers which then reassociate with ApoA1 in a manner that is not reflective of native conditions. Conducting CryoEM of NS1 stocks only in the presence of full-length mAbs or Fabs also severely biases what species of NS1 is visualized since any NS1 oligomers without the B-ladder domain exposed will not be visualized. If the residues obscured by their mAb are involved in formation of higher-order oligomers then this antibody would functionally inhibit these species from forming. The absence of critical controls, use of one mAb, and acid elution for protein purification severely limits the interpretation of these data and do not paint a clear picture of if NS1 produced during infection is structurally distinct from recombinant NS1. Certainly there is novelty in purifying NS1 from virally infected cells, but without using a few different NS1 antibodies to purify NS1 stocks (or better yet a polyclonal population of antibodies) it's unclear if the results of the authors are simply a consequence of the mAb they selected.

Data produced from numerous labs studying structure and function of flavivirus NS1 proteins provide diverse lines of evidence that the oligomeric state of NS1 is dynamic and can shift depending on context and environment. This means that the methodology used for NS1 production and purification will strongly impact the results of a study. The data in this manuscript certainly capture one of these dynamic states and overall support the general model of a dynamic NS1 oligomer that can associate with both host proteins as well as itself but the assertions of this manuscript are overall too strong given their data, as there is little evidence in this manuscript, and none available in the large body of existing literature, to support that NS1 exists only as a dimer associated with ApoA1. More likely the results of this paper are a result of their NS1 purification methodology.

Suggestions for the Authors:

Major:

(1) Because of the methodology used for NS1 purification, it is not clear from the data provided if NS1 from viral infection differs from recombinant NS1. Isolating NS1 from viral infection using a polyclonal antibody population would be better to answer their questions. On this point, Vero cells are also not the best candidate for their NS1 production given these cells do not come from a human. A more relevant cell line like U937-DC-SIGN would be preferable.

We performed an optimization of sNS1 secretion from DENV infection in different cell lines (Author response image 1 below) to identify the best cell line candidate to obtain relatively high yield of sNS1 for the study. As shown in Author response image 1, the levels of sNS1 in the tested human cell lines Huh7 and HEK 293T were at least 3-5 fold lower than in Vero cells. Although using a monocytic cell line expressing DC-SIGN as suggested by the reviewer would be ideal, in our experience the low infectivity of DENV in monocytic cell lines will not yield sufficient amount of sNS1 needed for structural analysis. For these practical reasons we decided to use the closely related non-human primate cell line Vero for sNS1 production supported by our optimization data.

Author response image 1.

sNS1 secretion in different mammalian and mosquito cell lines after DENV2 infection. The NS1 secretion level is measured using PlateliaTM Dengue NS1 Ag ELISA kit (Bio-Rad) on day 3 (left) and day 5 (right) post infection respectively.

(2) The authors need to support their interaction predictions and models via orthogonal assays like mutagenesis followed by HDL/ApoA1 complexing and even NS1 functional assays. The authors should be able to mutate NS1 at regions predicted to be critical for ApoA1/HDL interaction. This is critical to support the central conclusions of this manuscript.

In our previous publication (Chan et al., 2019 Sci Transl Med), we used similarly purified sNS1 (immunoaffinity purification followed by acid elution) from infected culture supernatants from both DENV2 wild-type and T164S mutant (both also studied in the present work) to carry out stimulation assay on human PBMCs as described by other leading laboratories investigating NS1 (Modhiran et al., 2015 Sci Transl Med). For reader convenience we have extracted the data from our published paper and present it as Author response image 2 below.

Author response image 2.

(A) IL6 and (B) TNFa concentrations measured in the supernatants of human PBMCs incubated with either 1µg/ml or 10µg/ml of the BHK-21 immunoaffinity-purified WT and TS mutant sNS1 for 24 hours. Data is adapted from Chan et al., 2019.

Incubation of immunoaffinity-purified sNS1 (WT and TS) with human PBMCs from 3 independent human donors triggered the production of proinflammatory cytokines IL6 and TNF in a concentration dependent manner (Author response image 2), consistent with the published data by Modhiran et al., 2015 Sci Transl Med. Interestingly the TS mutant derived sNS1 induced a higher proinflammatory cytokines production than WT virus derived sNS1 that appears to correlate with the more lethal and severe disease phenotype in mice as also reported in our previous work (Chan et al., 2019). Additionally, the functionality of our immune-affinity purified infection derived sNS1 (isNA1) is now further supported by our preliminary results on the NS1 induced endothelial cell permeability assay using the purified WT and mutant isNS1 (Author response image 3). As shown in Author response image 3, both the isNS1wt and isNS1ts mutant reduced the relative transendothelial resistance from 0 to 9 h post-treatment, with the peak resistance reduction observed at 6 h post-treatment, suggesting that the purified isNS1 induced endothelial dysfunction as reported in Puerta-Guardo et al., 2019, Cell Rep.) It is noteworthy that the isNS1 in our study behaves similarly as the commercial recombinant sNS1 (rsNS1 purchased from the same source used in study by Puerta-Guardo et al., 2019) in inducing endothelial hyperpermeability. Collectively our previous published and current data suggest that the purified isNS1 (as a complex with ApoA1) has a pathogenic role in disease pathogenesis that is also supported in a recent publication by Benfrid et al., EMBO 2022). The acid elution has not affected the functionality of NS1.

Author response image 3.

Functional assessment of isNS1wt and isNS1ts on vascular permeability in vitro. A trans-endothelial permeabilty assay via measurement of the transendothelial electrical resistance (TEER) on human umbilical vascular endothelial cells (hUVEC) was performed, as described previously (Puerta-Guardo et al., 2019, Cell Rep). Ovalbumin serves as the negative control, while TNF-α and rsNS1 serves as the positive controls.

We agree with reviewer about the suggested mutagnesis study. We will perform site-directed mutagenesis at selected residues and further structural and functional analyses and report the results in a follow-up study.

(3) The authors need to show that the NS1 stocks produced using acid elution are functional compared to standard recombinantly produced NS1. Do acidic conditions impact structure/function of NS1?

We are providing the same response to comments 1 & 2 above. We would like to reiterate that we have previously used sNS1 from immunoaffinity purification followed by acid elution to test its function in stimulating PBMCs to produce pro-inflammatory cytokines (Chan et al., 2019; Author response image 2). Similar to Modhiran et al. (2015) and Benfrid et al. (2022), the sNS1 that we extracted using acid elution are capable of activating PBMCs to produce pro-inflammatory cytokines. We have now further demonstrated the ability of both WT and TS isNS1 in inducing endothelial permeability in vitro in hUVECs, using the TEER assay (Author response image 3). Based on the data presented in the rebuttal figures as well as our previous publication we do not think that the acid elution has a significant impact on function of isNS1.

We performed affinity purification to enrich the complex for better imaging and analysis (Supp Fig. 1b) since the crude supernatant contains serum proteins and serum-free infections also do not provide sufficient isNS1. The major complex observed in negative stain is 1:1 (also under acidic conditions which implies that the complex are stable and intact). We agree that it is possible that other oligomers can form but we have observed only a small population (74 out of 3433 particles, 2.15%; 24 micrographs) of HDL:sNS1 complex at 1:2 ratio as shown in the Author response image 4 below and in the manuscript (p. 4 lines 114-117, Supp Fig. 1c). Other NS1 dimer:HDL ratios including 2:1 and 3:1 have been reported by Benfrid et al., 2022 by spiking healthy sera with recombinant sNS1 and subsequent re-affinity purification. However, this method used an approximately 8-fold higher sNS1 concentration (400 ug/mL) than the maximum clinically reported concentration (50 ug/mL) (Young et al., 2000; Alcon et al., 2002; Libraty et al., 2002). In our hands, the sNS1 concentration in the concentrated media from in vitro infection was quantified as 30 ug/mL which is more physiologically relevant.

We conclude that the integrity of the HDL of the complex is not lost during sample preparation, as we are able to observe the complex under the negative staining EM as well as infer from XL-MS. Our rebuttal data and our previous studies with our acid-eluted isNS1 from immunoaffinity purification clearly show that our protein is functional and biologically relevant.

Author response image 4.

(A) Representative negative stain micrograph of sNS1wt (B) Representative 2D averages of negative stained isNS1wt. Red arrows indicating the characteristic wing-like protrusions of NS1 inserted in HDL. (C) Data adapted from Figure 2 in Benfrid et al. (2022).

(4) Overall, the data obtained from the mutant NS1 (contrasted to WT NS1) reveals how dynamic the oligomeric state of NS1 proteins are but the authors do not provide any insight into how/why this is, some additional lines of evidence using either structural studies or mutagenesis to compare WT and their mutant and even NS1 from a different serotype of DENV would help the field to understand the dynamic nature of NS1.

The T164S mutation in DENV2 NS1 was proposed as the residue associated with disease severity in 1997 Cuban dengue epidemic (Halsted SB. “Intraepidemic increases in dengue disease severity: applying lessons on surveillance and transmission”. Whitehorn, J., Farrar. J., Eds., Clinical Insights in Dengue: Transmission, Diagnosis & Surveillance. The Future Medicine (2014), pp. 83-101). Our previous manuscript examined this mutation by engineering it into a less virulent clade 2 DENV isolated in Singapore and showed that sNS1 production was higher without any change in viral RNA replication. Transcript profiling of mutant compared to WT virus showed that genes that are usually induced during vascular leakage were upregulated for the mutant. We also showed that infection of interferon deficient AG129 mice with the mutant virus resulted in disease severity, increased complement protein expression in the liver, tissue inflammation and greater mortality compared to WT virus infected mice. The lipid profiling in our study (Chan et al., 2019) suggested small differences with WT but was overall similar to HDL as described by Gutsche et al. (2011). We were intrigued by our functional results and wanted to explore more deeply the impact of the mutation on sNS1 structure which at that stage was widely believed to be a trimer of NS1 dimers with a central channel (~ X Å) stuffed with lipid as established in several seminal publications (Flamand et al., 1999; Gutsche et al., 2011; Muller et al., 2012). In fact “This Week in Virology” netcast (https://www.microbe.tv/twiv/twiv-725/) discussed two back-to-back publications in Science (Modhiran et al., 371(6625)190-194; Biering et al., Science 371(6625):194-200)) which showed that therapeutic antibodies can ameliorate the NS1 induced pathogenesis and expert discussants posed questions that also pointed to the need for more accurate definition of the molecular composition and architecture of the circulating NS1 complex during virus infection to get a clearer handle on its pathogenic mechanism. Our current studies and also the recent high resolution cryoEM structures (Shu et al., 2022) do not support the notion of a central channel “stuffed with lipid”. Even in the rare instances where trimer of dimers are shown, the narrow channel in the center could only accommodate one molecule of lipoid molecule no bigger than a typical triglyceride molecule. This hexamer model cannot explain the lipid proeotmics data in the literature.

In our study we observed predominantly 1:1 NS1 dimer to HDL (~30 μg/mL) mirroring maximum clinically reported concentration of sNS1 in the sera of DENV patients (40-50 μg/mL) as we highlighted in our main text (P. 18, lines 461-471). What is often quoted (also see later) is the recent study of Flamand & co-workers which show 1-3 NS1 dimers per HDL (Benfrid et al, 2022) by spiking rsNS1 (400 μg/mL) with HDL. This should not be confused with the previous models which suggested a lipid filled central channel holding together the hexamer. The use of physiologically relevant concentrations is important for these studies as we have highlighted in our main text (P. 18, lines 461-471).

Our interpretation for the mutant (isNS1ts) is that it is possible that the hydrophilic serine at residue 164 located in the greasy finger loop may weaken the isNS1ts binding to HDL hence the observation of free sNS1 dimers in our immunoaffinity purified (acid eluted sample). The disease severity and increased complement protein expression in AG129 mice liver can be ascribed to weakly bound mutant NS1 with fast on/off rate with HDL being transported to the liver where specific receptors bind to free sNS1 and interact with effector proteins such as complement to drive inflammation and associated pathology. Our indirect support for this is that the XL-MS analysis of purified isNS1ts identified only 7 isNS1ts:ApoA1 crosslinks while 25 isNS1wt:ApoA1 crosslinks were identified from purified isNS1wt (refer to Fig. 4 and Supp. Fig. 8).

Taken together, the cryoEM and XL-MS analysis of purified isNS1ts suggest that isNS1ts has weaker affinity for HDL compared to isNS1wt. We welcome constructive discussion on our interpretation that we and others will hopefully obtain more data to support or deny our proposed explanation. Our focus has been to compare WT with mutant sNS1 from DENV2 and we agree that it will be useful to study other serotypes.

Reviewer #2:

CryoEM:

Some of the neg-stain 2D class averages for sNS1 in Fig S1 clearly show 1 or 2 NS1 dimers on the surface of a spherical object, presumably HDL, and indicate the possibility of high-quality cryoEM results. However, the cryoEM results are disappointing. The cryo 2D class averages and refined EM map in Fig S4 are of poor quality, indicating sub-optimal grid preparation or some other sample problem. Some of the FSC curves (2 in Fig S7 and 1 in Fig S6) have extremely peculiar shapes, suggesting something amiss in the map refinement. The sharp drop in the "corrected" FSC curves in Figs S5c and S6c (upper) indicate severe problems. The stated resolutions (3.42 & 3.82 Å) for the sNS1ts-Fab56.2 are wildly incompatible with the images of the refined maps in Figs 3 & S7. At those resolutions, clear secondary structural elements should be visible throughout the map. From the 2D averages and 3D maps shown in the figures this does not seem to be the case. Local resolution maps should be shown for each structure.

The same sample is used for negative staining and the cryoEM results presented. The cryoEM 2D class averages are similar to the negative stain ones, with many spherical-like densities with no discernible features, presumably HDL only or the NS1 features are averaged out. The key difference lies in the 2D class averages where the NS1 could be seen. The side views of NS1 (wing-like protrusion) are more obvious in the negative stain while the top views of NS1 (cross shaped-like protrusion) are more obvious under cryoEM. HDL particles are inherently heterogeneous and known to range from 70-120 Å, this has been highlighted in the main text (p. 8, lines 203 and 228). This helps to explain why the reviewer may find the cryoEM result disappointing. The sample is inherently challenging to resolve structurally as it is (not that the sample is of poor quality). In terms of grid preparation, Supp Fig 4b shows a representative motion-corrected micrograph of the isNS1ts sample whereby individual particles can be discerned and evenly distributed across the grid at high density.

We acknowledge that most of the dips in the FSC curves (Fig S5-7) are irregular and affect the accuracy of the stated resolutions, particularly for the HDL-isNS1ts-Fab56.2 and isNS1ts-Fab56.2 maps for which the local resolution maps are shown (Fig S7d-e). Probable reasons affecting the FSC curves include (1) the heterogeneous nature of HDL, (2) preferred orientation issue (p 7, lines 198 -200), and (3) the data quality is intrinsically less ideal for high resolution single particle analysis. Optimizing of the dynamic masking such that the mask is not sharper than the resolution of the map for the near (default = 3 angstroms) and far (12 angstroms) parameters during data processing, ranging from 6 - 12 and 14 - 20 respectively, did not help to improve the FSC curves. To report a more accurate global resolution, we have revised the figures S5-7 with new FSC curve plots generated using the remote 3DFSC processing server.

Regardless, the overall architecture and the relative arrangement of NS1 dimer, Fab, and HDL are clearly visible and identifiable in the map. These results agree well with our biochemical data and mass-spec data.

The samples were clearly challenging for cryoEM, leading to poor quality maps that were difficult to interpret. None of the figures are convincing that NS1, Ab56.2 or Fab56.2 are correctly fit into EM maps. There is no indication of ApoA1 helices. Details of the fit of models to density for key regions of the higher-resolution EM maps should be shown and the models should be deposited in the PDB. An example of modeling difficulty is clear in the sNS1ts dimer with bound Fab56.2 (figs 3c & S7e). For this complex, the orientation of the Fab56.2 relative to the sNS1ts dimer in this submission (Fig 3c) is substantially different than in the bioRxiv preprint (Fig 3c). Regions of empty density in Fig 3c also illustrate the challenge of building a model into this map.

We acknowledge the modelling challenge posed by low resolution maps in general, such as the handedness of the Fab molecule as pointed out by the reviewer (which is why others have developed the use of anti-fab nanobody to aid in structure determination among other methods). The change in orientation of the Fab56.2 relative to the sNS1ts dimer was informed by the HDX-MS results which was not done at the point of bioRxiv preprint mentioned. With regards to indication of ApoA1 helices, this is expected given the heterogeneous nature of HDL. To the best of our knowledge, engineered apoA1 helices were also not reported in many cryoEM structures of membrane proteins solved in membrane scaffold protein (MSP) nanodiscs. This is despite nanodiscs, comprised of engineered apoA1 helices, having well-defined size classifications.

Regions of weak density in Fig 3c is expected due to the preferred orientation issue acknowledged in the results section of the main text (p. 9, line 245). The cryoEM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-36483 (isNS1ts:Fab56.2) and EMD-36480 (Fab56.2:isNS1ts:HDL). The protein model files for isNS1ts:Fab56.2 and Fab56.2:isNS1ts:HDL model are available upon request. Crosslinking MS raw files and the search results can be downloaded from https://repository.jpostdb.org/preview/14869768463bf85b347ac2 with the access code: 3827. The HDX-MS data is deposited to the ProteomeXchange consortium via PRIDE partner repository51 with the dataset identifier PXD042235.

Mass spec:

Crosslinking-mass spec was used to detect contacts between NS1 and ApoA1, providing strong validation of the sNS1-HDL association. As the crosslinks were detected in a bulk sample, they show that NS1 is near ApoA1 in many/most HDL particles, but they do not indicate a specific protein-protein complex. Thus, the data do not support the model of an NS1-ApoA1 complex in Fig 4d. Further, a specific NS1-ApoA1 interaction should have evidence in the EM maps (helical density for ApoA1), but none is shown or mentioned. If such exists, it could perhaps be visualized after focused refinement of the map for sNS1ts-HDL with Fab56.2 (Fig S7d). The finding that sNS1-ApoA1 crosslinks involved residues on the hydrophobic surface of the NS1 dimer confirms previous data that this NS1 surface engages with membranes and lipids.

We thank the reviewer for the comment. The XL-MS is a method to identify the protein-protein interactions by proximity within the spacer arm length of the crosslinker. The crosslinking MS data do support the NS1-ApoA1 complex model obtained by cryo-EM because the identified crosslinks that are superimposed on the EM map are within the cut-off distance of 30 Å. We agree that the XL-MS data do not dictate the specific interactions between specific residues of NS1-ApoA1 in the EM model. We also do not claim that specific residue of NS1 in beta roll or wing domain is interacting with specific residue of ApoA1 in H4 and H5 domain. We claim that beta roll and wing domain regions of NS1 are interacting with ApoA1 in HDL indicating the proximity nature of NS1-ApoA1 interactions as warranted by the XL-MS data.

As explained in the previous response on the lack of indication of ApoA1 helical density, this is expected given the heterogeneous nature of HDL. It is typical to see lipid membranes as unstructured and of lower density than the structured protein. In our study, local refinement was performed on either the global map (presented in Fig S7d) or focused on the NS1-Fab region only. Both yielded similar maps as illustrated in the real space slices shown in Author response image 5. The mask and map overlay is depicted in similar orientations to the real space slices, and at different contour thresholds at 0.05 (Author response image 5e) and 0.135 (Author response image 5f). While the overall map is of poor resolution and directional anisotropy evident, there is clear signal differences in the low density region (i.e. the HDL sphere) indicative of NS1 interaction with ApoA1 in HDL, extending from the NS1 wing to the base of the HDL sphere.

Author response image 5.

Real Space Slices of map and mask used during Local Refinement for overall structure (a-b) and focused mask on NS1 region (c-d). The corresponding map (grey) contoured at 0.05 (e) and 0.135 (f) in similar orientations as shown for the real space slices of map and masks. The focused mask of NS1 used is colored in semi-transparent yellow. Real Space Slices of map and mask are generated during data processing in Cryosparc 4.0 and the map figures were prepared using ChimeraX.

Sample quality:

The paper lacks any validation that the purified sNS1 retains established functions, for example the ability to enhance virus infectivity or to promote endothelial dysfunction.

Please see detailed response for question 2 in Reviewer #1’s comments. In essence, we have showed that both isNS1wt and isNS1ts are capable of inducing endothelial permeability in an in vitro TEER assay (Rebuttal Fig 3) and also in our previous study that quantified inflammation in human PBMC’s (Rebuttal Fig 2).

Peculiarities include the gel filtration profiles (Fig 2a), which indicate identical elution volumes (apparent MWs) for sNS1wt-HDL bound to Ab562 (~150 kDa) and to the ~3X smaller Fab56.2 (~50 kDa). There should also be some indication of sNS1wt-HDL pairs crosslinked by the full-length Ab, as can be seen in the raw cryoEM micrograph (Fig S5b).

Obtaining high quality structures is often more demanding of sample integrity than are activity assays. Given the low quality of the cryoEM maps, it's possible that the acidification step in immunoaffinity purification damaged the HDL complex. No validation of HDL integrity, for example with acid-treated HDL, is reported.

Please see detailed response for question 3 in Reviewer #1’s comments.

Acid treatment is perhaps discounted by a statement (line 464) that another group also used immunoaffinity purification in a recent study (ref 20) reporting sNS1 bound to HDL. However the statement is incorrect; the cited study used affinity purification via a strep-tag on recombinant sNS1.

We thank the Reviewer for pointing this out and have rewritten this paragraph instead (p 18, line 445-455). We also expanded our discussion to highlight our prior functional studies showing that acid-eluted isNS1 proteins do induce endothelial hyperpermeability (p 18-19, line 470-476).

Discussion:

The Discussion reflects a view that the NS1 secreted from virus-infected cells is a 1:1 sNS1dimer:HDL complex with the specific NS1-ApoA1 contacts detected by crosslinking mass spec. This is inconsistent with both the neg-stain 2D class average with 2 sNS1 dimers on an HDL (Fig S1c) and with the recent study of Flamand & co-workers showing 1-3 NS1 dimers per HDL (ref 20). It is also ignores the propensity of NS1 to associate with membranes and lipids. It is far more likely that NS1 association with HDL is driven by these hydrophobic interactions than by specific protein-protein contacts. A lengthy Discussion section (lines 461-522) includes several chemically dubious or inconsistent statements, all based on the assumption that specific ApoA1 contacts are essential to NS1 association with HDL and that sNS1 oligomers higher than the dimer necessarily involve ApoA1 interaction, conclusions that are not established by the data in this paper.

We thank the Reviewer and have revised our discussion to cover available structural and functional data to draw conclusions that invariably also need further validation by others. One point that is repeatedly brought up by Reviewer 1 & 2 is the quality and functionality of our sample. Our conclusion now reiterates this point based on our own published data (Chan et al., 2019) and also the TEER assay data provided as Author response image 3.

Reviewer #1 (Recommendations For The Authors):

Minor:

(1) Fig. S3B, should the label for lane 4 be isNS1? In figure 1C you do not see ApoA1 for rsNS1 but for S3B you do? Which is correct?

This has been corrected in the Fig. S3B, the label for lane 4 has been corrected to isNS1 and lane 1 to rsNS1, where no ApoA1 band (25 kDa) is found.

(2) Line 436, is this the correct reference? Reference 43?

This has been corrected in the main text. (p 20, Line 507; Lee et al., 2020, J Exp Med).

Reviewer #2 (Recommendations For The Authors):

The cryoEM data analysis is incompletely described. The process (software, etc) leading to each refined EM map should be stated, including the use of reference structures in any step. These details are not in the Methods or in Figs S4-7, as claimed in the Methods. The use of DeepEMhancer (which refinements?) with the lack of defined secondary structural features in the maps and without any validation (or discussion of what was used as "ground truth") is concerning. At the least, the authors should show pre- and post-DeepEMhancer maps in the supplemental figures.

The data processing steps in the Methods section have been described with improved clarity. DeepEMhancer is a deep learning solution for cryo-EM volume post-processing to reduce noise levels and obtain more detailed versions of the experimental maps (Sanchez-Garcia, et al., 2021). DeepEMhancer was only used to sharpen the maps and reduce the noise for classes 1 and 2 of isNS1wt in complex with Ab56.2 for visualization purpose only and not for any refinements. To avoid any confusion, the use of DeepEMhancer has been removed from the supp text and figures.

Line 83 - "cryoEM structures...recently reported" isn't ref 17

This reference has been corrected in to Shu et al. (2022) in p 3, line 83.

Fig. S3 - mis-labeled gel lanes

This has been corrected in the Fig. S3B, the label for lane 4 has been corrected to isNS1 and lane 1 to rsNS1.

Fig S6c caption - "Representative 2D classes of each 3D classes, white bar 100 Å. Refined 3D map for classes 1 and 2 coloured by local resolution". The first sentence is unclear, and there is no white scale bar and no heat map.

Fig S6c caption has been corrected to “Representative 3D classes contoured at 0.06 and its particle distribution as labelled and coloured in cyan. Scale bar of 100 Å as shown. Refined 3D maps and their respective FSC resolution charts and posterior precision directional distribution as generated in crysosparc4.0”.

Reviewer #2 (Public Review):

Summary:

Chew et al describe interaction of the flavivirus protein NS1 with HDL using primarily cryoEM and mass spec. The NS1 was secreted from dengue virus infected Vero cells, and the HDL were derived from the 3% FBS in the culture media. NS1 is a virulence factor/toxin and is a biomarker for dengue infection in patients. The mechanisms of its various activities in the host are incompletely understood. NS1 has been seen in dimer, tetramer and hexamer forms. It is well established to interact with membrane surfaces, presumably through a hydrophobic surface of the dimer form, and the recombinant protein has been shown to bind HDL. In this study, cryoEM and crosslinking-mass spec are used to examine NS1 secreted from virus-infected cells, with the conclusion that the sNS1 is predominantly/exclusively HDL-associated through specific contacts with the ApoA1 protein.

Strengths: The experimental results are consistent with previously published data.

Weaknesses:

CryoEM:<br /> Some of the neg-stain 2D class averages for sNS1 in Fig S1 clearly show 1 or 2 NS1 dimers on the surface of a spherical object, presumably HDL, and indicate the possibility of high-quality cryoEM results. However, the cryoEM results are disappointing. The cryo 2D class averages and refined EM map in Fig S4 are of poor quality, indicating sub-optimal grid preparation or some other sample problem. Some of the FSC curves (2 in Fig S7 and 1 in Fig S6) have extremely peculiar shapes, suggesting something amiss in the map refinement. The sharp drop in the "corrected" FSC curves in Figs S5c and S6c (upper) indicate severe problems. The stated resolutions (3.42 & 3.82 Å) for the sNS1ts-Fab56.2 are wildly incompatible with the images of the refined maps in Figs 3 & S7. At those resolutions, clear secondary structural elements should be visible throughout the map. From the 2D averages and 3D maps shown in the figures, this does not seem to be the case. Local resolution maps should be shown for each structure.

The samples were clearly challenging for cryoEM, leading to poor quality maps that were difficult to interpret. None of the figures are convincing that NS1, Ab56.2 or Fab56.2 are correctly fit into EM maps. There is no indication of ApoA1 helices. Details of the fit of models to density for key regions of the higher-resolution EM maps should be shown and the models should be deposited in the PDB. An example of modeling difficulty is clear in the sNS1ts dimer with bound Fab56.2 (figs 3c & S7e). For this complex, the orientation of the Fab56.2 relative to the sNS1ts dimer in this submission (Fig 3c) is substantially different than in the bioRxiv preprint (Fig 3c). Regions of empty density in Fig 3c also illustrate the challenge of building a model into this map.

Mass spec:<br /> Crosslinking-mass spec was used to detect contacts between NS1 and ApoA1, providing strong validation of the sNS1-HDL association. As the crosslinks were detected in a bulk sample, they show that NS1 is near ApoA1 in many/most HDL particles, but they do not indicate a specific protein-protein complex. Thus, the data do not support the model of an NS1-ApoA1 complex in Fig 4d. Further, a specific NS1-ApoA1 interaction should have evidence in the EM maps (helical density for ApoA1), but none is shown or mentioned. If such exists, it could perhaps be visualized after focused refinement of the map for sNS1ts-HDL with Fab56.2 (Fig S7d). The finding that sNS1-ApoA1 crosslinks involved residues on the hydrophobic surface of the NS1 dimer confirms previous data that this NS1 surface engages with membranes and lipids.

Sample quality:<br /> The paper lacks any validation that the purified sNS1 retains established functions, for example the ability to enhance virus infectivity or to promote endothelial dysfunction. Peculiarities include the gel filtration profiles (Fig 2a), which indicate identical elution volumes (apparent MWs) for sNS1wt-HDL bound to Ab562 (~150 kDa) and to the ~3X smaller Fab56.2 (~50 kDa). There should also be some indication of sNS1wt-HDL pairs crosslinked by the full-length Ab, as can be seen in the raw cryoEM micrograph (Fig S5b).

Obtaining high quality structures is often more demanding of sample integrity than are activity assays. Given the low quality of the cryoEM maps, it's possible that the acidification step in immunoaffinity purification damaged the HDL complex. No validation of HDL integrity, for example with acid-treated HDL, is reported. Acid treatment is perhaps discounted by a statement (line 464) that another group also used immunoaffinity purification in a recent study (ref 20) reporting sNS1 bound to HDL. However the statement is incorrect; the cited study used affinity purification via a strep-tag on recombinant sNS1.

Discussion:<br /> The Discussion reflects a view that the NS1 secreted from virus-infected cells is a 1:1 sNS1dimer:HDL complex with the specific NS1-ApoA1 contacts detected by crosslinking mass spec. This is inconsistent with both the neg-stain 2D class average with 2 sNS1 dimers on an HDL (Fig S1c) and with the recent study of Flamand & co-workers showing 1-3 NS1 dimers per HDL (ref 20). It also ignores the propensity of NS1 to associate with membranes and lipids. It is far more likely that NS1 association with HDL is driven by these hydrophobic interactions than by specific protein-protein contacts. A lengthy Discussion section (lines 461-522) includes several chemically dubious or inconsistent statements, all based on the assumption that specific ApoA1 contacts are essential to NS1 association with HDL and that sNS1 oligomers higher than the dimer necessarily involve ApoA1 interaction, conclusions that are not established by the data in this paper.

Additional comments on the revised manuscript:

Comments on the structures:

The authors kindly provided their fitted atomic models for the 2 reported structures. The EM maps are available in the EMDB. Based on these materials, the derived structures are not well supported due to problems with the models, the maps, and the fit of models to maps.

Quick inspection revealed that the models for both structures are implausible due to a large steric clash of Fab56.2 and the end of the NS1. The Fab and NS1 protein backbones interpenetrate by nearly 20 Å. This substantial overlap exists for all 3 Fab56.2-NS1 interactions in the 2 structures, and is also visible in the perpendicular views of the NS1 dimer with 2 bound Fab56.2 in Fig. 2c. It appears that the Fab56.2 model was jammed into the NS1 model in order to bring all domains inside the density envelope at the threshold chosen to display the map. The poor fit of model to map is also clear in several protruding density regions without any model.

The fits of both atomic models to the maps are questionable because<br /> - The maps suffer from severe preferred orientation problems, as seen in the streaky tubes of density. The streaks in both maps do not match the NS1 beta strands of the fitted models.<br /> - The shape of the modeled ApoA1 helical ring surrounding the HDL does not match the shape of the EM density. In some regions, the ApoA1 helices are inside the rather strong density for the spherical HDL, but in other regions the helices are outside the density.<br /> - Both maps have regions of strong density that are adjacent to NS1 but lack any protein model, while other parts of the structure, including the beta-roll domain, lack density.<br /> - The claimed 4.3-Å resolution of the NS1-Fab56.2 complex is wildly overstated. The local resolution of ~2.5 Å for the "best" part of the structure (Supp Fig. 7E) appears to pertain to the beta strands at the center of the NS1 dimer. However, these density streaks do not match the beta strands of the fit model.<br /> - The manuscript lacks statistics on the fit of model to map. A standard cryo-EM "Table 1" should include more than is presented in Supp Table 1. The fitted model for at least the higher resolution structure should be deposited in the PDB.

Comments on the structure interpretation:

By now it should be abundantly clear that the oligomer state of NS1 is dynamic and highly sensitive to environmental conditions and to each sample's "history". For the reasons pointed out by reviewer 1, it is not clear that the immunoaffinity purification method captured all forms of sNS1 equally. Thus, the authors insistence that NS1 secreted from virus-infected cells is predominantly bound to HDL particles in a ratio of 1 NS1 dimer per HDL is not well supported. They employ similar arguments to challenge the discovery of sNS1 as a lipoprotein particle (PNAS 2011), contending that the 2011 finding was an artefact of recombinant NS1 production and is irrelevant to sNS1 from a virus infection. The several published structures of NS1 oligomers reveal a large degree of asymmetry in dimer-dimer interaction, consistent with the ability of NS1 to dynamically associate with a variety of hydrophobic entities.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This important study elucidates the molecular divergence of caspase 3 and 7 in the vertebrate lineage. Convincing biochemical and mutational data provide evidence that in humans, caspase 7 has lost the ability to cleave gasdermin E due to changes in a key residue, S234. However, the physiological relevance of the findings is incomplete and requires further experimental work.

Public Reviews:

Reviewer #1 (Public Review):

Summary

In this study, Xu et al. provide insights into the substrate divergence of CASP3 and CASP7 for GSDME cleavage and activation during vertebrate evolution vertebrates. Using biochemical assays, domain swapping, site-directed mutagenesis, and bioinformatics tools, the authors demonstrate that the human GSDME C-terminal region and the S234 residue of human CASP7 are the key determinants that impede the cleavage of human GSDME by human CASP7.

Strengths

The authors made an important contribution to the field by demonstrating how human CASP7 has functionally diverged to lose the ability to cleave GSDME and showing that reverse-mutations in CASP7 can restore GSDME cleavage. The use of multiple methods to support their conclusions strengthens the authors' findings. The unbiased mutagenesis screen performed to identify S234 in huCASP7 as the determinant of its GSDME cleavability is also a strength.

Weaknesses

While the authors utilized an in-depth experimental setup to understand the CASP7-mediated GSDME cleavage across evolution, the physiological relevance of their findings are not assessed in detail. Additional methodology information should also be provided.

Specific recommendations for the authors

(1) The authors should expand their evaluation of the physiological relevance by assessing GSDME cleavage by the human CASP7 S234N mutant in response to triggers such as etoposide or VSV, which are known to induce CASP3 to cleave GSDME (PMID: 28045099). The authors could also test whether the human CASP7 S234N mutation affects substrate preference beyond human GSDME by testing cleavage of mouse GSDME and other CASP3 and CASP7 substrates in this mutant.

(1) The physiological relevance was discussed in the revised manuscript (lines 328-340). Our study revealed the molecular mechanism underlying the divergence of CASP3- and CASP7-mediated GSDME activation in vertebrate. One of the physiological consequences is that in humans, CASP7 no longer directly participates in GSDME-mediated cell death, which enables CASP7 to be engaged in other cellular processes. Another physiological consequence is that GSDME activation is limited to CASP3 cleavage, thus restricting GSDME activity to situations more specific, such as that inducing CASP3 activation. The divergence and specialization of the physiological functions of different CASPs are consistent with and possibly conducive to the development of refined regulations of the sophisticated human GSDM pathways, which are executed by multiple GSDM members (A , B, C, D, and E), rather than by GSDME solely in teleost, such as Takifugu. More physiological consequences of CASP3/7 divergence in GSDME activation need to be explored in future studies.

With respect to the reviewer’s suggestion of assessing GSDME cleavage by the human CASP7 S234N mutant in response to triggers such as etoposide or VSV: (i) CASP7 S234N is a creation of our study, not a natural human product, hence its response to CASP7 triggers cannot happen under normal physiological conditions except in the case of application, such as medical application, which is not the aim of our study. (ii) CASP3/7 activators (such as raptinal) induced robust activation of the endogenous CASP3 (Heimer et al., Cell Death Dis. 2019;10:556) and CASP7 (Author response image 1, below) in human cells. Since CASP3 is the natural activator of GSDME, the presence of the triggers inevitably activates GSDME via CASP3. Hence, under this condition, it will be difficult to examine the effect of CASP7 S234N.

Author response image 1.

HsCASP7 activation by raptinal. HEK293T cells were transfected with the empty vector (-), or the vector expressing HsCASP7 or HsCASP7-S234N for 24 h. The cells were then treated with or without (control) 5 μM raptinal for 4 h. The cells were lysed, and the lysates were blotted with anti-CASP7 antibody.

(2) As suggested by the reviewer, the cleavage of other CASP7 substrates, i.e., poly (ADP-ribose) polymerase 1 (PARP1) and gelsolin, by HsCASP7 and S234N mutant was determined. The results showed that HsCASP7 and HsCASP7-S234N exhibited similar cleavage capacities. Figure 5-figure supplement 1 and lines 212-214.

(2) It would also be interesting to examine the GSDME structure in different species to gain insight into the nature of mouse GSDME, which cannot be cleaved by either mouse or human CASP7.

Because the three-dimensional structure of GSDME is not solved, we are unable to explore the structural mechanism underlying the GSDME cleavage by caspase. Since our results showed that the C-terminal domain was essential for caspase-mediated cleavage of GSDME, it is likely that the C-terminal domain of mouse GSDME may possess some specific features that render it to resist mouse and human CASP7.

(3) The evolutionary analysis does not explain why mammalian CASP7 evolved independently to acquire an amino acid change (N234 to S234) in the substrate-binding motif. Since it is difficult to experimentally identify why a functional divergence occurs, it would be beneficial for the authors to speculate on how CASP7 may have acquired functional divergence in mammals; potentially this occurred because of functional redundancies in cell death pathways, for example.

According to the reviewer’s suggestion, a speculation was added. Lines 328-340.

(4) For the recombinant proteins produced for these analyses, it would be helpful to know whether size-exclusion chromatography was used to purify these proteins and whether these purified proteins are soluble. Additionally, the SDS-PAGE in Figure S1B and C show multiple bands for recombinant mutants of TrCASP7 and HsCASP7. Performing protein ID to confirm that the detected bands belong to the respective proteins would be beneficial.

The recombinant proteins in this study are soluble and purified by Ni-NTA affinity chromatography. Size-exclusion chromatography was not used in protein purification.

For the SDS-PAGE in Figure 4-figure supplement 1B and C (Figure S1B and C in the previous submission), the multiple bands are most likely due to the activation cleavage of the TrCASP7 and HsCASP7 variants, which can result in multiple bands, including p10 and p20. According to the reviewer’s suggestion, the cleaved p10 was verified by immunoblotting. Figure 4-figure supplement 1B and C.

(5) For Figures 3C and 4A, it would be helpful to mention what parameters or PDB files were used to attribute these secondary structural features to the proteins. In particular, in Figure 3C, residues 261-266 are displayed as a β-strand; however, the well-known α-model represents this region as a loop. Providing the parameters used for these callouts could explain this difference.

For Figure 3C, in the revised manuscript, we used the structure of mouse GSDMA3 (PDB: 5b5r) for the structural analysis of HsGSDME. As indicated by the reviewer, the region of 261-266 is a loop. The description was revised in lines 172 and 174, Figure 3C and Figure 3C legend.

For Figure 4A, the alignment of CASP7 was constructed by using Esprit (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) with human CASP7 (PDB:1k86) as the template. The description was revised in the Figure legend.

(6) Were divergent sequences selected for the sequence alignment analyses (particularly in Figure 6A)? The selection of sequences can directly influence the outcome of the amino acid residues in each position, and using diverse sequences can reduce the impact of the number of sequences on the LOGO in each phylogenetic group.

In Figure 6A, the sequences were selected without bias. For Mammalia, 45 CASP3 and 43 CASP7 were selected; for Aves, 41 CASP3 and 52 CASP7 were selected; for Reptilia, 31CASP3 and 39 CASP7 were selected; for Amphibia, 11 CASP3 and 12 CASP7 were selected; for Osteichthyes, 40 CASP3 and 43 CASP7 were selected. The sequence information was shown in Table 1 and Table 2.

(7) For clarity, it would help if the authors provided additional rationale for the selection of residues for mutagenesis, such as selecting Q276, D278, and H283 as exosite residues, when the CASP7 PDB structures (4jr2, 3ibf, and 1k86) suggest that these residues are enriched with loop elements rather than the β sheets expected to facilitate substrate recognition in exosites for caspases (PMID: 32109412). It is possible that the inability to form β-sheets around these positions might indicate the absence of an exosite in CASP7, which further supports the functional effect of the exosite mutations performed.

According to the suggestion, the rationale for the selection of residues for mutagenesis was added (lines 216-222). Unlike the exosite in HsCASP1/4, which is located in a β sheet, the Q276, D278, and H283 of HsCASP7 are located in a loop region (Figure 5-figure supplement 2), which may explain the mutation results and the absence of an exosite in HsCASP7 as suggested by the reviewer.

Reviewer #2 (Public Review):

The authors wanted to address the differential processing of GSDME by caspase 3 and 7, finding that while in humans GSDME is only processed by CASP3, Takifugu GSDME, and other mammalian can be processed by CASP3 and 7. This is due to a change in a residue in the human CAPS7 active site that abrogates GSDME cleavage. This phenomenon is present in humans and other primates, but not in other mammals such as cats or rodents. This study sheds light on the evolutionary changes inside CASP7, using sequences from different species. Although the study is somehow interesting and elegantly provides strong evidence of this observation, it lacks the physiological relevance of this finding, i.e. on human side, mouse side, and fish what are the consequences of CASP3/7 vs CASP3 cleavage of GSDME.

Our study revealed the molecular mechanism underlying the divergence of CASP3- and CASP7-mediated GSDME activation in vertebrate. One of the physiological consequences is that in humans, CASP7 no longer directly participates in GSDME-mediated cell death, which enables CASP7 to be engaged in other cellular processes. Another physiological consequence is that GSDME activation is limited to CASP3 cleavage, thus restricting GSDME activity to situations more specific, such as that inducing CASP3 activation. The divergence and specialization of the physiological functions of different CASPs are consistent with and possibly conducive to the development of refined regulations of the sophisticated human GSDM pathways, which are executed by multiple GSDM members (A , B, C, D, and E), rather than by GSDME solely in teleost, such as Takifugu. More physiological consequences of CASP3/7 divergence in GSDME activation need to be explored in future studies. Lines 328-340.

Fish also present a duplication of GSDME gene and Takifugu present GSDMEa and GSDMEb. It is not clear in the whole study if when referring to TrGSDME is the a or b. This should be stated in the text and discussed in the differential function of both GSDME in fish physiology (i.e. PMIDs: 34252476, 32111733 or 36685536).

The TrGSDME used in this study belongs to the GSDMEa lineage of teleost GSDME. The relevant information was added. Figure 1-figure supplement 1 and lines 119, 271, 274-276, 287 and 288.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) For the chimeric and truncated constructs, such as HsNT-TrCT, TrNT-HsCT, Hsp20-Trp10, Trp20-Hsp10, etc., the authors should provide a table denoting which amino acids were taken from each protein to create the fusion or truncation.

According to the reviewer’s suggestion, the information of the truncate/chimeric proteins was provided in Table 4.

(2) Both reviewers agree that functional physiological experiments are needed to increase the significance of the work. Specifically, the physiological relevance of these findings can be assessed by using western blotting to monitor GSDME cleavage by the human CASP7 S234N mutant compared with wild type CASP7 in response to triggers such as etoposide or VSV, which are known to induce CASP3 to cleave GSDME (PMID: 28045099).

Additionally, the authors can assess cell death in HEK293 cells, HEK293 cells transfected with TrGSDME, HEK293 cells expressing TrCASP3/7 plus TrGSDME, and TrCASP3/7 plus the D255R/D258A mutant. These cells can be stimulated, and pyroptosis can be assessed by using ELISA to measure the release of the cytoplasmic enzyme LDH as well as IL-1β and IL-18, and the percentage of cell death (PI+ positive cells) may also be assessed.

(1) With respect to the physiological relevance, please see the above reply to Reviewer 1’s comment of “Specific recommendations for the authors, 1”.

(2) As shown in our results (Fig. 2), co-expression of TrCASP3/7 and TrGSDME in HEK293T cells induced robust cell death without the need of any stimulation, as evidenced by LDH release and TrGSDME cleavage. In the revised manuscript, similar experiments were performed as suggested, and cell death was assessed by Sytox Green staining (Figure 2-figure supplement 3A and B) and immunoblot to detect the cleavage of both wild type and mutant TrGSDME (Figure 2-figure supplement 3C). The results confirmed the results of Figure 2.

Reviewer #2 (Recommendations For The Authors):

Abstract:

Although the authors try to summarize the principal results of this study, please rewrite the abstract section to make it easier to follow and to empathise the implications of their results.

We have modified the Abstract as suggested by the reviewer.

Introduction:

The authors do not mention anything about the implication of the inflammasome activation to get pyroptosis by GSDM cleave by inflammatory caspases. Please consider including this in the introduction section as they do in the discussion section.

The introduction was modified according to the reviewer’s suggestion. Lines 58-61.

From the results section the authors name the human GSDM as HsGSDM and the human CASP as HsCASP, maybe the author could use the same nomenclature in the introduction section. The same for the fish GSDM (Tr) and CASP.

According to the reviewer’s suggestion, the same nomenclature was used in the introduction.

Line 39. Remove the word necrotic.

“necrotic” was removed .

Line 42. Change channels by pores. In the manuscript, change channels by pores overall.

“channels” was replaced by “pores”.

Line 42: Include that: by these pores can be released the proinflammatory cytokines and if these pores are not solved then pyroptosis occurs. Please rephrase this statement.

According to the reviewer's suggestion, the sentence was rephrased. Lines 46-48.

Line 45. GSDMF is not an approved gene name, its official nomenclature is PJVK (Uniprot Q0ZLH3). Please use PJVK instead GSDMF.

GSDMF was changed to PJVK.

Line 103: Can the authors explain better the molecular determinant?

The sentence was revised, line 109.

Results:

Line 110: Reference for this statement. The reference for this statement was added in line 116.

Figure 1A, B: Concentration or units used of HsCASP?

The unit (1 U) of HsCASPs was added to the figure legend (line 661).

Line 113: Add Hs or Tr after CASP would be helpful to follow the story.

“CASP” was changed to “HsCASP”.

Fig 1D: Why the authors do not use the DMPD tetrapeptide (HsGSDME CASP3 cut site) in this assay? Comparing with the data obtained in Fig 3B the TrCASP3 activity is going to be very closer to that obtained for VEID o VDQQD in the CASP3 panel.

The purpose of Figure 1D was to determine the cleavage preference of TrCASPs. For this purpose, a series of commercially available CASP substrates were used, including DEVD, which is commonly used as a testing substrate for CASP3. Figure 3B was to compare the cleavage of HsCASP3/7 and TrCASP3/7 specifically against the motifs from TrGSDME (DAVD) and HsGSDME (DMPD).

Figure 1D and Figure 3B are different experiments and were performed under different conditions. In Figure 1D, CASP3 was incubated with the commercial substrates at 37 ℃ for 2 h, while in Figure 3B, CASP3/7 were incubated with non-commercial DAVD (motif from TrGSDME) and DMPD (motif from HsGSDME) at 37 ℃ for 30 min. More experimental details were added to Materials and Methods, lines 443 and 447.

Fig 1H: What is the concentration used of the inhibitors?

The concentration (20 μM) was added to the figure legend (line 669).

Does the Hs CASP3/7 fail to cleave the TrGSDME mutants (D255R and D258A)? the authors do not show this result so they cannot assume that HsCASP3/7 cleave that sequence (although this is to be expected).

The result of HsCASP3/7 cleavage of the TrGSDME mutants was added as Figure 1-figure supplement 2 and described in Results, line 133.

Line 132-133: Can the author specify where is placed the mCherry tag? In the N terminal or C terminal portion of the different engineered proteins?

The mCherry tag is attached to the C-terminus. Figure 2 legend (line 676).

Fig 2A: Although is quite clear, a column histogram showing the quantification is going to be helpful.

The expression of TrGSDME-FL, -NT and -CT was determined by Western blot, and the result was added as Figure 2-figure supplement 1.

Fig 2A, B, C: After how many hours of expression are the pictures taken? Can the authors show a Western blot showing that the expression of the different constructions is similar?

The time was added to Figure 2 legend and Materials and Methods (line 466). The expression of TrGSDME-FL, -NT and -CT was determined by Western blot, and the result was added as Figure 2-figure supplement 1.

Fig 2C: Another helpful assay can be to measure the YO-PRO or another small dye internalization, to complete the LDH data.

According the reviewer’s suggestion, in addition to LDH release, Sytox Green was also used to detect cell death. The result was added as Figure 2-figure supplement 2 and described in Results, line 146.

Fig 2C: In the figure y axe change LHD by LDH.

The word was corrected.

Fig 2D: Change HKE293T by HEK293T in the caption.

The word was corrected.

Fig 2G: Please add the concentration used with the two plasmids co-transfection. A Western blot showing CASP3/7 expression vs TrGSDME is missing. Is that assay after 24h? please specify better the methodology.

The concentration of plasmid used in co-transfection and the time post transfection were added to the Materials and Methods (lines 422 and 424). In addition, the expression of CASP3/7 was added to Figure 2I.

Fig 2 J, K: Change HKE293T by HEK293T in the figure caption. The concentration of the caspase inhibitors is missing. Depending on the concentration used, these inhibitors used could provoke toxicity on the cells by themselves.

The word was corrected in the figure caption. The inhibitor concentration (10 μM) was added to the figure legend (line 690).

Line 151: TrCASP3/7 instead of CASP3/7

CASP3/7 was changed to TrCASP3/7.

Fig 3A, 3B: Please add the units used of the HsCASP

The unit was added to the figure legends (lines 697).

Fig 3A: Can the authors add the SDS-PAGE to see the Nt terminal portion as has been done in Fig 1A? Maybe in a supplementary figure.

The SDS-PAGE was added as Figure 3-figure supplement 1.

Fig 3B: If the authors could add some data about the caspase activity using any other CASP such as CASP2, CASP1 to compare the activity data with CASP3 and CASP7 would be helpful.

The proteolytic activity of TrCASP1 was provided as Figure 3-figure supplement 2.

Fig 3C: To state this (Line 160), the authors should use another prediction software to reach a consensus with the sequences of the first analysis. In fact, what happens when GSDME is modelled 3-dimensionally by comparing it to crystalized structures such as mouse GSDMA? If the authors add an arrow indicating where the Nt terminal portion ends and where Ct portion begins would make the figure clearer.

According to the suggestions of both reviewers, in the revised manuscript, we used mouse GSDMA3 (PDB: 5b5r) for the structural analysis of HsGSDME, which showed that the 261-266 region of HsGSDME was a loop. As a result, Figure 3C was revised. Relevant change in Results: lines 172 and 174.

As suggested by the reviewer, we modelled the three-dimensional structure of HsGSDME by using SWISS-MODEL with mouse GSDMA3 as the template (Author response image 2, below).

Author response image 2.

The three-dimensional structure model of HsGSDME. (A) The structure of HsGSDME was modeled by using mouse GSDMA3 (MmGSDMA3) as the template. The N-terminal domain (1-246 aa) and the C-terminal domain (279-468 aa) of HsGSDME are shown in red and blue, respectively. (B) The superposed structure of HsGSDME (cyan) and MmGSDMA3 (purple).

Fig 3F: if this is an immunoblotting why NT can be seen? In other Western blots only the CT is detected, why? The use of the TrGSDME mouse polyclonal needs more details (is a purify Ab, was produced for this study, what are the dilution used...)

Since the anti-TrGSDME antibody was generated using the full-length TrGSDME, it reacted with both the N-terminal and the C-terminal fragments of TrGSDME in Figure 3F. In Figure 3G, the GSDME chimera contained only TrGSDME-CT, so only the CT fragment was detected by anti-TrGSDME antibody. More information on antibody preparation and immunoblot was added to “Materials and Methods” (lines 390 and 391).

Fig 4B: Can the authors show in which amino acid the p20 finish for each CASP? (Similarly, as they have done in panel 3E)

Fig 4B was revised as suggested.

Fig 5F: With 4 units of WT CASP7 the authors show a HsGSDME Ct in the same proportion than when the S234N mutant is used (at lower concentrations). How do the authors explain this?

The result showed that the cleavage by 4U of HsCASP7 was comparable to the cleavage by 0.25U of HsCASP7-S234N, indicating that S234 mutation increased the cleavage ability of HsCASP7 by 16 folds.

Line 203: Can the authors show an alignment between this region of casp1/4 and 7? Maybe in supplementary figures.

As reported by Wang et. al (PMID: 32109412), the βIII/βIII’ sheet of CASP1/4 forms the exosite critical for GSDMD recognition. The structural comparison among HsCASP1/4/7 and the sequence alignment of HsCASP1/4 βIII/βIII’ region with its corresponding region in HsCASP7 were added as Figure 5-figure supplement 2.

Line 205: A mutation including S234N with the exosite mutations (S234+Q276W+D278E+H283S) is required to support this statement.

The sentence of “suggesting that, unlike human GSDMD, HsGSDME cleavage by CASPs probably did not involve exosite interaction” was deleted in the revised manuscript.

Fig 5I, 5J: which is the amount of HsGSDME and TrGSDME? I would place these figures in supplementary material.

The protein expression of TrGSDME/HsGSDME was shown in the figure. Fig 5I and 5J were moved to Figure 5-figure supplement 3.

Line 218: I would specify that this importance is in HUMAN CASP7 to cleavage Human GSDME.

“CASP7” and “GSDME” were changed to “HsCASP7” and “HsGSDME”, respectively.

Fig 6C: 4 units is the amount of S234N mutant needed to see an optimal HsGSDME cleavage in Fig 5F.

In Figure 6C, the cleavage efficacy of HsCASP3-N208S was apparently decreased compared to that of HsCASP3, and 4U of HsCASP3-N208S was roughly equivalent to 1U of HsCASP3 in cleavage efficacy. In Figure 5F, cleavage by 4U of HsCASP7 was comparable to the cleavage by 0.25U of HsCASP7-S234N. Together, these results confirmed the critical role of S234/N208 in HsCASP3/7 cleavage of HsGSDM.

Fig 6I: Could be the fact that the mouse GSDME has a longer Ct than human GSDME affect the interaction with CASP7? Less accessible to the cut site? Needs a positive control of mouse GSDME with mouse Caspase 3.

Although mouse GSDME (MmGSDME) (512 aa) is larger than HsGSDME (496 aa), the length of the C-terminal domain of MmGSDME (186 aa) is comparable to that of HsGSDME (190 aa).

Author response image 3.

Conserved domain analysis of mouse (upper) and human (lower) GSDME.

As suggested by the reviewer, the cleavage of MmGSDME by mouse caspase-3 (MmCASP3) was added as Figure 6-figure supplement 2 and described in Results, lines 258.

Material and Methods:

-Overall, concentrations or amounts used in this study regarding the active enzyme or plasmids used are missing and need to be added.

The missing concentrations of the enzymes and plasmids were added in Material and Methods (lines 421, 453, 457, and 470) or figure legends (Figure 1 and 3).

-It would be helpful if the authors label in the immunoblotting panels what is the GSDME that they are using. (Hs GSDME FL...).

As suggested, the labels were added to Figures 1A ,1B, and 3.

-Add the units of enzyme used.

The units of enzyme were added to figure legends (Figure 1A, 3A, 3D, and 3F) or Material and Methods (lines 453 and 457).

The GSDME sequence obtained for Takifugu after amplification of the RNA extracted should be shown and specified (GSDMEa or GSDMEb). From which tissue was the RNA extracted?

The details were added to Materials and Methods (lines 398 and 402).

Please remove from tag: 1. Social media & digital marketing 2. Sonic identity 3. Web design

1. The tag text will be in white.
2. I have provided a revised banner in the drive, plz use that, have also tagged you in it.

add caveat on timing if you need urgent answer:

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Reply to the reviewers

Point-by-point response to reviewers’ comments:

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Major comments: 1. Previous studies using HDR and donor templates have shown that mutating the PAM sites in donor templates can enhance repair efficiencies. It would be helpful to add a discussion about the fact that SpRY does not have a PAM sequence that could be mutated and the potential consequences on repair efficiency.

We find that one mismatch in the target sequence (i.e. albino wt v. albino[b4]) is enough to completely abolish activity of SpRY. We have stated this more clearly in the manuscript.

It is also unclear how the template for the induction of mutations in kcnj13 was chosen. From the experiment with SpRY it seems that an HDR template equivalent to the sequence of the sgRNA target strand was most efficient, while in this experiment the alternative strand was used. An explanation should be added to the text.

Oligonucleotides corresponding to both DNA strands were tested, only one of them yielded positive results. We do not know the mechanistic basis for this finding, but amended the manuscript accordingly.

Minor comments: 1. It is not directly evident what the difference between the OP2 and OP2* sgRNA is. A short explanation would help clarify this and make it easier for the reader to understand.

OP2 (now re-named: U6) targets the wild type sequence, whereas OP2* (now: U6*) targets the albino[b4] sequence, which has one mutation leading to a premature stop codon. As this mutation is in the target region, we need adapt the sgRNA accordingly. We have stated this in the text more clearly now.

Similarly, it would be helpful to add the length of the different donor templates to Figure 2.

We have added the lengths of the oligonucleotides (in nt) to Fig.2.

While the PAM sequences and their difference between guides is discussed for two of them (OP2 and U5), it would be helpful to add the PAM sequences for all guides to Table 1 or figure 1.

We have added a table with all target sites including PAM sequences.

For people who are unfamiliar with the obelix phenotype/pigment pattern, it would be helpful to add a picture of an obelix mutant to Figure 4, so they would know what the phenotype would look like and how obvious it would be.

We have added a panel showing an obelix mutant fish to Fig.4.

Reviewer #2:

While every new and improved method to generate stable allele swap lines is greatly needed in the community, the results are not sufficient to convince me that the new version is leading to better success than previous methods. While they found one successful founder event, a single one is not enough to calculate efficiencies. Could just be luck that they got one. It is already known that HDR is very locus-specific, so maybe the locus they chose is such a locus.

This comment is difficult to address; while we found that the improved HDR method we present in the paper leads to better success for the repair of the albinob4 mutation and the one specific allele exchange we performed, we, of course, agree that one founder event is not enough to calculate efficiencies. However, we would like to maintain that one founder will in almost all cases be better than none. We also think that the locus we chose, kcnj13, is not a particularly lucky one yielding positive results easily, because it used to be refractory to editing following published protocols for a long time.

Overall, the paper suffers from the problem that the authors initially set out to investigate a specific genetic mutation in zebrafish but, upon observing that the resultant mutant exhibited no discernible phenotype, they shifted their focus towards refining and showcasing their methodological approach. This dual identity results in a study that, while informative, lacks the comprehensive exploration typical of dedicated research papers or the focused, technical depth one might expect from methodological publications.

Overall, we feel that there might be a slight misunderstanding here. The reviewer states that ‘… the paper suffers from the problem that the authors initially set out to investigate a specific genetic mutation in zebrafish but, upon observing that the resultant mutant exhibited no discernible phenotype, they shifted their focus…’, which is quite the opposite of what actually lead to the writing of the manuscript. We had already suspected that the single amino acid difference in the protein sequence between the two sister species might not be responsible for the observed functional divergence of the gene. We had also already found allele-specific differences in expression levels in hybrids, which make cis-regulatory evolution more likely. So, the null-hypothesis of our experiments was that both protein sequences would be functionally equivalent. However, as we had difficulties with the allele exchange due to low HDR efficiencies we needed to improve the method before we could definitively show this.

We have re-written some parts of the manuscript to make it clearer that we do not claim to have invented a method for HDR that is superior to all previously published ones. Rather, we think that we offer a variation of these published methods, which other researchers, struggling with low editing efficiencies (as we did), might want to try. What we do show in the manuscript is that the addition of an aNLS to Cas9 or SpRY leads to an increase in the efficiency in the generation of albino k.o. alleles and in HDR to repair the albinob4 mutation (see Fig. 3). If this will also be the case when editing other genes in the zebrafish genome needs to be investigated, but is clearly beyond the scope of this manuscript. We investigated one other locus in the zebrafish genome and could get one founder fish for the allele exchange in kcnj13, as opposed to zero we obtained with previously tried methods (conventional Cas9 with long or short donor-DNAs, prime editing). One advantage of ’our method’ is the simplicity of implementation. The Cas9 and SpRY proteins are easy to express in E. coli and the purification using two affinity tags is highly efficient resulting in samples of sufficient purity and high enough concentration for immediate use in injection experiments. So, we think that other researchers could easily try out the aNLS tagged proteins without changing much else of the protocols they usually employ for genome editing in zebrafish.

Reviewer #3:

Major comments: • The Cas9SpRY has been previously analyzed for the efficiency in zebrafish (Liang et al, Nat Comm 2022). This becomes only clear after reading the discussion. A comparison of these previously published SpRYCas9 proteins containing the bpNLS is missing, also a comparison of the efficiencies. The same locus (Albino) has been used in the study, are the guides identical? This study has not efficiently put the results in perspective of published results of the afore mentioned paper. And it seems that addition of the aNLS is not providing any benefit, which is good to know for the community.

We have added information to the introduction making it clear that the SpRY protein has previously been used in zebrafish. We also expanded the discussion and added more details comparing our results to previously published ones. However, this comparison is not always easy because the evaluation methods are different, sequencing v. phenotypic read-out. While the addition of the aNLS to the SpRY protein did not significantly enhance the (already high) k.o. efficiency for the albino locus, it did result in a significant boost of the repair efficiency of the albino[b4] mutation (see Fig.3C). Therefore, we think that the general statement it ’is not providing any benefit’ might not be entirely accurate. We think that the use of SpRY could be beneficial in some instances, but it must be assessed one a case-by-case basis.

• The HDR numbers is relying on 1 germline founder fish and might not be representative. More loci and higher numbers would be desirable.

We completely agree with the reviewer on this point. However, we feel that this is beyond the scope of this manuscript; we are looking forward to seeing other labs using the aNLS tagged proteins and finding out about their experiences.

• The allele exchange in Obelix is an interesting approach to use HDR but should be explained a little bit more. The motivation behind this experiments rains unclear.

We have added some information on obelix to provide more context

minor points: • All y axes require a labeling: % of what?!

We have changed the labels to % of larvae.

• When showing the specific classes of phenotpes the reader would benefit if the classes were written directly into the fish picture rather than using B, C, D, etc...

We have added this information directly to the pictures.

• OP2 should be called U6 to avoid unnecessary confusion, or is there anything special about it, why does it have another name?

We have changed OP2 to U6, as requested. The naming was completely due to historic reasons, there is nothing special about this target site / sgRNA.

• Differences in efficiency could potentially attributed to the PAM sequence as discussed. Please list the different PAM sequences and discuss in more detail. Why are so many gRNAs not efficient in the KO approach (Figure1)?

We added a table with the different target sites and the corresponding PAM sequences.

While we cannot provide a satisfactory explanation for the low efficiencies of five from six sgRNAs in our experiments, we notice that in the published data from Liang et al., 2022, a sizeable proportion of the tested sgRNAs with the SpRY protein also show low efficiency or no activity at all (see Fig. 2B, Liang et al., 2022, https://doi.org/10.1038/s41467-022-31034-8). This phenomenon is likely to be locus-specific and more data will be needed to come to a mechanistic understanding. We also do acknowledge that there is the possibility that our assay, the albino mutant phenotype in larvae, is likely not as sensitive as sequencing-based approaches. For one, we rely on the bi-allelic k.o. of the target gene, and we only assess a small proportion of all larval cells. However, we think that our approach with a phenotypic read-out is still valid, as it will reflect the practical requirements for an HDR method in many laboratories, where low efficiencies will result in no or weak and variable F0 phenotypes and in very low probabilities for germ-line transmission, which in most cases researchers will want to avoid.

• Line 217: correct co.injected to co-injected

done

The scientific advancement is not clear. Readers would benefit if the advancement can be worked out better. Most readers would like to decide if it is worth changing their Cas9 design for genome editing in zebrafish and what efficiencies to expect.

We have modified the manuscript to better convey the scientific advancement it presents. We think it lies mainly in the fact that no other changes to the design of genome editing experiments is required, but to exchange the Cas9 protein usually employed for the aNLS tagged proteins. Both proteins, aNLS tagged Cas9 or SpRY, can easily be produced and purified in the lab following standard protocols. In less than one week enough protein for several hundred or thousands of injection experiments can be purified and aliquoted. We suggest that everybody uses their tried and tested method to produce knock-in alleles, and, as long as it works for them, don’t change it. If, however, the efficiencies are too low to get the desired allele, it will be very quick and simple to try our method. This is what we wanted to demonstrate with the editing of the obelix locus. In all cases we can envisage identifying one founder fish will be considerably better than not finding a single one.

Reviewer #4:

Major

1. The authors use a mutated version of the widely used Cas9 protein from Streptococcus pyogenes, SpRY which basically does not rely on a PAM motif adjacent to the sgRNA target site. While this has certain advantages which are properly described, lowering stringency also comes with disadvantages, i.e. enhanced off target site activity. While assessing these is of the scope of the paper, these considerations should be properly discussed. Under which circumstances do the authors suggest to use SpRY and at which the conventional Cas9 or TALENs?

This is an important point and we have expanded on this. We think that SpRY offers a possibility to target sites that are not accessible to conventional Cas9, but it should not be expected to work as well as Cas9 for all loci (see also Liang et al., 2022 Fig.2). Whether the reduced stringency leads to more off-target effects is unclear; we did not experience higher rates of deformations or mortality in the injected larvae. This is, admittedly, a very crude measure for potential off-target effects, but is also in good accordance with the findings of Liang et al., 2022. In contrast to this, all labs that produce their own Cas9 protein could easily switch to the aNLS tagged version. It does not seem to have any disadvantages.

The authors designed 6 guides against slc42a2/alb according to the text and to Fig1 U1-U5+OP. Table 1 contains 16 sequences fitting these criteria. Which ones where used? Why are they named differently (U vs OP)? What method was used to design them? Does their design include PAM requirements? Have these guides been used previously and confirmed to work efficiently using CAS9? If the authors intend to provide an improved method that can widely and easily be adopted by other labs, they should put special emphasis in describing the procedure properly possibly including a supplemental figure detailing the workflow.

We have added a table with the target sites and the corresponding PAMs (see response to reviewer #1). The oligonucleotides shown in Table 1, which is now Table 2, are the ones used to generate the plasmid templates for the in vitro transcription of the sgRNAs.

The naming of the target sites, which was solely due to ’historic’ reasons, has been changed to U1 - U6.

They were designed (basically by hand) to allow in vitro transcription with T7 RNA polymerase (i.e. 5’ with GG), to have a G/C content of 50 - 65% and to represent a variety of different PAM sequences, that should potentially result in high activity (according to the data published by Walton et al., 2020 DOI: 10.1126/science.aba8853).

These sgRNAs could not be tested with Cas9 as they lack the PAM (NGG) required for activity of this protein.

We think that the main advantage of ’our’ method lies in the fact that aNLS-Cas9 (and aNLS-SpRY) can easily incorporated into the experimental procedures and workflows already in place in other laboratories. There is no need to follow exactly our protocol, eg. regarding sgRNA production or target site selection. We think that we showed that SpRY can be as effective as conventional Cas9, but not for all target sites, and that the addition of an aNLS sequence to Cas9 or SpRY is beneficial for genome editing in zebrafish, even when the aNLS is not combined with a myc-tag, as is the case shown by Thumberger et al., 2022, i.e. hei-tag.

The authors use a recessive pigment mutant (albino) to validate and quantify precise genome editing by HDR applying their toolbox. This is very clever and probably the most robust readout possible. The authors found that adding an aNLS to CAS9 and SpRY improves rescue efficiency, possibly also for germ line transmission. The authors should compare their efficiency for accurate editing with that of other papers in the field to allow for a better comparison.

We have now included a more detailed comparison of our results with previously published data in the discussion. However, this comparison is not always easy because the evaluation methods are different, sequencing v. phenotypic read-out. In terms of accuracy of the methods, we found that the majority of the HDR events we detected were associated with additional mutations. Some of these were possibly due to synthesis errors in the donor oligonucleotides, which might be alleviated by different purification methods. Other mutations, however, most likely occurred during cellular repair of dsDNA breaks and are therefore not easily avoided, unless double strand breaks are avoided, which would be the case if base editors are used. However, with base editors it is so far not possible to introduce every possible DNA change, making HDR methods still useful.

Minor:

1. Fig.1A: Please indicate orientation of the gene

done

Line 168: ... tested sperm... à Method not explained in the methods section

The sperm samples extracted from anaesthetized males were used in exactly the same way as larvae were in other genotyping experiments; as is mentioned in the methods section. We have re-phrased this section a bit to make it clearer that we used larvae or sperm in exactly the same way for genotyping.

Kcnj13 editing. Explain obelix pigment phenotype to the non expert reader in pigmentation possibly illustrating D. aesculapii. This is a very powerful method allowing such comparisons, however it is not properly explained.

We have added some information on the obelix phenotype and included a panel of a mutant zebrafish in Fig.4.

Line 130: 'hei-tag' not properly explained

The hei-tag, published by Thumberger et al., 2022, consists of a myc-tag, a flexible linker and an aNLS in exactly that order. We have added some more details on the hei-tag to the text.

The co-editing of a restriction site for later identification of the edited allele is clever. However precise editing should be performed carefully and include splice site prediction algorithms to avoid enabling ectopic splice sites by silent mutagenesis. Also, an example of the analysis would be benefitial to Fig.4 or in the supplement.

We agree that this is an important point. We originally designed the edit in a way that would not result in the generation of a strong ectopic splice site by avoiding the creation of AG or GT di-nucleotide sequences.

We now also performed analysis with spliceator (http://www.lbgi.fr/spliceator/), a splice site prediction tool using convolutional neural networks, which confirmed that no ectopic splice site should be generated.

We could include this into a supplementary figure, if deemed necessary.

The manuscript is well written, the data are presented in an accessible way and the results look convincing. The work clearly shows a path to improvement of a fundamental method of gene editing in zebrafish and other species and clearly provides essential data on the topic. However, some aspects of the work are not properly described for the non-expert. Given the nature of the work which aims to improve an important, established method a more precisely described workflow in form of a table and workflow chart would certainly help the reader to focus on the essentials of the procedure.

As mentioned above, we think that it will be easy for other labs to incorporate our improvements into their existing protocols by exchanging normal Cas9 for aNLS-Cas9 or aNLS-SpRY. There should not be the need to strictly follow our protocols, e.g., for target site selection or sgRNA synthesis. The proteins can easily be expressed in bacteria and purified by standard methods using the His- and Strep-tags, as we published previously for conventional Cas9 (Podobnik et al. 2023).

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 #4

Evidence, reproducibility and clarity

In this manuscript, Dorner, Stratmann et al. developed a new variant of the homologous directed repair mediated genome editing technique in zebrafish using modified Cas9 proteins. They focus on the SpRY Cas9 protein variant, which offers a more relaxed PAM requirement for gene targeting. The requirement of a PAM has particularly hampered the feasabilty of HDR in the zebrafish model as the genomic sites of interest often do not meet the PAM requirements for conventional Cas9. Their improved method enhances the versatility of CRISPR/Cas methods in zebrafish, a crucial model organism in biomedical research. The authors also demonstrate that integrating an artificial nuclear localization signal (aNLS) into Cas9 variants not only improves gene knockout efficiency but also boosts homology-directed repair (HDR) frequency. This advancement allows for more precise genetic modifications, including single base pair changes, offering significant potential for research and other applications in genetics.

The manuscript is well written, the data are presented in an accessible way and the results look convincing. The work clearly shows a path to improvement of a fundamental method of gene editing in zebrafish and other species and clearly provides essential data on the topic. However, some aspects of the work are not properly described for the non-expert. Given the nature of the work which aims to improve an important, established method a more precisely described workflow in form of a table and workflow chart would certainly help the reader to focus on the essentials of the procedure.

Major comments:

1. The authors use a mutated version of the widely used Cas9 protein from Streptococcus pyogenes, SpRY which basically does not rely on a PAM motif adjacent to the sgRNA target site. While this has certain advantages which are properly described, lowering stringency also comes with disadvantages, i.e. enhanced off target site activity. While assessing these is of the scope of the paper, these considerations should be properly discussed. Under which circumstances do the authors suggest to use SpRY and at which the conventional Cas9 or TALENs?

2. The authors designed 6 guides against slc42a2/alb according to the text and to Fig1 U1-U5+OP. Table 1 contains 16 sequences fitting these criteria. Which ones where used? Why are they named differently (U vs OP)? What method was used to design them? Does their design include PAM requirements? Have these guides been used previously and confirmed to work efficiently using CAS9? If the authors intend to provide an improved method that can widely and easily be adopted by other labs, they should put special emphasis in describing the procedure properly possibly including a supplemental figure detailing the workflow.

3. The authors use a recessive pigment mutant (albino) to validate and quantify precise genome editing by HDR applying their toolbox. This is very clever and probably the most robust readout possible. The authors found that adding an aNLS to CAS9 and SpRY improves rescue efficiency, possibly also for germ line transmission. The authors should compare their efficiency for accurate editing with that of other papers in the field to allow for a better comparison.

Minor comments:

1. Fig.1A: Please indicate orientation of the gene

2. Line 168: ... tested sperm...  Method not explained in the methods section

3. Kcnj13 editing. Explain obelix pigment phenotype to the non expert reader in pigmentation possibly illustrating D. aesculapii. This is a very powerful method allowing such comparisons, however it is not properly explained.

4. Line 130: 'hei-tag' not properly explained

5. The co-editing of a restriction site for later identification of the edited allele is clever. However precise editing should be performed carefully and include splice site prediction algorithms to avoid enabling ectopic splice sites by silent mutagenesis. Also, an example of the analysis would be benefitial to Fig.4 or in the supplement.

Significance

The manuscript is well written, the data are presented in an accessible way and the results look convincing. The work clearly shows a path to improvement of a fundamental method of gene editing in zebrafish and other species and clearly provides essential data on the topic. However, some aspects of the work are not properly described for the non-expert. Given the nature of the work which aims to improve an important, established method a more precisely described workflow in form of a table and workflow chart would certainly help the reader to focus on the essentials of the procedure.

from What an abomination

to What an abomination

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For the Authors):

(1) While not absolutely necessary - it would be nice to see at least at the in-situ level what happens to the handful of other HC-important transcription factors in the Rbm24 KO (IKZF2, Barlh1, RFX) as the authors did look at Insm1.

Reply: Thanks for your suggested experiments. We agree that knowing whether the genes that are known to be involved in cell survival regulation are changed will provide insights into the mechanisms underlying cell death of Rbm24-/- HCs. Our data showed that Ikzf2 seemed to be upregulated when in the Rbm24-/- HCs, relative to Rbm24+/+ HCs at P5. We also tested Barlh1 and RFX, but we did not obtain confident data to present. Nonetheless, following the reviewer’s logic, we further tested Gata3, another gene involved in HC survival, and found that Gata3 was down-regulated in Rbm24 -/- HCs, compared to Rbm24+/+ HCs. Please refer to the text on lines 12-22 on page 12 and lines 1-10 on page 13, and Figure 3-figure supplement 1.

(2) Major comments: The nomenclature for mouse gene vs. mouse protein needs to be addressed throughout the manuscript. The nomenclature when referring to a mouse gene: gene symbols are italicized, with only the first letter in upper-case (e.g. Rbm24).

The nomenclature when referring to a mouse protein: Protein symbols are not italicized, and all letters are in upper-case (e.g. RBM24).

Reply: Thanks for pointing it out. In the entire manuscript, we have followed the reviewer’s comments to list gene and protein.

(3) Supplemental Figure 2D: Individual data points should be displayed on the bar graph via dots. SEM is not appropriate for this graph as SEM precision with only 3 samples is low. Furthermore, readers are more interested in knowing the variability within samples and not proximity of mean to the population mean, therefore standard deviation (SD) should be used instead.

Reply: We have edited the Figure 1-figure supplement 2D, as suggested. The Figure 1figure supplement 2 legend was updated, too. Please refer to line 21-22 on page 32.

(4) Red/Green should be avoided, especially when both are on the same image (merged immunofluorescence images that are found throughout the manuscript). I highly recommend changing to a color-blind friendly color scheme (such as cyan/green/magenta, cyan/magenta/yellow, etc.) for inclusivity.

Reply: Thanks for pointing it out. We have changed the red to magenta in all our Figures and figure supplements.

(5) Minor comments: As CRISPR-stop is a major method used throughout the paper, a brief explanation is needed for readers to understand what this methodology entails and why it was used. Something along the lines of," The CRISPR-stop technique allows for the introduction of early stop codons without the induction of DNA damage via Cas9 which can cause deleterious effects".

Reply: We have further elaborated how CRISPR-stop works and its advantages. Please refer to lines 8-13 on page 5.

(6) Page 5; line 5 - "Phenotypes occur earlier..." Grammar

Reply: The grammar error was corrected. Please refer to line 4, page 5.

(7) Page 5; line 5 - "Given Pou4f3 is the upstream regulator..." Not proven, rephrase

Reply: We have rephrased this sentence. Please refer to lines 5-6 on page 5.

(8) Supplemental 1A: Fine, Proof of knockout, I wouldn't mention INSM1 being "irregular"

Reply: We have rephrased this sentence. Please refer to lines 2-3 on page 6.

(9) Page 5; line21 - "Alignment of Insm1+ OHCs was not as regular..." Not a good description

Reply: We have rephrased this sentence. Please refer to lines 2-3 on page 6.

(10) Page 6; line11 - "Rbm24 was completely absent.." Redundancy with line 9

Reply: Thanks for pointing it out, and we have removed the redundant sentence.

(11) Page 7 - HA tag should be indicated originally as: Hemaglutinin (HA)

Reply: We have switched “HA” to “Hemaglutinin (HA)”. Please refer to line 15, page 7.

(12) Page 9, line 11- "Determine if autonomous/noncell autonomous." Disagree, cells still clustered in supplemental fig 4.

Reply: We have removed this sentence.

Reviewer #2 (Recommendations For The Authors):

The writing of the manuscript is adequate, but it would certainly be improved by professional editing.

Reply: Thanks for the reviewer’s encouraging comments. The revised version of our manuscript has been edited by an English native speaker.

Author Response

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1:

Summary:

In this study, Yan et al. investigate the molecular bases underlying mating type recognition in Tetrahymena thermophila. This model protist possesses a total of 7 mating types/sexes and mating occurs only between individuals expressing different mating types. The authors aimed to characterize the function of mating type proteins (MTA and MTB) in the process of self- and non-self recognition, using a combination of elegant phenotypic assays, protein studies, and imaging. They showed that the presence of MTA and MTB in the same cell is required for the expression of concavalin-A receptors and for tip transformation - two processes that are characteristic of the costimulation phase that precedes cell fusion. Using protein studies, the authors identify a set of additional proteins of varied functions that interact with MTA and MTB and are likely responsible for the downstream signaling processes required for mating. This is a description of a fascinating self- and non-self-recognition system and, as the authors point out, it is a rare example of a system with numerous mating types/sexes. This work opens the door for the further understanding of the molecular bases and evolution of these complex recognition systems within and outside protists.

The results shown in this study point to the unequivocal requirement of MTA and MTB proteins for mating. Nevertheless, some of the conclusions regarding the mode of functioning of these proteins are not fully supported and require additional investigation.

Strengths:

(1) The authors have established a set of very useful knock-out and reporter lines for MT proteins and extensively used them in sophisticated and well-designed phenotypic assays that allowed them to test the role of these proteins in vivo.

(2) Despite their apparent low abundance, the authors took advantage of a varied set of protein isolation and characterization techniques to pinpoint the localization of MT proteins to the cell membrane, and their interaction with multiple other proteins that could be downstream effectors. This opens the door for the future characterization of these proteins and further elucidation of the mating type recognition cascade.

Weaknesses:

The manuscript is structured and written in a very clear and easy-to-follow manner. However, several conclusions and discussion points fall short of highlighting possible models and mechanisms through which MT proteins control mating type recognition:

(1) The authors dismiss the possibility of a "simple receptor-ligand system", even though the data does not exclude this possibility. The model presented in Figure 2 S1, and on which the authors based their hypothesis, assumes the independence of MTA and MTB proteins in the generation of the intracellular cascade. However, the results presented in Figure 2 show that both proteins are required to be active in the same cell. Coupled with the fact that MTA and MTB proteins interact, this is compatible with a model where MTA would be a ligand and MTB a receptor (or vice-versa), and could thus form a receptor-ligand complex that could potentially be activated by a non-cognate MTA-MTB receptor-ligand complex, leading to an intracellular cascade mediated by the identified MRC proteins. As it stands, it is not clear what is the proposed working model, and it would be very beneficial for the reader for this to be clarified by having the point of view of the authors on this or other types of models.

We are very grateful that Reviewer #1 proposed the possibility that MTA and MTB form a receptor-ligand complex in which one acting as the ligand and the other as the receptor. We considered this hypothesis when asking how dose MTRC function, too. However, our current results do not support this idea. For instance, if MTA were a ligand and MTB a receptor, we would expect a mating signal upon treatment with MTAxc protein, but not with MTBxc. Contrary to this expectation, our experiments revealed that both MTAxc and MTBxc exhibit very similar effects (Figure 5, green and blue), and their combined treatment produces a stronger effect (Figure 5, teal). This suggests a mixed function for both proteins. (We incorporated this discussion into the revised version [line 120-121, 240-244].) It is pity that our current knowledge does not provide a detailed molecular mechanism for this intricate system. We are actively investigating the protein structures of MTA, MTB, and the entire MTRC, hoping to gain deeper insights into the molecular functions of MTA and MTB.

Additionally, we also realized that the expression we used in the previous version, “simple receptor-ligand model”, is not clearly defined. As Reviewer #1 pointed out, in this section, we examined whether the individual proteins of MTA and MTB act as a couple of receptor and ligand. We think this is the simplest possibility as a null hypothesis for Tetrahymena mating-type recognition. We have clarified it in the revised version (line 90-91, 104-106). According to this section, we proposed that MTA and MTB may form a complex that serves as a recognizer (functioning as both ligand and receptor) (line 117-118).

(2) The presence of MTA/MTB proteins is required for costimulation (Figure 2), and supplementation with non-cognate extracellular fragments of these proteins (MTAxc, or MTBxc) is a positive stimulator of pairing. However, alone, these fragments do not have the ability to induce costimulation (Figure 5). Based on the results in Figures 5 and 6 the authors suggest that MT proteins mediate both self and non-self recognition. Why do MTAxc and MTBxc not induce costimulation alone? Are any other components required? How to reconcile this with the results of Figure 2? A more in-depth interpretation of these results would be very helpful, since these questions remain unanswered, making it difficult for the reader to extract a clear hypothesis on how MT proteins mediate self- and non-self-recognition.

Several factors could contribute to the inability of MTA/Bxc to induce costimulation. It is highly likely that additional components are necessary, given that MTA/B form a protein complex with other proteins. Moreover, the expression of MTA/Bxc in insect cells, compared with Tetrahymena, might result in differences in post-translational modifications. Additionally, there are variations in protein conditions; on the Tetrahymena membrane, these proteins are arranged regularly and concentrated in a small area, while MTA/Bxc is randomly dispersed in the medium. The former condition could be more efficient. If there is a threshold required to stimulate a costimulation marker, MTA/Bxc may fail to meet this requirement. Much more studies are needed to fully answer this question. We acknowledged this limitation in the revised version (line 244-248).

Reviewer #2:

This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

Thanks for the valuable comments and suggestions. We are currently in the process of constructing deletion strains for these genes. As of now, we have successfully obtained ΔMRC1-3 and MRC4-6 knockdown strains. Our preliminary observations indicate that ΔMRC1-3 strains are unable to undergo mating. However, we prefer not to include these results in the current manuscript, as we believe that more comprehensive studies are still needed.

Reviewer #3:

The authors describe the role, location, and function of the MTA and MTB mating type genes in the multi-mating-type species T. thermophila. The ciliate is an important group of organisms to study the evolution of mating types, as it is one of the few groups in which more than two mating types evolved independently. In the study, the authors use deletion strains of the species to show that both mating types genes located in each allele are required in both mating individuals for successful matings to occur. They show that the proteins are localized in the cell membrane, not the cilia, and that they interact in a complex (MTRC) with a set of 6 associated (non-mating type-allelic) genes. This complex is furthermore likely to interact with a cyclin-dependent kinase complex. It is intriguing that T. thermophila has two genes that are allelic and that are both required for successful mating. This coevolved double recognition has to my knowledge not been described for any other mating-type recognition system. I am not familiar with experimental research on ciliates, but as far as I can judge, the experiments appear well performed and mostly support the interpretation of the authors with appropriate controls and statistical analyses.

The results show clearly that the mating type genes regulate non-self-recognition, however, I am not convinced that self-recognition occurs leading to the suppression of mating. An alternative explanation could be that the MTA and MTB proteins form a complex and that the two extracellular regions together interact with the MTA+MTB proteins from different mating types. This alternative hypothesis fits with the coevolution of MTA and MTB genes observed in the phylogenetic subgroups as described by Yan et al. (2021 iScience). Adding MTAxc and/or MTBxc to the cells can lead to the occupation of the external parts of the full proteins thereby inhibiting the formation of the complex, which in turn reduces non-self interactions. Self-recognition as explained in Figure 2S1 suggests an active response, which should be measurable in expression data for example. This is in my opinion not essential, but a claim of self-recognition through the MTA and MTB should not be made.

We express our gratitude to Reviewer #3 for proposing the occupation model and have incorporated this possibility into the manuscript. We believe it is possible that occupation may serve as the molecular mechanism through which self-recognition negatively regulates mating. If there is a physical interaction between mating-type proteins of the same type, but this interaction blocks the recognition machinery rather than initiating mating, it can be considered a form of self-recognition. This aligns with the observation that strains expressing MTA/B6 and MTB2 mate normally with WT cells of all mating types except for VI and II (line 203-204). A concise discussion on this topic is included in the manuscript (line 288-293, 659-661). We are actively investigating the downstream aspects of mating-type recognition, and we hope to provide further insights into this question soon.

The authors discuss that T. thermophila has special mating-type proteins that are large, while those of other groups are generally small (lines 157-160 and discussion). The complex formed is very large and in the discussion, they argue that this might be due to the "highly complex process, given that there are seven mating types in all". There is no argument given why large is more complex, if this is complex, and whether more mating types require more complexity. In basidiomycete fungi, many more mating types than 7 exist, and the homeodomain genes involved in mating types are relatively small but highly diverse (Luo et al. 1994 PMID: 7914671). The mating types associated with GPCR receptors in fungi are arguably larger, but again their function is not that complex, and mating-type specific variations appear to evolve easily (Fowler et al 2004 PMID: 14643262; Seike et al. 2015 PMID: 25831518). The large protein complex formed is reminiscent of the fusion patches that develop in budding or fission yeasts. In these species, the mating type receptors are activated by ligand pheromones from the opposite mating type that induce polarity patch formation (see Sieber et al. 2023 PMID: 35148940 for a recent review). At these patches, growth (shmooing) and fusion occur, which is reminiscent (in a different order) of the tip transformation in T. thermophilia. The fusion of two cells is in all taxa a dangerous and complex event that requires the evolution of very strict regulation and the existence of a system like the MTRC and cyclin-dependent complex to regulate this process is therefore not unexpected. The existence of multiple mating types should not greatly complicate the process, as most of the machinery (except for the MTA and MTB) is identical among all mating types.

We are very grateful that Reviewer #3 provide this insightful view and relevant papers. In response to the feedback, we removed the sentences regarding “multiple mating types greatly complicate the process” in the revised version. Instead, we have introduced a discussion section comparing the mating systems of yeasts and Tetrahymena (line 279-286).

The Tetrahymena/ciliate genetics and lifecycle could be better explained. For a general audience, the system is not easy to follow. For example, the ploidy of the somatic nucleus with regards to the mating type is not clear to me. The MAC is generally considered "polyploid", but how does this work for the mating type? I assume only a single copy of the mating type locus is available in the MAC to avoid self-recognition in the cells. Is it known how the diploid origin reduces to a single mating type? This does not become apparent from Cervantes et al. 2013.

In T. thermophila, the MIC (diploid) contains several mating-type gene pairs (mtGP, i.e., MTA and MTB) organized in a tandem array at the mat locus on each chromosome. In sexual reproduction, the new MAC of the progeny develops from the fertilized MIC through a series of genome editing events, and its ploidy increases to ~90 by endoreduplication. During this process, mtGP loss occurs, resulting in only one mtGP remaining on the MAC chromosome. The mating-type specificity of mtGPs on each chromosome within one nucleus becomes relatively pure through intranuclear coordination. After multiple assortments (possibly caused by MAC amitosis during cell fission), only mtGPs of one mating-type specificity exist in each cell, determining the cell’s mating type.

It is pity that the exact mechanisms involved in this complicated process remain a black box. The loss of mating-type gene pairs is hypothesized to involve a series of homologous recombination events, but this has not been completely proven. Furthermore, there is no clear understanding of how intranuclear coordination and assortment are achieved. While we have made observations confirming these events, a breakthrough in understanding the molecular mechanism is yet to be achieved.

We included more information in the revised version (line 672-683). Given the complexity of these unusual processes, we recommend an excellent review by Prof. Eduardo Orias (PMID: 28715961), which offers detailed explanations of the process and related concepts (line 685-686).

Also, the explanation of co-stimulation is not completely clear (lines 49-60). Initially, direct cell-cell contact is mentioned, but later it is mentioned that "all cells become fully stimulated", even when unequal ratios are used. Is physical contact necessary? Or is this due to the "secrete mating-essential factors" (line 601)? These details are essential, for interpretation of the results and need to be explained better.

Sorry that we didn’t realize the term “contact” is not precise enough. In Tetrahymena, physical contact is indeed necessary, but it can refer to temporary interactions. Unlike yeast, Tetrahymena cells exhibit rapid movement, swimming randomly in the medium. Occasionally, two cells may come into contact, but they quickly separate instead of sticking together. Even newly formed loose pairs often become separated. As a result, one cell can come into contact with numerous others and stimulate them. We have clarified this aspect in the revised version (line 50-51, 57).

Abstract and introduction: Sexes are not mating types. In general, mating types refer to systems in which there is no obvious asymmetry between the gametes, beyond the compatibility system. When there is a physiological difference such as size or motility, sexes are used. This distinction is of importance because in many species mating types and sexes can occur together, with each sex being able to have either (when two) or multiple mating types. An example are SI in angiosperms as used as an example by the authors or mating types in filamentous fungi. See Billiard et al. 2011 [PMID: 21489122] for a good explanation and argumentation for the importance of making this distinction.

We have clarified the expression in the revised version (line 20, 38, 40, 45).

Recommendations for the authors:

Reviewer #1:

I really enjoyed reading this manuscript and I think a few tweaks in the writing/data presentation could greatly improve the experience for the reader:

(1) The information about your previous work in identifying downstream proteins CDK19, CYC9, and CIP1 (lines 170-173) could be directly presented in the introduction.

We have moved this information in the introduction in the revised version (line 74-77).

(2) For a reader who is not familiar with Tetrahymena, a few more details on how reporter and knock-out lines are generated would be beneficial.

We introduced the knock-out method in Figure 2 – figure supplement 1B, HA-tag method in Figure 3A, and MTB2-eGFP construction method in Figure 4E. In addition, we introduced how co-stimulation markers observed in Materials and Methods (line 404-410)

(3) Figures 5 and 6: clarify the types of pairing and treatments that were done directly in the figure (eg. adding additional labels). As of now, it is necessary to go through the text and legend to try and understand in detail what was done.

Cell types and treatments were directly introduced in the revised figure (Figure 5 and 6).

(4) The logical transition in lines 136-142 is hard to follow.

We rewrote this paragraph in the revised version (lines 143-156). Additionally, we added a figure to illustrate the theoretical mating-type recognition model between WT cells and ΔCDK19, ΔCYC9 cells, MTAxc, MTBxc proteins, and ΔMTA, ΔMTB cells (Figure 2 – figure supplement 1D-G).

(5) Lines 191-196: the fact that cells expressing multiple mating types can self goes against an active self-rejection system - if this is the case there should be self-rejection among all expressed mating types. Unless non-self recognition is an active process and self-recognition is simply the absence of non-self recognition. The authors briefly mention this in lines 263-265, but it would be interesting to expand and clarify this.

We appreciate that Reviewer #1 notice the interesting selfing phenotype of the MTB2-eGFP (MTVI background) strain. We further discussed it in the revised manuscript (line 298-306).

(6) The authors briefly mention the possibility of different mating types using different recognition mechanisms (lines 255-260), based on the big differences in the size of the mating-specific region of MT proteins. Following this and the weakness nr. 2, I think it would be pertinent to gather and present more information on the properties and structures of the mating-type specific regions of MT proteins. Simple in silico analysis of motifs, structure, etc. could help clarify the role of these regions. It seems more parsimonious that MT proteins would have variable mating type specific regions that account for the recognition of the different mating types, and conserved cytoplasmic functions that could trigger a single downstream signaling cascade. It would be interesting to know the authors' opinion on this.

We are very grateful for this suggestion. Actually, we are currently working on determining the 3D structure of MTRC. The Alphafold2 prediction indicates that the MT-specific region is comprised of seven global β-sheets, resembling the structure of immunoglobulins (Ig). Our most recent cryo-EM results have revealed a ~15Å structure, aligning well with the prediction. However, the main challenge lies in the low expression levels, both in Tetrahymena and insect/mammal cells. We anticipate obtaining more detailed results soon. Therefore, we prefer to present the MT recognition model with robust experimental evidence in the future, and didn’t discuss too much on this aspect in the current manuscript.

(7) Adding a figure including a proposed model, as well as expanding the discussion on the points presented as "weaknesses" would help clarify the ideas/hypothesis on how the mating recognition works. I think this would really elevate the paper and help highlight the results.

We added a figure to introduce the model and the weaknesses in the revised version (Figure 7, line 656-665).

(8) Line 202-203: It is far-fetched to infer subcellular localization based on the data presented here, couterstaining with other dyes and antibodies specific to certain cell components, as well as negative control images, are required.

Thanks for the suggestion. We attempted to stain cell components using various dyes and antibodies. Unfortunately, we found that cell surface and cilia (especially oral cilia) is very easy to give a false positive signal. We think this issue seriously affects the credibility of the results. It may seem like splitting hairs, but we are trying to be precise.

Meanwhile, we still believe the mating-type proteins localizes to cell surface because MTA-HA is identified in the isolated cell surface proteins.

Regarding negative control, as shown in Fig. 4G, where a MTB2-eGFP cell is pairing with a WT cell, no GFP signal is observed in the WT cell.

(9) Lines 131: clarify the sentence - expression of Con-A receptors requires both MTA and MTB (MTA to receive the signal).

We modified the sentence in the revised version (line 139-140).

Reviewer #2:

Minor points.

(1) Line 194-196. Why are these cells able to self?

These cells able to self may because the MTRC contain heterotypic mating-type proteins (MTA6 and MTB2), which activate mating when they interact with another heterotypic MTRC (line 207-208).

(2) Line 232. What do the authors mean by the term synergistic effect here? Definition and statistics?

Sorry about the confusion. The synergistic effect refers to the effect of MTAxc and MTBxc become stronger when using together. We clarified it in the revised version (line 232).

(3) For Figure 4 panel D, are there antibodies that are available as a control for cilia? If so, then blotting this membrane would show that cilia-associated proteins are in the cilia preparation, which is a standard control for sub-cellular fractionation.

Thanks for the suggestion. Unfortunately, we didn’t find a suitable cilia-specific antibody yet. Instead, we employed MS analysis to confirm the presence of cilia proteins in this sample (line 195-196, Figure 4–Source data 1). We also observed the sample under the microscope, which directly revealed the presence of cilia (Figure 4C).

(4) At least one reference cited in the text was not present in the reference list. The authors should go through the references cited to ensure that all have made it into the reference list.

We have checked all the references.

Some minor edits:

(1) MTA and MTB are presented in both roman and italics (e.g. line 209) in the manuscript. Maybe all should be in italics? Or is this a distinction between the gene and the protein?

The italics word (MTA) refers to gene, and non-italics word (MTA) refers to protein.

(2) Line 251. Change "achieving" to "achieve".

We have corrected this word (line 266).

Reviewer #3:

Line 101. It would help to explain this expectation earlier in this paragraph.

We explained the expectation in the revised version (line 92-97, 104-106).

Line 109. How is a co-receptor different from the MTRC complex?

We have rewritten the relevant sentences to enhance clarity (line 116-119). The molecular function of the MTRC complex could involve acting as a co-receptor or recognizer (functioning as both ligand and receptor). Based on the results presented in this section, we propose that MTA and MTB may function as a complex, but the confirmation of this hypothesis (MTRC) is provided in a later section. Therefore, we did not use the term “MTRC” here. These sentences briefly discuss the molecular function of this complex and explain why MTRC does not appear to function as a co-receptor.

Line 251: which "dual approach" is referred to?

Dual approach is referred to both self and non-self recognition. We explained it in the revised version (line 265-266).

Line 258: what "different mechanisms" do the authors have in mind? Why would a different mechanism be expected? The different sizes could have evolved for (coevolutionary?) selection on the same mechanism.

Sorry about the confusion. We clarified it in the revised version (line 269-278).

What we intended to express is that we are uncertain whether the mating-type recognition model we discovered in T. thermophila is applicable to all Tetrahymena species due to significant differences in the length of the mating-type-specific region. We believe it is important to highlight this distinction to avoid potential misinterpretations in future studies involving other Tetrahymena species. At the same time, we look forward to future research that may provide insights into this question.

Fig 2 C&D. Is it correct that these figures show the strains only after 'preincubation'? This is not apparent from the caption of the text. Additionally, the order of the images is very confusing. Write in the figures (so not just in the caption) what the sub-script means.

These panels are re-organized in the revised version (Fig. 2C&D). There are three kinds of pictures: “not incubated”, “WT pre-incubated by mutant” and “mutant pre-incubated by WT”.

The methods used to generate Figure 5 are not clearly described. I understand that the obtained xc proteins were added to the cells, and then washed, after which a test was performed mixing WT-VI and WT-VII cells. Were both cells treated? Or only one of the strains? The explanation for the reused washing medium is not clear and the method is not indicated.

Both cells are treated. More details are provided in the revised manuscript (line 230-231, 633-634, 637-639, Fig. 5). To prepare the starvation medium containing mating-essential factors, cells were starved in fresh starvation medium for ~16 hours. Subsequently, cells were removed by three rounds of centrifugation (1000 g, 3 min) (line 330-332).

In general, the figures are difficult to understand without repeated inquiries in the captions. Give more information in the figures themselves.

More information is introduced in the figure (Fig. 2C, Fig. 3B, Fig. 4A, B, D, Fig. 5 and Fig. 6).

tag yourself soy ésta

Author Response

Note to the editor and reviewers.

All the authors would like to thank the editorial team and the two anonymous reviewers for their efforts and thoughtfulness in assessing our manuscript. We very much appreciate it and we all believe that the manuscript has been much improved in addressing the comments and suggestions made.

General considerations on the revised manuscript

We have applied extensive modifications to the manuscript with our main goal being the improvement of clarity. The Introduction has been changed mainly to introduce precisely our terminology and we have stuck to it in the rest of the manuscript. The Results section has been divided up into more defined sections. The discussion has been extensively re-written to improve clarity, following the suggestion of the reviewers. Main figures 1 and 4 have been modified with clearer schematics. Supplementary figures and legends have been modified and several supplementary schematic figures have been added to clearly present our interpretations for various data. We have added a Supplementary Discussion where the most detailed technical parts of our discussion are presented to avoid unnecessarily weighing down the main discussion, where our main conclusions are outlined. We have presented our mass photometry mixing experiment in a new supplementary figure, with detailed explanation. We have also expanded our discussion of in vivo and general relevance of our study.

Response to manuscript evaluation

Our manuscript has been evaluated as a valuable study and presenting solid experimental evidence. We appreciate the recognition of our work.

Two weaknesses were identified by reviewers: 1) our experiments do not completely exclude the possibility of an alternative nucleophile. This relates to the evaluation of our experimental evidence. 2) Our study does not address the in vivo relevance of the interface swapping phenomenon, which relate to the value of the study for the community.

Response to the evaluation of experimental evidence (Weakness #1):

We argued in the original manuscript that we have excluded completely the presence of an alternative nucleophile. This conclusion is based on a series of experiments which were presented in the originally submitted manuscript. These experiments are not discussed by the reviewers in relation to this main conclusion and therefore we suggest that they have not been properly evaluated. We believe our conclusion to be appropriately supported by these data (see our response to reviewer #1). In addition, the criticism of our gel-filtration data by reviewer #2 was based on a misinterpretation of Supplementary figure 1 b. We accept of course that the way the data was presented could be misleading and we assume responsibility for this. We have attempted to correct this by changing the main text and the figures legends and annotation. In conclusion, we believe that the evaluation of experimental evidence as presented in the revised manuscript could be upgraded to “convincing”.

Response to our study general relevance evaluation (weakness #2):

We agree with both reviewers about the in vivo relevance of our observation being an important question, not addressed so far. Indeed, the value of our study would be greatly increased by in vivo data and be of interest to a wider audience. However, we would like to argue that our study would interest a wider audience than initially stated for the following reasons: 1) Our study is the first evidence of interface swapping in vitro and will constitute a base to investigate this phenomenon both in vivo and in vitro. It will therefore interest a wide audience due to the potential involvement of interface swapping in a wide range of processes, such as recombination, evolution, and drug targeting (see also below). 2) DNA cleavage is the central mode of action of antibiotics targeting bacterial type II topoisomerases (i.e. topoisomerases “poisons”). This already established target is one of the few having produced new scaffolds and too few new antibacterial are in production to fulfill medical needs. The role of interface stability is also emerging as a modulator of the efficiency of topoisomerase poisons. See for instance (Germe, Voros et al. 2018, Bandak, Blower et al. 2023). By shedding light on interface dynamics, our study will be of interest to scientist interested in the development of these drugs. In addition, the heterodimer system can potentially produce detailed mechanistic information (Gubaev, Weidlich et al. 2016, Hartmann, Gubaev et al. 2017, Stelljes, Weidlich et al. 2018) not only on gyrase but also on other, dimeric type II topoisomerases or even other dimeric enzyme in general. We have amended the manuscript to make these points clearer. Therefore, we believe that the evaluation of the revised manuscript’s relevance could be upgraded to “important”.

Point-by-point response to the reviewer

Reviewer #1 (Public Review):

Germe and colleagues have investigated the mode of action of bacterial DNA gyrase, a tetrameric GyrA2GyrB2 complex that catalyses ATP-dependent DNA supercoiling. The accepted mechanism is that the enzyme passes a DNA segment through a reversible double-stranded DNA break formed by two catalytic Tyr residues-one from each GyrA subunit. The present study sought to understand an intriguing earlier observation that gyrase with a single catalytic tyrosine that cleaves a single strand of DNA, nonetheless has DNA supercoiling activity, a finding that led to the suggestion that gyrase acts via a nicking closing mechanism. Germe et al used bacterial co-expression to make the wild-type and mutant heterodimeric BA(fused). A complexes with only one catalytic tyrosine. Whether the Tyr mutation was on the A side or BA fusion side, both complexes plus GyrB reconstituted fluoroquinolone-stabilized double-stranded DNA cleavage and DNA supercoiling. This indicates that the preparations of these complexes sustain double strand DNA passage. Of possible explanations, contamination of heterodimeric complexes or GyrB with GyrA dimers was ruled out by the meticulous prior analysis of the proteins on native Page gels, by analytical gel filtration and by mass photometry. Involvement of an alternative nucleophile on the Tyr-mutated protein was ruled unlikely by mutagenesis studies focused on the catalytic ArgTyrThr triad of residues. Instead, results of the present study favour a third explanation wherein double-strand DNA breakage arises as a consequence of subunit (or interface/domain) exchange. The authors showed that although subunits in the GyrA dimer were thought to be tightly associated, addition of GyrB to heterodimers with one catalytic tyrosine stimulates rapid DNA-dependent subunit or interface exchange to generate complexes with two catalytic tyrosines capable of double-stranded DNA breakage. Subunit exchange between complexes is facilitated by DNA bending and wrapping by gyrase, by the ability of both GyrA and GyrB to form higher order aggregates and by dense packing of gyrase complexes on DNA. By addressing a puzzling paradox, this study provides support for the accepted double strand break (strand passage) mechanism of gyrase and opens new insights on subunit exchange that may have biological significance in promoting DNA recombination and genome evolution.

The conclusions of the work are mostly well supported by the experimental data.

Strengths:

The study examines a fundamental biological question, namely the mechanism of DNA gyrase, an essential and ubiquitous enzyme in bacteria, and the target of fluoroquinolone antimicrobial agents.

The experiments have been carefully done and the analysis of their outcomes is comprehensive, thoughtful and considered.

The work uses an array of complementary techniques to characterize preparations of GyrA, GyrB and various gyrase complexes. In this regard, mass photometry seems particularly useful. Analysis reveals that purified GyrA and GyrB can each form multimeric complexes and highlights the complexities involved in investigating the gyrase system.

The various possible explanations for the double-strand DNA breakage by gyrase heterodimers with a single catalytic tyrosine are considered and addressed by appropriate experiments.

The study highlights the potential biological importance of interactions between gyrase complexes through domain-or subunit-exchange

We thank the reviewer for their support, effort, and comments. The above is a great summary.

Weaknesses:

The mutagenesis experiments described do not fully eliminate the perhaps unlikely participation of an alternative nucleophile.

We agree that the mutagenesis experiment on its own does not fully eliminate the possibility of an alternative nucleophile. The number of residues mutated is limited, and therefore it is possible we have missed a putative alternative nucleophile.

However, we have other data and experiments supporting the conclusion that no alternative nucleophile exists. Therefore, we want to stress that our conclusion that no such alternative exist is based on these extra data. These data and experiments are not discussed by either reviewer despite being present in the original manuscript. This puzzled us and we have modified the manuscript and the figures in the hope that they, and their significance, would not be missed.

Briefly:

1) We have performed cleavage-based labeling of the nucleophile responsible for cleavage. This experiment is depicted in Figure 4. The nucleophilic activity of the residue involved results in covalent link between the polypeptide (that includes the residue) and radiolabeled DNA. Therefore, a polypeptide that includes an active nucleophile will be radiolabeled and visible, whereas a polypeptide that is missing an active nucleophile will remain unlabeled and invisible. We can distinguish the BA and the A polypeptide from their size. In the case of the BA.A complex both the BA polypetide and the A polypetide are radiolabeled and therefore both have an active nucleophile. In the case of the BAF.A complex, the unmutated A polypeptide is labeled, meaning that a nucleophile is still active. In contrast, the BAF polypeptide shows no detectable labeling. This result means that removing the hydroxyl group from the catalytic tyrosine abolishes any protein-DNA covalent link, suggesting that no other nucleophile from the BA polypetidic chain can substitute for the catalytic tyrosine hydroxyl group. This experiment excludes the possibility of an alternative nucleophile coming from the polypeptidic chain of either GyrA or GyrB. This experiment, described in figure 4, is not discussed by the reviewer. This experiment is similar in principle to early experiments identifying catalytic tyrosine in topoisomerases. See for instance, (Shuman, Kane et al. 1989).

2) The experiment above does not exclude a nucleophile coming from the solvent. To exclude this possibility, we have used T5 exonuclease (which needs a free 5’ DNA end to digest) and ExoIII (which need a free 3’ DNA end to digest). We have shown the reconstituted cleavage is not sensitive to T5 and sensitive to ExoIII. This shows that the 5’ end of the cleaved sites are protected by a bulky polypeptide impairing T5 activity, which is active in our reaction as shown by the digestion of a control DNA fragment. This experiment shows that the reconstituted cleavage is very unlikely to come from a small nucleotide potentially provided by the solvent. This experiment is described in the main text and the results are shown in supplementary figure 5. It is not mentioned by either reviewer.

3) Finally, we would like to emphasize our experiment comparing the BAF.A59 to BALLL.A59. The BALLL.A59 complex displays increased cleavage compared to BAF.A59. If this increased cleavage was due to an alternative nucleophile on the BALLL side, we would expect an accompanying increase in supercoiling activity since the BALLL.A59 possesses one CTD, which is sufficient for supercoiling. The fact that no increased supercoiling activity is observed strongly suggests subunit exchange reconstituting an A59 dimer, inactive for supercoiling but active for cleavage. We believe this somewhat complex observation to be quite significant and we have attempted to clarify the manuscript and discuss its full significance in several places.

Reviewer #1 (Recommendations For The Authors):

An interesting paper on DNA gyrase that explains a puzzling paradox in terms of the double-strand break mechanism.

Major points

1) The authors consider several mechanisms that could potentially explain their data. On page 15, the authors present the evidence against the nicking closing mechanism proposed by Gubaev et al. Throughout the manuscript, they indicate where their experimental results agree with this earlier work but should also indicate and account for differences. For example, Gubaev et al describe cross linking experiments that they claim rule out subunit exchange. These aspects should be clearly explained.

Thank you for the suggestion. We have re-written the discussion to address this point. We are extensively discussing experiments from (Gubaev, Weidlich et al. 2016), and offer our interpretation of apparently conflicting results. We suggest that their experiments are basically consistent with our data when correctly interpreted. To keep the main manuscript clear, we have added a supplementary discussion where experiments from (Gubaev, Weidlich et al. 2016) are discussed further in relation to our data.

2) Page 9. The experiments done to rule out the perhaps unlikely alternative nucleophile hypothesis relate to the possible role of the Arg and Threonine of the RYT triad. These residues are close to the DNA and therefore are prime candidates and attractive targets for mutagenesis. However, strictly speaking, the mutant enzyme data presented do not rule all possibilities. For example, Serine is often the nucleophile used by resolvases to effect DNA recombination via subunit exchange. The ideal experiment to rule out/rule in other nucleophiles would be to identify the residue(s) that become attached to DNA in the cleavage reaction.

Please see above. We have effectively ruled an alternative nucleophile with our cleavage-based labeling experiment and others that were present and discussed in the original manuscript but were missed. We have modified the manuscript and figures in order to make this point clearer than before.

3) p17. The readout for subunit exchange used by the authors is double-stranded DNA cleavage. Attempts to directly detect the formation of the DNA cleaving complexes GyrA2B2 and (GyrBA)2 (arising from subunit exchange between heterodimers) by mass photometry were not successful. Perhaps FRET would have been another approach to try as it could also detect interface and domain interchanges.

Directly detecting interface exchange directly by proximity experiment would be extremely useful. FRET would have to be done in the BAF.A + GyrB configuration where the amount of interface exchange is important. Now, we do not have the tools to do that and developing them would be outside the scope of the study. We propose cross linking experiment to be done in the future. We argue that the manuscript is convincing without these for now. This will be addressed in the future. This point, and other possible future experiments are now discussed in the discussion section.

4) The underlying canvas of this paper is the strand passage mechanism of gyrase. It would seem appropriate to include the papers first proposing it - Brown P.O and Cozzarelli N.R. (1979) and Mizuuchi K et al (1980).

We very much agree. These papers have now been added in the introduction as appropriate, highlighting the relationship between double-strand cleavage and the strand-passage mechanism.

5) Figure 1. The quality of the insets is poor. It is difficult to pick out the key catalytic residues and their disposition vis-a-vis DNA.

We agree, Figure 1 has been re-done and the schematic theme has been harmonized throughout the whole manuscript. We very much hope that clarity has improved. Thank you for the suggestion.

6) The experimental work is a very detailed analysis of a specific feature of engineered gyrase heterodimers. Making the work accessible to the general reader will be important. Using shorter paragraphs each with a specific theme might help. In particular, the second paragraph of the Results on p7, the section on p9 and bottom of p11, p13 and the first paragraph of the Discussion on p14 are each a page or more long. A shorter manuscript that avoids overinterpretation of the smaller details would also help.

We agree. We have now split long paragraphs into individual sections, with titles, in the Results. This structure is recapitulated at the beginning of the discussion, and we have split the discussion into shorter paragraphs, each with a unique point being made.

7) The impact of the Gubaev et al (2016) paper for the field in general, and as the catalyst for the present work should be better documented. Mention of this earlier paper and its significance at the beginning of the Abstract and elsewhere e.g in the Introduction might also help with a more logical organization of the current findings and result in a shorter paper (which would be easier to read).

We have added a reference to (Gubaev, Weidlich et al. 2016) in the abstract and have expanded our introduction

Minor points

1) Legends for Figs 2 and 6; Supplementary Figs 1 and 8. The designation of subfigures as a, b, c, d , e etc appears to be incorrect. Check throughout and in the text.

The manuscript has been checked for such errors.

2) Figure 2, and first paragraph p8. Peaks in Fig 2c should be labelled to facilitate discussion on p8.

Agreed, this has been done.

3) Supplementary Fig 4 and elsewhere in the manuscript. A variety of notations are used to denote phenylalanine mutants e.g. AsubscriptF, AsuperscriptF and AF. Check and use one format throughout.

Done

4) Figures showing gels include the label '+EtBr, +cipro'. This is somewhat confusing because EtBr was contained in the gel (not the samples) whereas cipro was included in the reaction. Modify or describe in the legend..

We have re-written the figure legend.

5) Supplementary Fig 4b describes a small effect on the ratio of linear to nicked DNA for the triple LLL mutant. Is this significant? How many times was the measurement made?

This has been addressed in the original manuscript in the supplementary data. In term of quantification, the experiment has been done 3 times for each prep, with the same GyrB prep and concentration. The standard error is displayed on the figure. This result is very reproducible and have been reproduced more than 3 times. No LLL cleavage assay showed more single-strand than double-strand cleavage. For the phenylalanine mutant, no cleavage assay showed more double-strand than single-strand cleavage.

6) Supplementary Fig 5 legend. Should 'L' read 'size markers' (and give their sizes)?

Yes indeed, we have modified the figure to clarify.

7) p11 line 5. Is this statement correct?

Yes, it is correct. Although we hope we are on the same line. When the Tyrosine is mutated on one side only of the heterodimer, both single- and double-strand cleavage are protected from T5 exonuclease digestion.

8) 12 last line should read...and supercoiling activity (not shown)..were

Thank you, done.

There are a number of typos throughout the text, for example:

Page 3 line..Difficult to conclude...what?

Page 3 para 3...Lopez....and Blazquez

We have corrected these typos and checked the whole manuscript.

Reviewer #2 (Public Review):

DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification. The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity.

The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. incriminate in this earlier study the potential subunit swapping of the recombinant protein with the endogenous enzyme, that would be responsible for the detected negative supercoiling activity.

Accordingly, the authors also conclude that they cannot completely exclude the presence of endogenous subunits in their samples as well.

Strengths

The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).

Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :

• the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.

• DNA relaxation, cleavage and supercoiling assays

• biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

Together the combination of experimental approaches provides solid evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

We thank the reviewer for their supportive and considered comments.

Weaknesses

The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria, as proposed by Ideka et al, 2004. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs.

Thank you for this comment. Indeed, whether this interface exchange can happen in vivo and lead to recombination is a very important question. However, we believe that this is outside the scope of this study simply because of the amount of work one can fit into one paper. Proving that interface exchange can happen in vitro has already necessitated a number of non-trivial experiments and likewise investigating interface exchange in vivo will require a careful, long-term study (see our reply to reviewer #2 comment, who also raised this point). We can’t address it with one additional experiment with the tools we have. However, we very much hope to do it in the future.

Reviewer #2 (Recommendations For The Authors):

Specific questions and comments for the authors:

1) Complex identification during purification

The statement line 236-237 that "Our heterodimer preparation showed a single-peak on a gel-filtration column, distinct from the GyrA dimer peak" is not entirely clear. In Fig supp 1 b, how can the authors conclude from the superose 6 that GyrBA is separated from the GyrA dimer? Since they seem close in size 160/180kDa, they are unlikely to be well separated in a superose 6 gel filtration column. The SDS-PAGE seems to show both species in the same fractions #15-17 therefore it would not be possible to distinguish GyrBA. A from A2.

There appears to be some confusion about what Supp Fig. 1b shows. First, in all our gel filtration conditions both GyrBA and GyrA can’t exist as monomers at a significant concentration. Therefore, we can never observe the GyrBA monomer on a gel filtration column. Supp Fig. 1b shows the gel filtration profile of the BA.A heterodimer only. This is the output of the last, polishing step in the reaction. We analyze these results using SDS-PAGE. Therefore, the BA.A heterodimer will be denatured and separated into 2 polypeptides: GyrBA and GyrA, which migrates according to their size in an SDS-PAGE and forms two bands. These two bands do not represent two separate species in solution. They represent the separation of one species only, the BA.A heterodimer into its two, denatured, subunits: GyrA and GyrBA. We do not conclude from Supp Fig. 1 as a whole that GyrBA and the GyrA dimer are well separated, and this is not stated in the manuscript. We conclude that the BA.A dimer is fairly well separated from the GyrA dimer. They have significant different size (~260 kDa and ~180 kDa respectively) and form different peaks on a gel filtration column. The BA.A heterodimer has a GyrA subunit and therefore will shows a GyrA band on an SDS-PAGE, like the GyrA dimers but the two are obviously distinct in their quaternary structure. We are hoping that our new schematics and re-write of some of the results and figure legends will clarify this.

Panel 6 shows a different elution volume for the 2 species BA.A and A2 on an analytical S200 column, which appears better at separating the complexes in this size range.

Did the authors consider using a S200 column instead of superose 6 for the sample preparation, to optimize the separation of GyrBA. A from A2?

This is not a necessarily true statement (see above). We have not run the GyrA dimer on a Superose 6 column. The analysis was done on an s200 because extensive data for the GyrA dimer was already available with this, already calibrated column. We do not expect the Superose 6 to be worse in this size range. In fact, it might even be better. The Superose 6 profile in Supp. Fig. 1b shows BA.A only and no GyrA dimer. We have clarified the annotations in the figure to make this clearer.

Regarding the analytical gel filtration experiment, there is however an overlap in the elution volume in the analytical column, therefore how can the authors ensure there is no excess free A2 complex in the GyrBA. A sample?

Indeed, there is an overlap, but we argue that it is overstated. The important part of the overlap is where the maximum height of the GyrA peak is positioned compared to the BA.A trace, not where the traces intersect. This overlap is minimal. If a contaminating GyrA peak was hidden in the BA.A peak, it would have to be at least 10 times less intense than the BA.A peak. Since BA.A and GyrA dimer have roughly the same extinction coefficient, this means that a contamination would detectable at 10 % or even less. Our mass photometry further excludes such contamination.

Alternatively, the addition of a larger (cleavable) tag at the C-terminal end of the BA construct (therefore not disturbing dimer association) could allow to better distinguish the 2 populations already at the size exclusion step.

This is true and could allow cleaner purification. There are also other ways to achieve cleaner purification, like adding a secondary tag. However, like we argue in the manuscript, our contaminations are already minimal. It is questionable what benefits could be gained in changing the protocol. We also argue that the tandem tag method does not completely exclude contamination (Supplementary Discussion) and therefore we are not sure if this would be worth the time and expenditure.

2) GyrA and GyrB Oligomers:

In the mass photometry experiment, the authors explain that the low concentration of the proteins promotes dissociation of GyrA dimers, hence the detection of GyrA monomers instead of GyrA dimers, which are also detected in the GyrBA.A sample.

However, it cannot be concluded that the GyrA dimer is not formed in the condition of the gel filtration chromatography, at higher concentration.

In our mass photometry experiment, The BA.A sample is not as diluted as the GyrA dimer and much closer to our experimental condition. Since we have calculated the dissociation constant, we can calculate the expected level of dissociation (or reassociation). The level of dissociation is minimal in these conditions. If some dissociation is expected from the BA.A heterodimers, a very low amount of GyrBA monomer should also be present and yet they are not observed. We presume that it is because mass photometry is much more sensitive to GyrA (see our mixing mass photometry experiment that we have added). If the GyrA would reassociate at higher concentration, it would do so either with itself (forming a GyrA dimer) or with the GyrBA monomer, reforming the heterodimer. Assuming both GyrA dimer and heterodimer have the same dissociation constant, roughly one third of the GyrA monomer would reassociate with themselves. Assuming even complete reassociation of the GyrA dimer, this would leave only GyrA dimer accounting for 2% of the prep.

Another interpretation would be to assume that GyrBA monomers are not present at all and that GyrA monomer are reassociating only with themselves. This is not valid because of the following thermodynamic reason:

Since the profile for the GyrA dimer are collected at equilibrium, we should expect a ratio between GyrA monomer and dimers that follow the dissociation constant. In other words, if the GyrA monomer were in equilibrium with GyrA dimer we should expect a much higher dimer concentration already as the GyrA monomers are not as dilute. We do not observe a GyrA dimer peak in the BA.A profile, even though we can detect a low amount of GyrA dimer mixed with BA.A. Therefore, we conclude that the observed GyrA monomer must be in equilibrium with another dimerization partner, which is most probably the GyrBA monomer (see above). Therefore, only a minimal amount of GyrA dimer is expected to be formed at higher concentration by direct reassociation. This could probably increase if we let this solution-based exchange carry on for a long time at dissociation equilibrium. We have actually shown that this solution-based exchange is very slow and take several days because of the low dissociation at equilibrium.

The mass spectrometry analysis in Fig 2 confirms the presence of (monomeric) GyrA in the sample, despite different experimental conditions.

The concentration of heterodimer in the mass spectrometry experiment is actually higher than in the mass photometry experiment. This shows that self-reassociation of the GyrA monomer as suggested above is undetectable with mass spectrometry at higher concentration.

We considered that the “GyrA monomer” peak could be a contaminating GyrB monomer, which is ~90 kDa, which would explain the lack of reassociation. However, the mass spectrometry peak shows precisely the expected molecular weight of GyrA so we interpret this peak as arising from very limited dissociation of the BA.A heterodimer. The reassociation is limited at high concentration due simply to the fact that the difference in concentration between the mass photometry and our other experimental conditions is not that high. The GyrA dimer had to be diluted 400 times to see significant dissociation and yet even at this very low concentration the dissociation is far from complete.

Our general conclusions on the couple of point above is that we cannot completely exclude the presence of GyrA dimers being present, although they are undetectable in our working conditions either by mass photometry (lower concentration), Mass spectrometry (higher concentration) and even gel filtration (even higher concentration, see above). For the mass photometry, we have established that our detection threshold for a contamination is very low (see our mixing experiment).

Figure 2A: the authors state in the introduction that GyrB is a monomer in solution and then explain that the upper bands in the native gel are multimer of GyrB. Could the authors comment and provide the size exclusion profile of the Gyr B purification?

We have expanded our discussion of this. However, we have not been successful in collecting a gel filtration profile for GyrB. This is likely due to excessive oligomerization at the concentration we are using for gel filtration. We suggest that our mass photometry and Blue-Native PAGE experiment shows clearly that GyrB can be detected as a monomer in solution at the appropriate dilution. However, GyrB tends to oligomerize in a regular fashion (Consider especially Supp Fig. 8a), which suggest that it could align heterodimers on DNA in a linear, regular orientation. We have added a discussion of this.

Together the relevance of the oligomeric state of purified GyrA or GyrB should be clarified, relative to their role in subunit swapping.

We have added explanation in our discussion, while also trying to not be too speculative. Basically, we believe that GyrB oligomerization is likely to be involved. It is difficult to conclude for GyrA since no experiment has allowed us to test it. Therefore, the role of GyrA oligomerization, if any, is unclear. The GyrA tetramer is very prominent though and forms very easily. GyrB on the contrary forms longer oligomers more readily than GyrA and we surmise that this would help interface exchange. However, the structure of these GyrA and GyrB oligomers is not clear, which make it difficult to go beyond speculation on this. It would be a very interesting experiment if we were able to suppress GyrB oligomerization whilst conserving its ability to promote strand-passage and cleavage. Same goes for GyrA. Unfortunately, we are unable to do that at this time.

4) Subunit exchange

Line 320: the concept of subunit exchange in this context should be clearly explained. If one understands correctly, the authors mean that the BAF polypeptide, part of the BAF.A complex, could be replaced by a combination of B+A therefore forming a fully functional WT A2B2 gyrase complex.

Thank you for the suggestion. We have harmonized and clearly defined our terminology for interface swapping and subunit exchange in the introduction and attempted to be much more rigorous when referring to it.

A great effort has been done in this study to explain all the pros and cons of the experimental design but the length of the explanations may prevent readers outside of the field to fully appreciate the conclusions. This article would benefit from the addition of a few schematics to summarize the working hypothesis.

Thanks for the suggestion. We have added a series of schematics to illustrate our interpretation for each construct. As mentioned above the terminology has been more rigorously defined and updated throughout the manuscript.

5) Presence of endogenous GyrA

Line 419-425: it is quite difficult to follow the explanations regarding the possible contamination of the sample by endogenous GyrA.

Maybe these points should rather be addressed in the discussion, when debating the conclusions of Gubaev et al.

We agree. We have re-organized the Discussion doing just that. We added a Supplementary Discussion in which we further discuss the contamination problem in relation to (Gubaev, Weidlich et al. 2016).

Production of the subunits in another (non bacterial) expression system or a cell free system may prevent the association of endogenous protein.

Absolutely. We are planning on addressing this in the future, using the yeast expression system.

6) Mechanism for subunit swapping

Lines 588-595: As described by the authors the BA fusion shows decreased activity when compared with the WT probably due to limited conformational flexibility in absence of an additional linker sequence between the fused subunits.

The affinity of BA for A may possibly be reduced compared to the free A2B2 complex, due to a relative stiffness of the fusion upon full association with a free B subunit, as rightfully pointed by the authors.

If subunit exchange do happen in vitro, at least in the conditions of this study, the authors could assess the affinity of BA for A, when compared to the association of free B and A subunits

Experiments using analytical ultracentrifugation or surface plasmon resonance (SPR) may allow to determine the relative affinity of the BA +(A+B) compared to the A2B2 complex. This could be done also for the BALLL mutant and association with A59.

It would be extremely useful to measure the affinity of BA for A. However, this is difficult because of the high affinity of the interface. To measure a dissociation constant, one has to be able to measure the concentration of the monomer and the dimer at equilibrium. Because of this, the complex must be diluted enough to see any dissociation, making detection difficult. In practice, this also means that we cannot purify monomeric versions of these subunits. We therefore can’t perform “on-rate” study on an SPR surface, which would require flowing monomers on its partner subunit tethered to the SPR surface. However, we could perform “off-rate” studies, but the dissociation time is likely to be very long, making the measurement difficult. We have not tried it though, and it could turn out to be informative. An analysis of antibodies off-rate done in the past could provide a guideline for us to perform this experiment. Analytical ultracentrifugation is an excellent technique and could in theory provide information. In practice however it would be still necessary to dilute the complex enough to obtain significant dissociation at equilibrium, making detection difficult. As far as we are aware, analytical ultracentrifugation rely on UV absorbance for protein detection and therefore we probably would not detect our material at the necessary dilution. We are however open-minded about technique with very sensitive detection methods that could be used.

9) In vivo relevance

The study does not conclude on the subunits exchange in vivo, which have been suggested by earlier studies by Ikeda et al. To elaborate further on the relevance of such mechanism in the bacteria, experiments involving the fluorescent labeling of endogenous / exogenous mutant subunits may be required to provide further information on this phenomenon.

We completely agree that the in vivo relevance of such phenomena is the central question. Addressing this directly is not trivial though. Expressing both BA and A in vivo will results in random partnering and lead to a mix of dimers: A2 (1/4), BA2(1/4) and BA.A (1/2), assuming equal interface affinity. Therefore, to see subunit exchange in the same way as in vitro, one would have to get rid of the BA2 and A2 dimer together, or the BA.A dimer only. Our initial strategy to do that would be to engineer a specific dimer as being uniquely targeted for degradation. This could allow us to “get rid” of for instance the BA.A dimer. Subsequently, we would turn off the degradation and translation together and observe the rate of subunit exchange. This is not trivial though and would be the subject of a further study.

10) Figure 3: I guess the "intact" label refers to the supercoiled DNA (SC) ? It also appears as "uncleaved" in supp Figure 6. The same label for this topoisomer should be used throughout.

Thank you for pointing that out. It has now been corrected.

Bandak, A. F., T. R. Blower, K. C. Nitiss, R. Gupta, A. Y. Lau, R. Guha, J. L. Nitiss and J. M. Berger (2023). "Naturally mutagenic sequence diversity in a human type II topoisomerase." Proceedings of the National Academy of Sciences 120(28).

Germe, T., J. Voros, F. Jeannot, T. Taillier, R. A. Stavenger, E. Bacque, A. Maxwell and B. D. Bax (2018). "A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance." Nucleic Acids Res.

Gubaev, A., D. Weidlich and D. Klostermeier (2016). "DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism." Nucleic Acids Res 44(21): 10354-10366.

Hartmann, S., A. Gubaev and D. Klostermeier (2017). "Binding and Hydrolysis of a Single ATP Is Sufficient for N-Gate Closure and DNA Supercoiling by Gyrase." J Mol Biol 429(23): 3717-3729. Shuman, S., E. M. Kane and S. G. Morham (1989). "Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I." Proc Natl Acad Sci U S A 86(24): 9793-9797.

Stelljes, J. T., D. Weidlich, A. Gubaev and D. Klostermeier (2018). "Gyrase containing a single C-terminal domain catalyzes negative supercoiling of DNA by decreasing the linking number in steps of two." Nucleic Acids Res.

eLife assessment

This study presents two useful new mouse models that individually tag proteins from the SMAD family to identify distinct roles during early pregnancy. Solid evidence is provided that SMAD1 and SMAD5 target many of the same genomic regions as each other and the progesterone receptor. Given the broad effect of these signaling pathways in multiple systems, these new tools will most likely interest readers across biological disciplines.

"bevor die AFD die verfassung abschafft, müssen wir die verfassung abschaffen" -- klingt doch logisch...<br /> die sind einfach voll hängen geblieben in ihrer opferrolle, und erfinden jeden tag neue strohmänner, neue false flag attacks, neue lügen... 9/11 ist quasi zum dauerzustand geworden<br /> aber die grundprobleme bleiben: pazifismus und übervölkerung

Reviewer #2 (Public Review):

Summary:<br /> Verma et al. provide a short technical report showing that endogenously tagged dynein and dynactin molecules localize to growing microtubule plus-ends and also move processively along microtubules in cells. The data are convincing, and the imaging and movies very nicely demonstrate their claims. I don't have any large technical concerns about the work. It is perhaps not surprising that dynein-dynactin complexes behave this way in cells due to other reports on the topic, but the current data are among some of the nicest direct demonstrations of this phenomenon. It may be somewhat controversial since a separate group has reported that dynein does not move processively in mammalian cells (https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). Because of this, it might be nice for the authors to comment on this discrepancy in the field, although the aforementioned work is still in pre-print form.

Strengths:<br /> Using state-of-the-art methods to endogenously tag dynein/dynactin subunits and performing live-cell imaging is convincing and useful for the field.

Weaknesses:<br /> The claims are perhaps not surprising or novel given the extensive data already published in the field. However, there aren't many similar studies using endogenously tagged subunits to date.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Verma et al. set out to visualize cytoplasmic dynein in living cells and describe their behaviour. They first generated heterozygous CRISPR-Cas9 knock-ins of DHC1 and p50 subunit of dynactin and used spinning disk confocal microscopy and TIRF microscopy to visualize these EGFP-tagged molecules. They describe robust localization and movement of DHC and p50 at the plus tips of MTs, which was abrogated using SiR tubulin to visualize the pool of DHC and p50 on the MTs. These DHC and p50 punctae on the MTs showed similar, highly processive movement on MTs. Based on comparison to inducible EGFP-tagged kinesin-1 intensity in Drosophila S2 cells, the authors concluded that the DHC and p50 punctae visualized represented 1 DHC-EGFP dimer+1 untagged DHC dimer and 1 p50-EGFP+3 untagged p50 molecules.

Strengths:<br /> The idea and motivation behind this work are commendable.

Weaknesses:<br /> There are several major issues with the characterization of the knock-in lines generated, the choice of imaging and analysis methods, and inadequate discussion of prior findings.

The specific points are below:

1. CRISPR-edited HeLa clones:<br /> (i) The authors indicate that both the DHC-EGFP and p50-EGFP lines are heterozygous and that the level of DHC-EGFP was not measured due to technical difficulties. However, quantification of the relative amounts of untagged and tagged DHC needs to be performed - either using Western blot, immunofluorescence or qPCR comparing the parent cell line and the cell lines used in this work.<br /> (ii) The localization of DHC predominantly at the plus tips (Fig. 1A) is at odds with other work where endogenous or close-to-endogenous levels of DHC were visualized in HeLa cells and other non-polarized cells like HEK293, A-431 and U-251MG (e.g.: OpenCell (https://opencell.czbiohub.org/target/CID001880), Human Protein Atlas (https://www.proteinatlas.org/ENSG00000197102-DYNC1H1/subcellular#human), https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). The authors should perform immunofluorescence of DHC in the parental cells and DHC-EGFP cells to confirm there are no expression artifacts in the latter. Additionally, a comparison of the colocalization of DHC with EB1 in the parental and DHC-EGFP and p50-EGFP lines would be good to confirm MT plus-tip localisation of DHC in both lines.<br /> (iii) It would also be useful to see entire fields of view of cells expressing DHC-EGFP and p50-EGFP (e.g. in Spinning Disk microscopy) to understand if there is heterogeneity in expression. Similarly, it would be useful to report the relative levels of expression of EGFP (by measuring the total intensity of EGFP fluorescence per cell) in those cells employed for the analysis in the manuscript.<br /> (iv) Given that the authors suspect there is differential gene regulation in their CRISPR-edited lines, it cannot be concluded that the DHC-EGFP and p50-EGFP punctae tracked are functional and not piggybacking on untagged proteins. The authors could use the FKBP part of the FKBP-EGFP tag to perform knock-sideways of the DHC and p50 to the plasma membrane and confirm abrogation of dynein activity by visualizing known dynein targets such as the Golgi (Golgi should disperse following recruitment of EGFP-tagged DHC-EGFP or p50-EGFP to the PM), or EGF (movement towards the cell center should cease).

2. TIFRM and analysis:<br /> (i) What was the rationale for using TIRFM given its limitation of visualization at/near the plasma membrane? Are the authors confident they are in TIRF mode and not HILO, which would fit with the representative images shown in the manuscript?<br /> (ii) At what depth are the authors imaging DHC-EGFP and p50-EGFP?<br /> (iii) The authors rely on manual inspection of tracks before analyzing them in kymographs - this is not rigorous and is prone to bias. They should instead track the molecules using single particle tracking tools (eg. TrackMate/uTrack), and use these traces to then quantify the displacement, velocity, and run-time.<br /> (iv) It is unclear how the tracks that were eventually used in the quantification were chosen. Are they representative of the kind of movements seen? Kymographs of dynein movement along an entire MT/cell needs to be shown and all punctae that appear on MTs need to be tracked, and their movement quantified.<br /> (v) What is the directionality of the moving punctae?<br /> (vi) Since all the quantification was performed on SiR tubulin-treated cells, it is unclear if the behavior of dynein observed here reflects the behavior of dynein in untreated cells. Analysis of untreated cells is required.

3. Estimation of stoichiometry of DHC and p50<br /> Given that the punctae of DHC-EGFP and p50 seemingly bleach on MT before the end of the movie, the authors should use photobleaching to estimate the number of molecules in their punctae, either by simple counting the number of bleaching steps or by measuring single-step sizes and estimating the number of molecules from the intensity of punctae in the first frame.

4. Discussion of prior literature<br /> Recent work visualizing the behavior of dyneins in HeLa cells (DOI: 10.1101/2021.04.05.438428), which shows results that do not align with observations in this manuscript, has not been discussed. These contradictory findings need to be discussed, and a more objective assessment of the literature in general needs to be undertaken.

Reviewer #2 (Public Review):

Summary:<br /> Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:<br /> One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:<br /> While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

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I know you're trying to steer them towards the annual membership... but could you lead with the monthly price tag here to make the mental hurdle even lower?

I'd also include that it's month-to-month without lock in contract

Die Bauernproteste haben zu Revisionen von Maßnahmen zur Dekarbonisierung (und Pestizidreduktion) in europäischen Ländern und auf EU-Ebene geführt, obwohl die Klimaziele der EU ohne eine deutliche Reduktion der Emissionen der Landwirtschaft nicht zu erreichen sind. Der Arikel der New York Times beschäftigt sich mit der besonderen Rolle der Landwirtschaft in der EU-Politik und mit der Notwendigkeit, Klimapolitik als just transition zu gestalten. https://www.nytimes.com/2024/02/06/climate/europe-farming-protests-policy.html

Mehr zu den EU-Emissionszielen für 2040: https://hypothes.is/search?q=tag%3A%22EU%20emission%20goals%202040%22

Die Europäische Kommission plant, die Treibhausgas-Emissionen bis 2040 um 90% im Vergleich zu 1990 zu reduzieren. Dabei soll CCS eine große Rolle spielen. Der Plan ist eine Empfehlung für die nächste Kommission. Aufgrund der Proteste von Landwirt:innen wurden einige der vorgesehenen Regelungen, etwa zu Methan und Lachgas, in letzter Minute zurückgenommen. https://www.theguardian.com/environment/2024/feb/06/eu-lays-out-plan-to-cut-greenhouse-emissions-by-90-by-2040

Mehr dazu: https://hypothes.is/search?q=tag%3A%22EU%20emission%20goals%202040%22

Die Hitzeperioden des vergangenen Sommers haben in Frankreich 5167 Todesopfer gefordert. Das ist eine der höchsten Zahlen der vergangenen Jahre. https://www.liberation.fr/environnement/climat/plus-de-5-000-morts-lies-a-la-chaleur-lors-de-lete-2023-20240208_UFBI7UKESFFJ7MHLRKWS4DPUM4/

Mehr zu Hitzetoten: https://hypothes.is/search?q=tag%3A%22heat%20deaths%22

Kurzer Überblicksartikel der taz zur Krise in der Windindustriebranche. Sie hängt unter anderem mit Lieferkettenproblemen, Preissteigerungen und Genehmigungsverfahren zusammen, aber auch mit eigenen Fehlern der drei westlichen Marktführer #Siemens Energy, #Ørsted und #Vestas. https://taz.de/Windenergiekonzerne-in-der-Bredouille/!5987469/

Mehr zu Ørsted: https://hypothes.is/search?q=tag%3A%22Orsted%22

Mehr zu Siemens Energy: https://hypothes.is/search?q=tag%3A%22Siemens%20Energy%22

Mehr zu Vestas: https://hypothes.is/search?q=tag%3A%22Vestas%22

Author Response

The following is the authors’ response to the original reviews.

Reviewer #2 (Public Review):

Making state-of-the-art (super-resolution) microscopy widely available has been the subject of many publications in recent years as correctly referenced in the manuscript. By advocating the ideas of open-microscopy and trying to replace expensive, scientific-grade components such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested researchers nowadays have a number of different frameworks to choose from. In the iteration of the theme presented here, the authors used the existing modular UC2 framework, which consists of 3D printable building blocks, and combined a cheapish laser, detector and x,y,(z) stage with expensive filters/dichroics and a very expensive high-end objective (>15k Euros). This particular choice raises a first technical question, to which extent a standard NA 1.3 oil immersion objective available for <1k would compare to the chosen NA 1.49 one.

Measurement of the illumination quality (e.g. the spectral purity) of low budget lasers convinced us of the necessity to use spectral filtering. These cannot be replaced with lower budget alternatives, to sill retain the necessary sensitivity to image single molecules. As expected, the high-quality objectives are able to produce high-quality data. Lower budget alternatives (<500 €) to replace the objective have been tried out. Image quality is reduced but key features in fluorescent images can be identified (see figure S1). The usage of a low budget objective for SMLM imaging is possible, but quality benchmarks such as identifying railroad tracks along microtubule profiles is not possible. Their usage is not optimal for applications aiming to visualize single molecules and might find better application in teaching projects.

The choice of using the UC2 framework has the advantage, that the individual building blocks can be 3D printed, although it should be mentioned that the authors used injection-molded blocks that will have a limited availability if not offered commercially by a third party. The strength of the manuscript is the tight integration of the hardware and the software (namely the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms for fast sub-pixel localisation and access to Napari).

The injection-molded cubes can be acquired through the OpenUC2 platform. Alternatively, the 3D printable version of the cubes is freely available and just requires the user to have a 3D printer. https://github.com/openUC2/UC2-GIT/tree/master/CAD/CUBE_EmptyTemplate

The presented experimental data is convincing, demonstrating (1) extended live cell imaging both using bright-field and fluorescence in the incubator, (2) single-particle tracking of quantum dots, and (3) and STORM measurements in cells stained against tubulin. In the following I will raise two aspects that currently limit the clarity and the potential impact of the manuscript.

First, the manuscript would benefit from further refinement. Elements in Figure 1d/e are not described properly. Figure 2c is not described in the caption. GPI-GFP is not introduced. MMS (moment scaling spectrum) could benefit from a one sentence description of what it actually is. In Figure 6, the size of the STORM and wide-field field of views are vastly different, the distances between the peaks on the tubuli are given in micrometers rather than nanometers. (more in the section on recommendations for the author)

Second, and this is the main criticism at this point, is that although all the information and data is openly available, it seems very difficult to actually build the setup due to a lack of proper documentation (as of early July 2023).

1) The bill of materials (https://github.com/openUC2/UC2-STORM-and-Fluorescence#bill-of-material) should provide a link to the commercially available items. Some items are named in German. Maybe split the BoM in commercially available and 3D printable parts (I first missed the option to scroll horizontally).

2) The links to the XY and Z stage refer to the general overview site of the UC2 project (https://github.com/openUC2/) requiring a deep dive to find the actual information.

3) Detailed building instructions are unfortunately missing. How to assemble the cubes (pCad files showing exploded views, for example)? Trouble shooting?

4) Some of the hardware details (e.g. which laser was being used, lenses, etc) should be mentioned in the manuscript (or SI)

I fully understand that providing such level of detail is very time consuming, but I hope that the authors will be able to address these shortcomings.

1) The bill of materials has been and will also in future still be improved. The items have been sorted into UC2 printed parts and externally acquired parts. The combination of part name as well as provider enables users to find and acquire the same parts. Additionally, depending on the country where the user is located, different providers of a given part might be advantageous as delivery means and costs might vary.

2) The Z-stage now has a specific repository with different solutions, offering different solutions with different levels of movement precision. According to the user and their budget, different solutions can be optimal for the endeavor.

https://github.com/openUC2/UC2-Zstage

The XY stage now also has a detailed repository, as the motorizing of the stage requires a fair amount of tinkering. The video tutorials and the detailed instructions on stage motorizing should help any user to reproduce the stage shown within this manuscript. https://github.com/openUC2/UC2-Motorized-XY-Table

3) The updated repository has a short video showing the general assembly of the cubes and the layers. Additionally, figure S2 shows all the pieces that are included in every layer (as a photograph as well as CAD). An exploded view of the complete setup would certainly be a helpful visualization of the complete setup. We however hope that the presented assembly tutorials and documents are sufficient to successfully reproduce the U.C.STORM setup.

First, we want to thank the reviewers for their effort to help us improving our work. We apologize for any trivial mistakes we had overlooked. Please find below our answers to the very constructive and helpful comments of the editors.  

Recommendations for the authors:

Reviewer #1 (Recommendations for The Authors):

To complement the current data set:

Figure 2(a & b): Panels i & ii, were chosen on the area where the distribution of the laser appears to be flatter. Can the authors select microtubules from a different section? Otherwise, it is reasonable to also crop the field-of-view along the flatter area (as done in Fig 6).

Figure 2 was changed to according to the reviewer’s suggestions. The profiles of microtubules from a different section have similar profiles, but the region with best illumination thus best SNR of the profile have been used for the figure.

Figure 2(c): The current plot shows the gaussian distribution which does not appear to be centered. Instead of a horizontal line, can the authors provide a diagonal profile across the field of view and update the panel below?

A diagonal cross-section of the illuminated FOV is provided in figure 2 to replace the previous horizontal profile. The pattern seems not to be perfectly radially symmetric, and more light seems to be blocked at the bottom of the illumination pattern compared to the top. A possible improvement can be provided by a fiber-coupled laser, that could provide a more homogeneous illumination while being easier to handle in the assembly process.

Author response image 1.

Diagonal cross-section of the illuminated FOV. Pixel-size (104nm) is the same as in figure 2. Intensity has been normalized according to the maximal value.

Figure 2(d): The system presents a XY drift of ~500nm over the course of a couple of hours. However, is not clear how the focus is being maintained. Can the authors clarify this point and add the axial drift to the plot?

The axial position of the sample could be maintained over a prolonged period of time without correcting for drift. Measurements where an axial shift was induced by tension pulses in the electronics have been discarded, but the stability of the stage seems to be sufficient to allow for imaging without lateral and axial drift correction. The XY drift measurement displayed in Figure 2(d) can be extended by measuring the σ of the PSF over time. The increase of σ would suggest an axial displacement in relation to the focus plane. In these measurements, a slight axial drift can be seen, the fluorescent beads however can still be localized over the whole course of the measurement.

A separate experiment was performed, using the same objective on the UC2 setup and on a high-quality setup equipped with a piezo actuator able to move in 10 nm steps. The precise Z steps of the piezo allows to reproducibly swipe through the PSF shape and to give an estimate of the axial displacement of the sample, according to the changes in PSF FWHM (Full Width at Half Maximum). When superimposing the graph with the UC2 measurement of fluorescent beads with the smallest possible Z step, an estimate about the relative axial position of the sample can be provided. The accuracy of the stage however remains limited.

Author response image 2.

Drift Figure: a. Drift of fluorescent TS beads on the UC2 setup positioned upon an optical table over a duration of two hours. Beads are localized and resulting displacement in i. and ii. are plotted in the graphs below. The procedure is repeated in b. with the microscope placed on a laboratory bench instead. c. (for the optical table i.) and d. (for the laboratory bench i.) show the variation in the sigma value of the localized beads over the measurement duration. As the sigma values changes when the beads are out of focus, the stability of the setup can be confirmed, as it remains practically unchanged over the measurement duration.

Author response image 3.

Z-focus Figure: Estimation of the axial position of TS beads on the UC2 setup. a. The change in PSF FWHM was quantified by acquiring a Z stack of a beads sample. The homebuilt high-quality setup (HQ) was used as a reference, by using the same objective and TS sample. The PSF FWHM on the UC2 setup was measured using the lowest possible axial stage displacement. A Z-position can thus be estimated for single molecules, as displayed in b.

Addressing the seemingly correlated behavior of the X and Y drift:

Further measurement show less correlation between drift in X and in Y. Simultaneous motion in X and Y seems to indicate that the stage or the sample is tilted. The collective movement in X and Y seems accentuated by bigger jumps, probably originating from vibrations (as more predominantly shown in the measurements on the laboratory bench compared to the optical table). Tension fluctuations inducing motion of the stage are possible but are highly unlikely to have induced the drift in the displayed measurements.

Figure 3: Can the authors comment on the effect or otherwise potential effect of the incubator (humidity, condensation etc) may have on the system (e.g., camera, electronics etc)?

When moving the microscope into the incubator, the first precaution is to check if the used electronics are able to perform at 37° C. Then, placing the microscope inside the incubator can induce condensation of water droplets at the cold interfaces, potentially damaging the electronics or reducing imaging quality. This can be prevented by preheating the microscope in e.g. an incubator without humidity, for a few hours before placing it within the functional incubator. The used incubator should also be checked for air streams (to distribute the CO2), and a direct exposure of the setup to the air stream should be prevented. The usage of a layer of foam material (e.g. Polyurethane) under the microscope helps to reduce possible effects of incubator vibrations on the microscope. The hydrophilic character of PLA makes its usage within the incubator challenging due to its reduced thermal stability. The temperature also inherently reduces the mechanical stability of 3D printed parts. Using a less hydrophilic and more thermally stable plastic, such as ABS, combined with a higher percentage of infill are the empirical solution to this challenge. Further options and designs to improve the usage of the microscope within the incubator are still in developement.

Figure 5: Can the authors perform single molecule experiments with an alternative tag such as Alexa647?

The SPT experiments were performed with QDs to make use of their photostability and brightness. The dSTORM experiment suggests that imaging single AF647 molecules with sufficient SNR is possible. The usage of AF647 for SPT is possible but would reduce the accuracy of the localization and shorten the acquired track-lengths, due to the blinking properties of AF647 when illuminated. The tracking experiment with the QDs thus was a proof of concept that the SPT experiments are possible and allow to reproduce the diffusion coefficients published in common literature. The usage of alternative tags can be an interesting extension of the capabilities that users can perform for their applications.

Figure 6: The authors demonstrate dSTORM of microtubules. It would enhance the paper to also demonstrate 3D imaging (e.g., via cylindrical lens).

The usage of a cylindrical lens for 3D imaging was not performed yet. The implementation would not be difficult, given the high modularity of the setup in general. The calibration of the PSF shape with astigmatism might however be challenging as the vertical scanning of the Z-stage lacks reliability in its current build. Methods such as biplane imaging might also be difficult to implement, as the halved number of photons in each channel leads to losses in the accuracy of localization. As a future improvement of the setup, the option of providing 3D information with single molecule accuracy is definitely desirable and will be tried out. In the following figure, two concepts for introducing 3D imaging capabilities in the detection layer of the microscope are presented.

Author response image 4.

3D concept Figure: Two possible setup modifications to provide axial information when imaging single molecules. a. A cylindrical lens can be placed to induce an asymmetry between the PSF FWHM in x and in y. Every Z position can be identified by two distinct PSF FWHM values in X and Y. b. By splitting the beam in two and defocusing one path, every PSF will have a specific set of values for its FWHM on the two detectors.

Imaging modalities section: Regarding the use of cling film to diffuse; can the authors comment on the continual use of this approach, including its degradation over time?

The cling foil was only used as a diffuser for broadening the laser profile. A detailed analysis of the constitution of the foil was not done, as no visible changes could be seen on the illumination pattern and the foil itself. The piece of cling foil is attached to a rotor. Detaching of the cling foil or vibrations originating from the rotor need to be minimized. By keeping the rotation speed to a necessary minimum and attaching the cling foil correctly to the rotor, a usable solution can be created. The low price of the cling foil provides the possibility to exchange the foil on a regular basis, allowing to keep the foil under optimal conditions.

Author response image 5.

Profile Figure: By moving a combination of pinhole and photometer to scan through the laser profile with a translational mount, the shape of the laser beam can be estimated. The cling foil plays the same role as a diffuser in other setups.

Reviewer #2 (Recommendations for The Authors):

lines

20, add "," after parts

110, rotating cling foil?

112/116, "custom 3D printed" I thought they were injection molded, please finalize

113, "puzzle pieces" rephrase and they are also barely visible

119, not clear that the stage is a manual stage that was turned into a motorised one by adding belts

123-126, detail for SI,

132, replace Arduino-coded with Arduino-based

143, add reference to Napari

146, (black) cardboard seems to be a cheaper and quicker alternative

153, dichroic

151-155, reads more like a blog post than a paper (maybe add a section on trouble shooting)

156, antibody?

167/189, moderate, please be specific

194, layer of foam material, specify

221, add description/reference to GPI. What is that? why is it relevant?

226: add one sentence description of MMS

318, add "," after students

332-334, as mentioned earlier, not clear, you bought a manual stage and connected belts, correct?

376-377, might be difficult to understand for the layman

391, what laser was used?

Figure 1, poor contrast between components, components visible should be named as much as possible, maybe provide the base layer in a different shade. To me, the red and blue labels look like fluorophores.

Figure 1. looks like d is the excitation layer and not e, please fix.

Figure 2, caption a-c, figure 1-d!, btw, why is the drift so anti-correlated?

Figure 6 (line 259) nanometer I guess, not micrometer

We now incorporated all the above-mentioned changes in the manuscript. Furthermore we added the supplementary Figures as below.

Author response image 6.

Basic concept of the UC2 setup: Left: Cubes (green) are connected to one another via puzzle pieces (white). Middle: 3D printed mounts have been designed to adapt various optics (right) to the cube framework. Combined usage of cubes and design of various mounts allows to interface various optics for the assembly.

Author response image 7.

Building the UC2 widefield microscope: a. Photograph of the complete setup. b. All pieces necessary to build the setup. A list of the components can be found in the bill of materials. c. Bottom emission layer of the microscope before assembly. d. Emission layer after assembly. Connection between cubes is doubled by using a layer of puzzles on the top and the bottom of the emission layer. e. CAD schematic of the emission layer and the positioning of the optics. f. Middle excitation layer of the microscope before assembly. Beam magnifier and homogenizer have been left out for clarity. g. Excitation layer after assembly is also covered by a puzzle layer. h. CAD schematic of the excitation layer and the positioning of the optics. i. Z-stage photograph and corresponding CAD file. Motor of the stage is embedded within the bottom cube. j. A layer of empty cubes supports the microscope stage. k. At this stage of the assembly, the objective is screwed into the objective holder. l. Finally, the stage is wired to the electronics and can then be mounted on top of the microscope (see a.).

Author response image 8.

Measurements performed on the UC2 setup with lower budget objectives. The imaged sample is HeLa cells, stably transfected to express CLC-GFP, then labelled with AF647 through immunostaining. The setup has been kept identical except for the objectives. Scale bar respectively represents 30 µm.

DOI: 10.1002/ctm2.1531

Resource: Addgene (RRID:SCR_002037)

Curator: @olekpark

SciCrunch record: RRID:SCR_002037

---

What is this?

Consolidated peer review report (22 January 2024)

GENERAL ASSESSMENT

Nanobodies (Nbs) are small antibody fragments that function similarly to antibodies. The smaller size of nanobodies makes them useful tools for studying biology and potentially useful as therapeutics. Nanobodies have had a significant impact on research related to the structure and function of G protein-coupled receptors (GPCRs), a family of proteins that are the target of approximately 30% of approved drugs. Nanobodies fused to peptide agonists can potentially increase the potency and selectivity of ligands.

The manuscript by Nayara Braga Emidio and Ross Cheloha describes the fusion of peptide agonists to Nbs to create chimeric ligands that differentially modulate the molecular pharmacology of the Neurokinin 1 receptor (NK1R), a potential therapeutic target for the treatment of pain. The authors observe that Nb-peptide fusions display divergent pharmacology to that of the unfused peptides via extensive characterisation at multiple signalling pathways (cAMP, Ca2+ mobilization, direct Gq TruPATH measurements, and β-arrestin recruitment), receptor binding assays, and measures of downstream transcriptional activation. The pharmacology results show that these conjugates exhibit diverse and unexpected signalling properties, including enhanced receptor binding, high potency partial agonism, prolonged cAMP production, and altered transcriptional outputs.

However, the degree to which signalling was altered was highly dependent on the location of the epitope tag and the utilized Nbs with small alterations in the relative distance and orientation between the Nb epitopes and peptide binding sites, causing significantly different outcomes. These findings highlight the potential of nanobody conjugation for creating compounds with biased agonism, extended duration of action, and improved transcriptional responses, suggesting their promise for research on GPCR signal transduction mechanisms. This study also lays the groundwork for important considerations regarding optimising nanobody-peptide fusions. Importantly, for peptide discovery, these approaches may afford improved properties regarding selectivity and duration of action. Overall, this work suggests an opportunity to create long-acting agonists with enhanced signalling properties using nanobody-peptide conjugates. However, this would require further experiments to validate the mechanism of the altered pharmacology responses of the Nb conjugates.

RECOMMENDATIONS

Essential revisions:

1. Because no known Nbs bind to WT NK1R, the authors have fused the epitopes of three different Nbs (6e, alpha, BC2) to the N-terminus of NK1R. These epitope tags could alter the pharmacology of the endogenous ligand Substance P (SP) with respect to the WT receptor. A comparison of signalling for WT versus the epitope-tagged NK1R in Figure 1C would alleviate these concerns.

2. The expected masses of the Nb conjugates after sortagging are sometimes over or under the expected masses (Table S2). Could the authors clarify the reason for these differences in the text.

3. Based on the results of Figure 2, all Nb conjugates, including the negative control NbGFP-peptide, negatively impact signaling by reducing efficacy in cAMP production (which is completely abolished for Nb-SP6-11) and reducing potency in β-arrestin and Gq activation. This could be due to differences in the binding of conjugated NKA compared to non-conjugated NKA due to conformational constraints or by hindering access of the peptides to the orthosteric pocket. In addition, it is unclear if the Nbs alter NK1R signaling on their own or if they act as allosteric modulators. These concerns could be experimentally addressed in both functional and binding experiments using 1) unconjugated Nbs and 2) unconjugated Nbs and peptides.

4. Compared to the Nbalpha-NKA and Nb6e-NKA conjugates, NbBC2-NKA has no effect in the cAMP assays (no increase in potency or effect in washout experiments). This is despite NbBC2-NKA having the greatest effect in binding experiments (Figure 3A,B). Can the authors discuss these differences, particularly with respect to the conclusion that a bitopic binding mechanism may contribute to prolonged signalling.

5. Regarding biased agonism as a potential advantage of Nb-peptide conjugates, the kinetics of β-arrestin recruitment or activation should also be measured (Figure 3) to determine if there is prolonged arrestin activation or receptor internalization.

6. The impact of Nb conjugation on ligand competition binding assays was assessed in Figures 3A and 3B. However, it would be useful to include the unconjugated Nbs as a control to determine if the enhanced inhibition is due to increased hindrance to the orthosteric pocket (see comment #3) or due to increased binding of the Nb-peptide conjugates as suggested. Similarly, in Fig S10, the lack of inhibition by spantide with the Nb6e-NKA could be due to reduced access of spantide to the orthosteric pocket in the presence of the Nb conjugate due to steric hindrance and testing with unconjugated Nb6e would strengthen the results.

7. The kinetics of cAMP signaling are assessed in Figures 3C and 3D. An EC10 concentration of G3-NKA was compared to an EC100 concentration of the other ligands, which may not be appropriate for comparisons of kinetics in this washout experiment. Do the authors have an explanation for comparing different concentrations?

8. In the cAMP washout experiments, cAMP production was still increased after washout (Figure 3C). Can the authors discuss why this was observed (Figure 3C).

9. In the transcriptional reporter assays (Figure 4), can the authors clarify why ~35nM was chosen as the concentration of peptides?

10. There are significant caveats with the Figure 5A model provided that the authors do not mention or address. Importantly, whilst AlphaFold 2 is useful for predicting the structure of well-ordered proteins, the relative location & orientation of these domains is unreliable when there are large flexible linkers between them; as is the case with the NK1R N-terminus. It would be at least worth mentioning this, and at best doing additional MD simulations to show the relative orientation of these two "domains". In addition, the authors should discuss the effects various linker lengths between the Nbs and peptide would have.

11. If the author's suggestions are true about the relative position of the epitope tag in relation to the peptide binding pocket, this could be demonstrated by making a construct where the epitope positions are swapped. Alternatively, instead of using 3x epitope-tagged constructs, single-tagged epitope NK1R constructs should demonstrate this.

12. There is no mention of the relative affinity of the Nb:epitope pairs and how this might influence the observed pharmacology, in particular the binding experiments with washout and readouts of transcriptional activation. This should be considered by the authors.
13. With respect to therapeutic relevance, how does the prolonged cAMP production or enhanced transcription correlate with their activity in pain? NK1R is a pro-nociception receptor, does this mean we need the reversed compounds or antagonists to inhibit the receptor activity? Clarification would be appreciated.

Optional suggestions:

1. In several instances, the authors have chosen to show a representative concentration-response curve rather than showing data that are grouped across multiple replicates (e.g. Fig S4). Consider grouping data, as is often done in the field.

2. In Figure S2, it is unclear which receptor construct was used in these experiments to validate the signalling of the G3-peptides. In some of the concentration-response curves (Gs Glo, Gq TRUPATH), the maximal response is not reached, and this could affect the estimation of EC50 values reported. In any case, the authors report that G3-SP6-11 has a 100-fold increased potency, indicating that the truncation of SP might already be unfavourable for signalling. Untruncated SP could be added for comparison and may have been a better choice of ligand to see whether Nb conjugation can, in fact, improve the natural NK1R agonist.

3. In Figure 1D, the binding of the unconjugated Nbs to the tagged receptor was tested. It would be useful to compare the binding of unconjugated Nbs with peptide-conjugated Nbs to see whether a second binding point by the peptide increases total binding.

4. Fig S5 shows a lot of variability between replicates in the association of the unconjugated Nbs, is this of concern?

5. The opposite effect of nanobody-fusion with SP6-11 in regard to the washout experiments compared to NKA are striking but somewhat confusing. Ideally, a longer linker between the fusion should be used to show that this is indeed due to steric restraint altering the peptide binding pose.

6. Despite the quicker washout of the Nb-fused SP6-11 peptide, there was no significant decrease in Gq-mediated transcriptional response (Fig S11). This is difficult to reconcile, given the conclusions drawn for Nb-fused NKA (opposing effects). Is this a dose issue? The authors should explain this further.

7. At the end of the article, there is an emphasis on the potential usefulness of translational therapeutics. It would be ideal if the authors could further expand upon the likelihood and criteria for a nanobody that would recognize the WT NK1R and could act as a peptide fusion tool i.e. how much solvent accessible surface away from the peptide binding pocket is available for NK1R, and how likely it would provide the relative distance given the findings of this work.

REVIEWING TEAM

Reviewed by:

Reviewer #1: molecular pharmacology of pain-related GPCRs

Reviewer #2: structure and pharmacology of GPCRs

Reviewer #3: structure and pharmacology of pain-related GPCRs

Curated by:

David Thal, Senior Research Fellow, Monash University, Australia

(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)

Your zettelkasten, having a perfect memory of your "past self" acts as a ratchet so that when you have a new conversation on a particular topic, your "present self" can quickly remember where you left off and not only advance the arguments but leave an associative trail for your "future self" to continue on again later.

Many thoughts and associations occur when you're having conversations with any text, whether it's with something you're reading by another author or your own notes in your zettelkasten or commonplace book. For more conversations on this topic, perhaps thumb through: https://hypothes.is/users/chrisaldrich?q=tag%3A%27conversations+with+the+text%27

If you view conversations broadly as means of finding and collecting information from external sources and naturally associating them together, perhaps you'll appreciate this quote:

No piece of information is superior to any other. Power lies in having them all on file and then finding the connections. There are always connections; you have only to want to find them.—Umberto Eco in Foucault's Pendulum (Secker & Warburg)

(Reply to u/u/Plastic-Lettuce-7150 at https://www.reddit.com/r/Zettelkasten/comments/1ae2qf4/communicating_with_a_zettelkasten/)

When looking at these questions I would say that for certain people these are questions that would plague someone's mind. It most certainly depend on the person though. For those who's entire life revolves around social media these are critical questions and I can definitely see when looking at the different areas of social media and how people would respond to the same picture differently across different social media

Reviewer #3 (Public Review):

Summary:<br /> It has been proposed in the literature, that the ATP release channel Panx1 can be activated in various ways, including by tyrosine phosphorylation of the Panx1 protein. The present study reexamines the commercial antibodies used previously in support of the phosphorylation hypothesis and the presented data indicate that the antibodies may recognize proteins unrelated to Panx1. Consequently, the authors caution about the use and interpretation of results obtained with these antibodies.

Strengths:<br /> The manuscript by Ruan et al. addresses an important issue in Panx1 research, i.e. the activation of the channel formed by Panx1 via protein phosphorylation. If the authors' conclusions are correct, the previous claims for Panx1 phosphorylation on the basis of the commercial anti-phospho-Panx1 antibodies would be in question.

This is a very detailed and comprehensive analysis making use of state-of-the-art techniques, including mass spectrometry and phos-tag gel electrophoresis.

In general, the study is well-controlled as relating to negative controls.

The value of this manuscript is, that it could spawn new, more function-oriented studies on the activation of Panx1 channels.

Weaknesses:<br /> Although the manuscript addresses an important issue, the activation of the ATP-release channel Panx1 by protein phosphorylation, the data provided do not support the firm conclusion that such activation does not exist. The failure to reproduce published data obtained with commercial anti-phospho Panx1 antibodies can only be of limited interest for a subfield.

1. The title claiming that "Panx1 is NOT phosphorylated..." is not justified by the failure to reproduce previously published data obtained with these antibodies. If, as claimed, the antibodies do not recognize Panx1, their failure cannot be used to exclude tyrosine phosphorylation of the Panx1 protein. There is no positive control for the antibodies.

2. The authors claim that exogenous SRC expression does not phosphorylate Y198. DeLalio et al. 2019 show that Panx1 is constitutively phosphorylated at Y198, so an effect of exogenous SRC expression is not necessarily expected.

3. The authors argue that the GFP tag of Panx1at the COOH terminus does not interfere with folding since the COOH modified (thrombin cleavage site) Panx1 folds properly, forming an amorphous glob in the cryo-EM structure. However, they do not show that the COOH-modified Panx1 folds properly. It may not, because functional data strongly suggest that the terminal cysteine dives deep into the pore. For example, the terminal cysteine, C426, can form a disulfide bond with an engineered cysteine at position F54 (Sandilos et al. 2012).

4. The authors dismiss the additional arguments for tyrosine phosphorylation of Panx1 given by the various previous studies on Panx1 phosphorylation. These studies did not, as implied, solely rely on the commercial anti-phospho-Panx1 antibodies, but also presented a wealth of independent supporting data. Contrary to the authors' assertion, in the previous papers the pY198 and pY308 antibodies recognized two protein bands in the size range of glycosylated and partial glycosylated Panx1.

5. A phosphorylation step triggering channel activity of Panx1 would be expected to occur exclusively on proteins embedded in the plasma membrane. The membrane-bound fraction is small in relation to the total protein, which is particularly true for exogenously expressed proteins. Thus, any phosphorylated protein may escape detection when total protein is analyzed. Furthermore, to be of functional consequence, only a small fraction of the channels present in the plasma membrane need to be in the open state. Consequently, only a fraction of the Panx1 protein in the plasma membrane may need to be phosphorylated. Even the high resolution of mass spectroscopy may not be sufficient to detect phosphorylated Panx1 in the absence of enrichment processes.

6. In the electrophysiology experiments described in Figure 7, there is no evidence that the GFP-tagged Panx1 is in the plasma membrane. Instead, the image in Figure 7a shows prominent fluorescence in the cytoplasm. In addition, there is no evidence that the CBX-sensitive currents in 7b are mediated by Panx1-GFP and are not endogenous Panx1. Previous literature suggests that the hPanx1 protein needs to be cleaved (Chiu et al. 2014) or mutated at the amino terminus (Michalski et al 2018) to see voltage-activated currents, so it is not clear that the currents represent hPANX1 voltage-activated currents.

This example has every tag category represented, as well as a healthy list of personal names.

Certainly! Let's break down the explanation step by step.

Purpose of pull in Mongoose:

In Mongoose, the pull method is used to remove items from an array field in a document. It is designed to work atomically, meaning it ensures consistency even if multiple operations are being performed simultaneously.

Syntax:

javascript doc.array.pull(...args);

How it works:

• Equality Check: The method uses an equality check by casting the provided value to an embedded document and comparing using the Document.equals() function.

• Atomic Operation: When you call pull, it performs an atomic operation on the database, ensuring that the removal is done in a single step.

• Example: javascript doc.array.pull(ObjectId); // Removes an item by matching ObjectId doc.array.pull({ _id: 'someId' }); // Removes an item by matching the _id field doc.array.pull(36); // Removes an item by matching the value 36 doc.array.pull('tag 1', 'tag 2'); // Removes items with values 'tag 1' and 'tag 2'

Removing from Subdocument Array:

You can use pull to remove items from a subdocument array as well.

• Example: ```javascript // Removing by passing an object with a matching _id doc.subdocs.push({ _id: 4815162342 }); doc.subdocs.pull({ _id: 4815162342 }); // removes the subdocument

// Removing by passing the _id directly doc.subdocs.push({ _id: 4815162342 }); doc.subdocs.pull(4815162342); // works in the same way ```

Atomic Operation and $set:

• The first pull call results in an atomic operation on the database.
• If pull is called repeatedly without saving the document, a $set operation is used on the complete array instead. This means it overwrites any possible changes that happened on the database in the meantime.

Usage in Mongoose:

In Mongoose, you can use pull on an array field of a document. Here's a simple example:

```javascript const mongoose = require('mongoose');

const schema = new mongoose.Schema({ items: [{ type: String }] });

const Model = mongoose.model('Example', schema);

// Example usage Model.findOne({ _id: 'someId' }, (err, doc) => { if (err) throw err;

// Removing 'unwantedItem' from the 'items' array doc.items.pull('unwantedItem');

// Save the document to persist the changes to the database doc.save((saveErr) => { if (saveErr) throw saveErr; console.log('Item removed successfully.'); }); }); ```

In this example, pull is used to remove an item from the 'items' array of a document, and then the changes are saved to the database.

33:50 basisdemokratie, feedback, fragen<br /> warum ist die verfassung unfehlbar?<br /> wenn migranten-invasion und links-extremismus und gift-impfungen und pestizide und schulmedizin und cannabis-verbot und kriege und und und so "falsch" sind, wie sollen wir sonst die globale bevölkerung reduzieren? wie sollen wir sonst die todesrate steigern?<br /> oder, wenn es die übervölkerung gar nicht gibt, was ist dann die "carrying capacity" von diesem planet? aktuell haben wir circa 1E10 menschen global, also circa 5000 m² fläche für landwirtschaft pro person = 1000 m² für pflanzen und 4000 m² für tiere. wären 1000 mal mehr menschen "zu viel"? was dann? warum ist das "recht auf leben" unfehlbar? warum haben tiere und pflanzen kein "recht auf leben"? was ist so toll am pazifismus, der langfristig nur übervölkerung und degeneration bringt? spätestens wenn das erdöl "endlich" aus ist, wird klar, wir haben zu viele menschen.

spoiler: ich sehe mich selber nicht "links" und nicht "rechts", sondern "oben". ich beobachte dieses spiel von "oben" und denke mir: die sind alle irgendwie dumm. einerseits dieses "linke" regime, das den ganzen tag nur lügen verbreitet, und heimlich die bevölkerung austauscht, und einen bürgerkrieg vorbereitet, damit die bevölkerung reduziert wird. andererseits eine "rechte" opposition, die wahrheit und ehrlichkeit fordert, aber die selber keine bessere lösung hat zur depopulation. ich selber habe das gleiche ziel wie die "linken" (also bürgerkrieg, depopulation, deindustrialisierung, zurück zu tribalismus), und ich habe den gleichen weg wie die "rechten" (also ehrlichkeit, transparenz, wahrheit, ...)

zurück zu tribalismus

das ist auch ein thema in meinem buch:<br /> pallas. wer sind meine freunde. gruppenaufbau nach persönlichkeitstyp.<br /> ich suche eine "weltformel" für die frage: wie müssen wir verschiedene persönlichkeitstypen verbinden, damit stabile gruppen entstehen?<br /> spoiler: die konsequenz ist sicher kein pazifismus, sondern tribalismus = kleinstaaten je 150 menschen, und dazu gehören auch regelmäßige stammkriege, für natürliche selektion.<br /> auch diese "unschöne" konsequenz ist ein grund, warum meine hypothese bisher ignoriert wird... die leute wollen es schön haben, aber nichts dafür opfern, und nicht dafür kämpfen, sondern wollen alles geschenkt kriegen. ist wohl so ein merkmal der "boomer" generation, die ihr ganzes leben lang diesen "billige energie" rausch erlebt haben, der "einfach weitergehen" soll

Author Response

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Nitrogen metabolism is of fundamental importance to biology. However, the metabolism and biochemistry of guanidine and guanidine containing compounds, including arginine and homoarginine, have been understudied over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new type of guanidine forming enzyme. It was previously known that 2-oxogluturate oxygenase catalysis in bacteria can produce guanidine via oxidation of arginine. Interestingly, the same enzyme that produces guanidine from arginine also oxidises 2-oxogluturate to give the plant signalling molecule ethylene. Funck et al show that a mechanistically related oxygenase enzyme from plants can also produce guanidine, but instead of using arginine as a substrate, it uses homoarginine. The work will stimulate interest in the cellular roles of homoarginine, a metabolite present in plants and other organisms including humans and, more generally, in the biochemistry and metabolism of guanidines.

1) Significance

Studies on the metabolism and biochemistry of the small nitrogen rich molecule guanidine and related compounds including arginine have been largely ignored over the last few decades. Very few guanidine forming enzymes have been identified. Funck et al define a new guanidine forming enzyme that works by oxidation of homoarginine, a metabolite present in organisms ranging from plants to humans. The new enzyme requires oxygen and 2oxogluturate as cosubstrates and is related, but distinct from a known enzyme that oxidises arginine to produce guanidine, but which can also oxidise 2-oxogluturate to produce the plant signalling molecule ethylene.

Overall, I thought this was an exceptionally well written and interesting manuscript. Although a 2-oxogluturate dependent guanidine forming enzyme is known (EFE), the discovery that a related enzyme oxidises homoarginine is really interesting, especially given the presence of homoarginine in plant seeds. There is more work to be done in terms of functional assignment, but this can be the subject of future studies. I also fully endorse the authors' view that guanidine and related compounds have been massively understudied in recent times. I would like to see the possibility that the new enzyme makes ethylene explored. Congratulations to the authors on a very nice study.

Response: We thank the reviewer for the positive evaluation of our manuscript. In the revised version, we have emphasized more clearly that we found no evidence for ethylene production by the recombinant enzymes. The other suggestions of the reviewer are also considered in the revised version as detailed below.

Reviewer #2 (Public Review):

In this study, Dietmar Funck and colleagues have made a significant breakthrough by identifying three isoforms of plant 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) as homo/arginine-6-hydroxylases, catalyzing the degradation of 6-hydroxyhomoarginine into 2aminoadipate-6-semialdehyde (AASA) and guanidine. This discovery marks the very first confirmation of plant or eukaryotic enzymes capable of guanidine production.

The authors selected three plant 2-ODD-C23 enzymes with the highest sequence similarity to bacterial guanidine-producing (EFE) enzymes. They proceeded to clone and express the recombinant enzymes in E coli, demonstrating capacity of all three Arabidopsis isoforms to produce guanidine. Additionally, by precise biochemical experiments, the authors established these three 2-ODD-C23 enzymes as homoarginine-6-hydroxylases (and arginine-hydroxylase for one of them). Furthermore, the authors utilized transgenic plants expressing GFP fusion proteins to show the cytoplasmic localization of all three 2-ODD-C23 enzymes. Most notably, using T-DNA mutant lines and CRISPR/Cas9-generated lines, along with combinations of them, they demonstrate the guanidine-producing capacity of each enzyme isoform in planta. These results provide robust evidence that these three 2-ODD-C23 Arabidopsis isoforms are indeed homoarginine-6-hydroxylases responsible for guanidine generation.

The findings presented in this manuscript are a significant contribution for our understanding of plant biology, particularly given that this work is the first demonstration of enzymatic guanidine production in eukaryotic cells. However, there are a couple of concerns and potential ways for further investigation that the authors should (consider) incorporate.

Firstly, the observation of cytoplasmic and nuclear GFP signals in the transgenic plants may also indicate cleaved GFP from the fusion proteins. Thus, the authors should perform Western blot analysis to confirm the correct size of the 2-ODD-C23 fusion proteins in the transgenic protoplasts.

Secondly, it may be worth measuring pipecolate (and proline?) levels under biotic stress conditions (particularly those that induce transcript changes of these enzymes, Fig S8). Given the results suggesting a potential regulation of the pathway by biotic stress conditions (eg. meJA), these experiments could provide valuable insights into the physiological role of guanidine-producing enzymes in plants. This additional analysis may give a significance of these enzymes in plant defense mechanisms.

Response: We thank also reviewer 2 for the positive evaluation and useful suggestions. We performed the proposed GFP Western blot, which indeed indicated the presences of both, fulllength fusion proteins and free GFP, which can explain the partial nuclear localization. We fully agree that further experiments with biotic and abiotic stress will be required to determine the physiological function of the 2-ODD-C23 enzymes. However, the list of potential experiments is long and they are beyond the scope of the present manuscript.

Reviewer #1 (Recommendations For The Authors):

Specific points

Overall, I thought this was a very interesting study, comprising biochemical, cellular, and in vivo studies. Of course more could be done on each of these, and likely will be, but I think the assignment of biochemical function is very strong, across all three approaches. The one new experiment I would like to see is a clear demonstration of whether ethylene is produced - unlikely but should be tested.

We had mentioned our failure to detect ethylene production by the plant enzymes in the previous version and have made it more prominent and reliable by including ethylene production as positive control in the new supplementary figure S5.

Abstract

Delete 'hitherto overlooked' - this is implicit 'but is more likely' to 'is likely'?

Agreed and modified

Introduction

Second sentence - what about relevant small molecule primary metabolites including precursors of proteins/nucleic acids.

We modified the sentence accordingly.

Paragraph 2 - maybe also note EFE produces glutamate semi aldehyde, via arginine C-5 oxidation.

Paragraph 2 has been re-phrased according to your suggestion.

Overall, I thought the introduction was exceptionally well written.

Perhaps either in the introduction, or later, note there are other 2OG oxygenases that oxidise arginine/arginine derivatives in various ways, e.g. clavaminate synthase/arginine hydroxylases/desaturases.

We added a sentence mentioning the arginine hydroxylases VioC and OrfP to the introduction and included VioC into the sequence comparison in supplementary figure 2 to show that these enzymes, as well as NapI, are very different from EFE and the plant hydroxylases.

Results

Paragraph 1 - qualify similarity and refer to/give a structurally informed sequence alignment, including EFE

A new supplemental figure S2 was added with sequence identity values and a structurally informed alignment. The text has been modified accordingly.

Paragraph 2 - briefly state method of guanidine analysis

We included a reference to the M&M section and mentioned LC-MS in paragraph 2.

Figure 1 - trivial point - proteins are not expressed/genes are

We have modified the legend to figure 1. However, we would like to point out that terms like “recombinant protein expression” are widely used in the field. A quick search with google Ngram viewer shows that “protein expression” started to appear in the mid-80ies and its use stayed constantly at 1/8th of “gene expression”.

Define errors clearly in all figure legends, clearly defining biological/technical repeats<br /> Page 6 - was the His-tag cleared to ensure no issues with Ni contamination?

We treat individual plants or independent bacterial cultures as biological replicates. Only in the case of enzyme activity assays with NAD(P)H, technical replicates were used and this has been indicated in the legend of figure 6.

Lower case 'p' in pentafluorobenzyl corrected

In Figure 2 make clear the hydroxylated intermediates are not observed

We now use grey color for the intermediates and have put them in brackets. Additionally we state in the figure legend that these intermediates were not detected.

Pages 6-7 - I may have missed this but it's important to investigate what happens to the 2OG. Is succinate the only product or is ethylene also produced? This possibility should also be considered in the plant studies, i.e. is there any evidence for responses related to perturbed ethylene metabolism. The authors consider a signalling role relating to AASA/P6C, but seem to ignore a potential ethylene connection.

As stated above, we checked for ethylene production with negative result. EFE produced 6 times more guanidine than the plant enzymes under the same condition, but even 100-fold lower ethylene production would have been clearly detected.

Page 12 - 'plants have been shown to....' Perhaps note how hydroxy guanidine is made?

We now mention the canavanine-γ-lyase that cleaves canavanine into hydroxyguanidine and homoserine.

Overall, I thought the discussion was good, but perhaps a bit long/too speculative on pages 12/13 and this detracted from the biochemical assignment of the enzyme. I'd suggest shortening the discussion somewhat - the precise roles of the enzyme can be the subject of future work. As indicated above, some discussion on potential links to ethylene would be appreciated.

Since reviewer 2 wanted more (speculative) discussion on the role of the 2-ODD-C23 enzymes and there was no detectable ethylene production, we took the liberty to leave the discussion largely unaltered.

I'd also like to see some more consideration/metabolic analyses of guanidine related metabolism in the genetically modified plants.

Such analyses will certainly be included in future experiments once we get an idea about the physiological role of the 2-ODD-C23 enzymes.

Page 16 - mass spectrometry

Corrected.

Please add a structurally informed sequence alignment with EFE and other 2OG oxygenases acting on arginine/derivatives.

An excerpt of the alignment is now presented in supplementary figure S2.

Reviewer #2 (Recommendations For The Authors):

I would like to see more discussion in the manuscript about the possible interconnection/roles between 2-ODD-C23 guanidine-producing, lysine- ALD1-Pipecolate producing, and proline metabolism pathways during both biotic and abiotic stresses.

Since we were unable to detect pipecolate in any of our plant samples and also our preliminary results with biotic stress did not produce any evidence for a function of the 2ODD-C23 enzymes in the tested defense responses, we would like to postpone such extended discussion until we find a condition where the physiological function of these enzymes is evident.

Fig. 4: Authors should change colors for Col-0, 0.2 HoArg and ctrl? They look too similar in my pdf file.

We changed the colors in figure 4 and hope that the enhanced contrast is maintained during the production of the final version of our article.

Reviewer #1 (Public Review):

Summary:

Bartolome et al. report adaptation of proximity labeling using BirA and TurboID fusions to proteasome subunits to identify the proteasome-proximal proteome both in cultured cells and also in a newly developed mouse model. Using this approach, the authors demonstrate identification of many known proteasome-interacting proteins, as well as several new proteins, some of which are validated directly. The authors further evaluate the proteasome-proximal proteome in most mouse organs, and find substantial agreement with the proteome identified from cultured cells, as well as between tissues. This represents one of the first studies of the "proteasome-ome" in vivo, and sets the stage for addressing numerous important future questions regarding how the proteasome's environment changes over time, in response to different stimuli, and in distinct disease conditions.

Strengths:

Generally speaking, the approach provided is rigorous and supported by several complementary lines of evidence, such as demonstration that the interactome is enriched for known proteasome-binding proteins and co-purification or co-elution experiments. Similarly, the high agreement between the outcomes in cultured cells and in the mouse model developed by the authors provides further confidence in the results.

Weaknesses:

The major weakness of the work is arguably the choice of proteasome subunits for tagging with biotinylating enzymes. In most cases, the subunits and termini chosen for tagging are known to either protrude toward functionally important regions (such as the substrate-processing pore of the ATPase component), to have important functional roles likely to be disrupted via tagging, or are subunits known to be substituted by others in some conditions. Thus, the interactome reported may conflate those of normal proteasomes with those harboring tag-induced functional or structural defects. Although the authors made a commendable attempt to demonstrate minimal impacts of tagging, the conclusions would be greatly further strengthened by contrasting the impacts of tagging subunits less likely to cause perturbations and by more rigorously demonstrating normal proteolysis of a broader array of known proteasome substrates.

Reviewer #3 (Public Review):

Summary:

Bartolome et al. present ProteasomeID, a novel method to identify components, interactors, and (potentially) substrates of the proteasome in cell lines and mouse models. As a major protein degradation machine that is highly conserved across eukaryotes, the proteasome has historically been assumed to be relatively homogeneous across biological scales (with few notable exceptions, e.g., immunoproteasomes and thymoproteasomes). However, a growing body of evidence suggests that there is some degree of heterogeneity in the composition of proteasomes across cell tissues, and can be highly dynamic in response to physiologic and pathologic stimuli. This work provides a methodological framework for investigating such sources of variation. The authors start by adapting the increasingly popular biotin ligation strategy for labelling proteins coming into close proximity with one of three different subunits of the proteasome, before proceeding with PSMA4 for further development and analysis based on their preliminary labelling data. In a series of well-constructed and convincing validation experiments, the authors go on to show that the tagged PSMA4 construct can be incorporated into functional proteasomes, and is able to label a broad set of known proteasome components and interacting proteins in HEK293T cells. They also attempt to identify novel proteasomal degradation substrates with ProteasomeID; while this was convincing for known substrates with particularly short half-lives (exemplified by the transcription factor c-myc), follow-up validation experiments with other substrates were less clear. One of the most compelling results was from a similar experiment to confirm proteasomal degradation induced by a BRD-targeting PROTAC, which I think is likely to be of keen interest to the targeted degradation community. Finally, the authors establish a ProteasomeID mouse model, and demonstrate its utility across several tissues.

Strengths:

1) ProteasomeID itself is an important step forward for researchers with an interest in protein turnover across biological scales (e.g., in sub-cellular compartments, in cells, in tissues, and whole organisms). I especially see interest from two communities: those studying fundamental proteostasis in physiological and pathologic processes (e.g., ageing; tissue-specific protein aggregation diseases), and those developing targeted protein degradation modalities (e.g., PROTACs; molecular glues). All the datasets generated and deposited here are likely to provide a rich resource to both. The HEK293T cell line data are a valuable proof-of-concept to allow expansion into more biologically-relevant cell culture settings; however, I envision the greatest innovation here to be the mouse model. For example, in the targeted protein degradation space, two major hurdles in early-stage pre-clinical development are (i) evaluation of degradation efficacy across disease-relevant tissues, and (ii) toxicity and safety implications caused by off-target degradation, e.g., of newly-identified molecular glues and/or in particularly-sensitive tissues. The ProteasomeID mouse allows early in vivo assessment of both these questions. The results of the BRD PROTAC experiment in 293T cells provides an excellent in vitro proof-of-concept for this approach.

2) The mass spectrometry-based proteomics workflows used and presented throughout the manuscript are robust, rigorous, and convincing. For example, the algorithm the authors use for defining enrichment score cut-offs are logical and based on rational models, rather than on arbitrary cut-offs that are common for similar proteomics studies. The construction (and subsequent validation) of both BirA*- and miniTurbo- tagged PSMA4 variants also increases the utility of the method, allowing researchers to choose the variant with the labelling time-scale required for their particular research question.

3) The optimised BioID and TurboID protocol the authors develop (summarised in Fig. S2A) and validate (Fig. S2B-D) is likely to be of broad interest to cell and molecular biologists beyond the protein degradation field, given that proximity labelling is a current gold-standard in global protein:protein interaction profiling.

Limitations:

I think the authors do an excellent job in highlighting the limitations of ProteasomeID throughout the Results and Discussion. I do have some specific comments that might provide additional context for the reader.

1) The authors do a good job in showing that a substantial proportion of PSMA4-BirA* is incorporated into functional proteasome particles; however, it is not immediately clear to me how much background (false-positive IDs) might be contributed by the ~40 % of PSMA4-BirA* that is not incorporated into the mature core particle (based on the BirA* SEC-MS traces in Fig. 2b and S3b, i.e., the large peak ~ fraction 20). Are there any bands lower down in the native gel shown in Fig. 2c, i.e., corresponding to lower molecular weight complexes or monomeric PSMA4-BirA*? The enrichment of proteasome assembly factors in all the ProteasomeID experiments might suggest the presence of assembly intermediates, which might themselves become substrates for proteasomal degradation (as has been shown for other incompletely-assembled protein complexes, e.g., the ribosome, TRiC/CCT).

2) Although the authors attempt to show that BirA* tagging of PSMA4 does not interfere with proteasome activity (Fig. 2e-f), I think the experimental evidence for this is incomplete. They show that the overall chymotrypsin-like activity (attributable to PSMB5) in cells expressing PSMA4-BirA* is not markedly reduced compared with control BirA*-expressing cells. However, they do not show that the activity of the specific proteasome sub-population that contains PSMA4-BirA* is unaffected (e.g., by purifying this sub-population via the Flag tag). The proteasome activity of the sub-population of wild-type proteasome complexes that do not contain the PSMA4-BirA* (~50%, based on the earlier immunoblots) could account for the entire chymotrypsin-like activity-especially in the context of HEK293T cells, where steady-state proteasome levels are unlikely to be limiting. It would also be useful to assess any changes in tryspin- and caspase- like activities, especially as tagging of PSMA4 could conceivably interfere with the activity of some PSMB subunits, but not others.

3) I was left unsure of the general utility of ProteasomeID for identifying novel proteasomal substrates in homeostatic or stressed conditions. The immunoblots for the two candidates the authors follow up in Fig. 4g was not especially clear; the reduction in the bands are modest, at best. Furthermore, classifying candidates based on enrichment following proteasome inhibition with MG-132 have the potential to lead to a high number of false positives. ProteasomeID's utility in identifying potential substrates in more targeted settings (e.g., molecular glues, off-target PROTAC substrates) is far more apparent.

Reviewer #1 (Public Review):

Summary:<br /> In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some DDC proteins are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further demonstrate that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2, or the mitotic exit network (MEN) component Bub2.

Strengths:<br /> The manuscript dissects the specific role that different components of the DDC and the SAC have during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution to the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are also generally well supported by the experimental data. The observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:<br /> The main weakness of the study is that it is fundamentally based only on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This is a widely used method that allows a very efficient depletion of proteins. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic. Verification of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

[Banken-Rettung um jeden Preis]

19:39<br /> Glauben Sie nicht, dass die Notenbanken bald gegensteuern?<br /> Weil sie vielleicht sogar gezwungen werden?

19:58<br /> Danke für die Frage.<br /> Es ist nicht nur so, dass die Frage berechtigt ist,<br /> "glauben Sie nicht, Herr Krall, dass die Banken gegensteuern werden?"<br /> Ich bin sicher, dass sie gegensteuern werden!<br /> Darauf läuft die ganze Sache ja hinaus.

20:07<br /> Schon beim nächsten Mal?

Die steuern ja schon gegen.<br /> Die FED [Federal Reserve] hat ja gerade zwei Billionen [US-Dollar]<br /> Liquiditätsspritze ins Bankensystem gegeben.

20:16<br /> Ist das jetzt QE [Quantitative Easing]?

Im Grunde genommen ist das QE [Quantitative Easing].<br /> Natürlich ist das QE.<br /> Wenn ich zwei Billionen zur Verfügung stelle,<br /> dann muss sie im Zweifel ja auch<br /> tatsächlich "drucken" oder elektronisch schaffen.<br /> Das heißt<br /> die Fiat-Geldmaschine wird ja gerade schon wieder angeworfen,<br /> das ist ja das, worauf es rausläuft.

20:30<br /> Natürlich gehen die Banken nicht pleite,<br /> die können die gar nicht pleite gehen lassen.<br /> Die Bankenkrise ist da, das Eigenkapital ist weg,<br /> wahrscheinlich ist es drei Mal weg, vielleicht ist es auch fünf Mal weg.<br /> Das war auch schon weg, bevor es jetzt geknallt hat,<br /> weil die Zombies, die waren ja schon vorher da,<br /> aber die sind halt nicht in die Knie gegangen ohne Zinserhöhung.

20:49<br /> Aber ökonomisch betrachtet,<br /> wenn man eine korrekte Risikobetrachtung gemacht hätte,<br /> und einen richtigen Stresstest gemacht hätte,<br /> dann hätte man schon längst wissen können,<br /> wie schlecht es den Banken geht in Wahrheit.

21:00<br /> Die ganze Fata Morgana von dem erhöhten Eigenkapital,<br /> das ist für die Leichtgläubigen unter uns.

21:05<br /> Und jetzt ist es natürlich so,<br /> dass die die Banken nicht abstürzen lassen werden.<br /> Ihr Geld ist auf dem Sparkonto vollkommen sicher,<br /> da müssen sie sich keine Sorgen machen,<br /> die werden gerettet werden, und zwar kostet es was es wolle.

21:15<br /> Weil wenn das nicht gerettet wird...<br /> das haben die Jungs aus Lehman gelernt...<br /> also wenn man so eine Bank in der Kampfklasse<br /> einfach untergehen lässt,<br /> und die nicht rettet, dann ist Achterbahn.

Und da gilt der schöne Satz:<br /> "ich zähle jetzt bis Eins, und dann ist Achterbahn."<br /> Und also bis zum "Eins zählen" kommen sie da gar nicht.

Also werden die gerettet,<br /> und die werden dafür die Geldmenge ausdehnen müssen,<br /> das ist das worauf es rausläuft.

21:39

Aber wer bezahlt dann die Rechnung?

Die Rechnung ist doch ganz klar.<br /> Wenn ich die Geldmenge ausdehne,<br /> in Europa um vier bis sechs,<br /> oder vielleicht sogar acht Billionen,<br /> in Amerika auch...<br /> denn wir stehen ja am Anfang,<br /> und zwei Billionen haben die Amerikaner jetzt schon dahingestellt,<br /> bevor es überhaupt irgendwie richtig los geht.

00:22:00<br /> [Dann steigt Inflation auf 30%]

21:55<br /> Also wenn da 4... 6... 8 Billionen auf beiden Seiten des Atlantiks<br /> an Zentralbank-Geldmenge frisch geschaffen wird,<br /> dann wird die Inflation zum nächsten Auf-Galopp ansetzen,<br /> geht dann aufs nächste Plateau,<br /> von jetzt 15 Prozent auf dann 30 Prozent.

22:14<br /> Und dann stehen die Zentralbanken wieder wie der Ochs vorm Berg,<br /> weil die werden dann sagen: "Hmm, 30 Prozent, schlecht..."<br /> weil dann wird es nämlich da draußen ungemütlich.

22:24<br /> Wir haben jetzt schon ein Drittel aller Haushalte in Deutschland<br /> (und nicht nur in Deutschland)<br /> die mit ihrem Geld nicht mehr klarkommen.

22:29<br /> Die weichen jetzt aus,<br /> da gibts jetzt eben kein Hühnchen mehr, und kein Schweinesteak,<br /> da gibts jetzt Haferflocken.<br /> Also die stellen Ihren Warenkorb um,<br /> deswegen erzählt man ihnen, sie sollen Insekten essen,<br /> was ich ja für die blödeste Idee seit Adam und Eva halte.<br /> Das ist schon spektakulär, wenn man darüber nachdenkt.

22:47<br /> Also ein Drittel der Haushalte ist jetzt schon in Schwierigkeiten,<br /> und zwar in ernsthaften Schwierigkeiten.<br /> Wenn dann noch Anpassungen bei den Hypothekenzahlungen kommen,<br /> dann wirds auch ganz viele "Häuslebauer" erwischen,<br /> die nicht drauf eingestellt sind,<br /> dass sich ihre Zinsbelastung vervierfacht oder verfünffacht.

23:02<br /> Wenn wir dann 30 Prozent [Inflation] haben,<br /> dann werden wir sehen,<br /> dass 80 bis 90 Prozent der Haushalte Schwierigkeiten haben,<br /> am Ende des Geldes ist dann noch zu viel Monat übrig,<br /> und ich würde mal sagen, die Hälfte ernsthafte Schwierigkeiten,<br /> das heißt, da wird es zu Zahlungsunfähigkeit kommen.

23:18<br /> Und wenn sie die Hälfte der Haushalte<br /> in die Zahlungsunfähigkeit schreiben<br /> durch eine inkompetente Geldpolitik,<br /> dann wage ich die Prognose,<br /> dass sich die Leute das nicht gefallen lassen werden,<br /> und dass das zu sozialen Verwerfungen führt.

23:28<br /> Aber jetzt sagen Sie "inkompetente Geldpolitik".<br /> Was würden Sie denn jetzt machen?<br /> Also Sie würden die Zinsen nicht senken,<br /> und die Banken pleite gehen lassen?<br /> Das ist ja auch nicht besser, oder?

23:35<br /> Roland Tichy hat mich mal gefragt,<br /> was ich tun würde,<br /> wenn ich Zentralbank-Chef wäre,<br /> wenn ich EZB-Chef wäre,<br /> und meine Antwort war ganz einfach:<br /> Zum Glück bin ich das nicht.<br /> Weil die haben sich so in die Falle manövriert,<br /> dass sie da nicht mehr rauskommen.

23:51<br /> Also es gibt keinen guten Ausweg?

23:52<br /> Es gibt keine gute Lösung.<br /> Der Mittelweg, den haben sie ja jetzt versucht.<br /> Diese dreieinhalb Prozent [Zinsen]<br /> waren ja eigentlich der Versuch des Mittelwegs,<br /> nämlich zu sagen:<br /> "Wasch mich, aber mach mich nicht nass."<br /> Den kleinen Fußzeh ins Wasser gestreckt,<br /> und dann mit furchtbarem Geheule festgestellt,<br /> wie kalt das Wasser ist.

24:10<br /> Eigentlich bräuchte man ja einen viel höheren Zinssatz,<br /> um 10% Inflation einzudämmen.<br /> Man braucht positive Realzinsen, um das Ding auf Schiene zu setzen,<br /> die haben wir aber bei weitem nicht,<br /> wir haben negative Realzinsen von 7 Prozent.

24:20<br /> Das heißt also,<br /> es werden gewaltige Mengen an Vermögen verbrannt durch die Inflation,<br /> und wenn ich die [Inflation] wirklich bekämpfen würde...

24:31<br /> und natürlich auch Einkommen... das Einkommen schrumpft.<br /> Wir haben seit Beginn dieser Krise<br /> einen Einkommensrückgang in Europa um 14 Prozent.<br /> Das ist mehr als zu Beginn der großen Depression 1929.

24:40<br /> Wenn sie da 30% draus machen, oder 40% oben drauf,<br /> dann sind sie schon bei minus 30, minus 40 Prozent.<br /> Das heißt die Einkommen schrumpfen im gleichen Ausmaß wie 1929/30.

24:51<br /> Und der Fehler ist nicht darin zu sehen,<br /> was die jetzt tun können oder nicht tun können,<br /> da habe ich keinen guten Rat.<br /> "damned if you do, damned if you dont."

25:00<br /> Der Fehler liegt darin:<br /> Die Inkompetenz der Geldpolitik reicht jetzt 20 Jahre zurück.<br /> Von Tag eins an war der Euro so konstruiert,<br /> dass er die Verschwendung alimentiert,<br /> dass er die Staatsfinanzen in Südeuropa finanziert,<br /> und so war er auch gedacht.

25:13<br /> Und man hat nicht ans Ende gedacht,<br /> als man das angefangen hat.<br /> Man hat gedacht, die Deutschen zahlen die Party,<br /> bis zum Sankt Nimmerleinstag.

Aber hier gilt jetzt der Satz von Maggie Thatcher:<br /> Die EU ist am Ende, wenn ihr das Geld der Deutschen ausgeht.

Analog zu dem Satz:<br /> Der Sozialismus ist am Ende, wenn ihm das Geld andere Leute ausgeht.

Nämlich das ist das, was die EU ist, sie ist Staats-Sozialismus.<br /> Und jetzt ist der Weg zu Ende gegangen,<br /> und die Reserven sind verbraucht.

— Markus Krall bei Mario Lochner, 2023-04-05

The good: Every image on the website has a descriptive alt tag, limited to 125 characters or less. This requirement ensures that screen readers can effectively convey the content of images. The descriptions are concise yet detailed enough to accurately represent the images, avoiding the use of random letters and numbers that often appear in file names (e.g., n83zeo1234q.jpg). This practice aligns well with the Perceivable principle of the Web Content Accessibility Guidelines (WCAG), ensuring that information is accessible to all users, regardless of their sensory abilities.

The image in this new article has a descriptive <alt> tag of 125 characters or less that a screen reader can read. This description is also concise while still containing enough detail to describe the image. Additionally, it does not contain random letters and numbers assigned to many image files.

deine website ist ein trauriger witz : /

schonmal was gehört von "zensurresistent"?

iss nur ne frage der zeit, bis die bullen deine domain abschalten

wie bei...

torrent.to https://www.digitaltrends.com/web/germany-sentences-torrent-site-owner-to-3-years-in-jail/

spickmich.de https://de.wikipedia.org/wiki/Spickmich

lernsieg.at https://www.w24.at/News/2019/11/Umstrittene-Lernsieg-App-wieder-offline

rarbg.to https://de.wikipedia.org/wiki/RARBG

didw*.onion https://de.wikipedia.org/wiki/Deutschland_im_Deep_Web

https://tarnkappe.info/artikel/it-sicherheit/neues-deepweb-forum-germania-will-didw-3-beerben-261558.html

oder... die zensurliste ist lang, und wird jeden tag länger

https://tarnkappe.info/artikel/szene/warez/piraterie-bekaempfung-nimmt-zu-gerichte-setzen-neue-massstaebe-286932.html

https://tarnkappe.info/artikel/szene/warez/ace-riesige-liste-von-piraterieseiten-zur-schliessung-im-jahr-2024-285883.html

https://www.anonymousnews.org/international/die-bruesseler-zensur-krake/

auch telegram ist zu zentral, wir brauchen eher was wie ssb (secure scuttlebutt), gittorrent, zeronet, berty, briar, sneakernet, ... also "offline first", was auch innem ad-hoch bluetooth/wlan p2p netzwerk funktioniert... dann braucht man schon nen stromausfall (oder ne EMP) um das system zu blockieren

remember, china ist das vorbild für die NWO, also die gleiche aggressive internet-zensur wird auch hier kommen, völlig egal mit welcher "begründung", polizeigewalt braucht keine gründe, und wenn irgendwelche richter 5 jahre später sagen "das war eigentlich doch legal" dann hilft das auch nix

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

The apicoplast, a non-photosynthetic vestigial chloroplast, is a key metabolic organelle for the synthesis of certain lipids in apicomplexan parasites. Although it is clear metabolite exchange between the parasite cytosol and the apicoplast must occur, very few transporters associated with the apicoplast have been identified. The current study combines data from previous studies with new data from biotin proximity labeling to identify new apicoplast resident proteins including two putative monocarboxylate transporters termed MCT1 and MCT2. The authors conduct a thorough molecular phylogenetic analysis of the newly identified apicoplast proteins and they provide compelling evidence that MCT1 and MCT2 are necessary for normal growth and plaque formation in vitro along with maintenance of the apicoplast itself. They also provide indirect evidence for a possible need for these transporters in isoprenoid biosynthesis and fatty acid biosynthesis within the apicoplast. Finally, mouse infection experiments suggest that MCT1 and MCT2 are required for normal virulence, with MCT2 completely lacking at the administered dose. Overall, this study is generally of high quality, includes extensive quantitative data, and significantly advances the field by identifying several novel apicoplast proteins together with establishing a critical role for two putative transporters in the parasite. The study, however, could be further strengthened by addressing the following aspects:

Response: We thank very much the reviewer for his/her positive evaluation of our work. To address the detailed function of the transporters, in the past three months, we have re-constructed plasmids (with codon-optimized DNA sequences of the genes) for expression of the transporters in a regular expression E. coli strain (BL21DE3) and in a pyruvate import knockout E. coli strain (a gift from Prof. Kirsten Jung), to examine the transport capability in vitro. And, we have also re-constructed a new plasmid containing a new leading peptide for targeting the pyruvate sensor PyronicSF to the apicoplast in the parasite, to probe the possible substrate pyruvate. However, we did not successfully observe expression of the transporters in the above E. coli strains, and we were unable to target the sensor to the correct localization (the apicoplast) in the parasite. As a result, all efforts have led the study to the current version of manuscript on the functional identification of transporters. We will keep working on this aspect, attempting to dissect out the exact transport function of the transporters in the future. In the current manuscript, we have discussed the limitations of our study in the last part of the manuscript.

Main comments

1) The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of IPP is only supported by indirect measurements (effects on host GFP uptake or trafficking, possibly due to effects on IPP dependent proteins such as rabs, and mitochondrial membrane potential, possibly due to effects on IPP dependent ubiquinone). This conclusion would be more strongly supported by directly measuring levels of IPP. If there are technical limitations that prevent direct measurement of IPP then the author should note such limitations and acknowledge in the discussion that the conclusion is based on indirect evidence.

Response: We thank the reviewer very much for the suggestions. We have tried to establish the measurement of IPP using a commercial company in recent months, yet we have not been successful in making the assay work. Considering the problem of indirect evidence, we have discussed this limitation in the discussion.

2) The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of fatty acids is also poorly supported by the data. The authors do not distinguish between the lower fatty acid levels being due to reduced synthesis of fatty acids, reduced salvage of host fatty acids, or both. Indeed, the authors provide evidence that parasite endocytosis of GFP is dependent on AMT1 and AMT2. Host GFP likely enters the parasite within a membrane bound vesicle derived from the PVM. The PVM is known to harbor host-derived lipids. Hence, it is possible that some of the decrease in fatty acid levels could be due to reduced lipid salvage from the host. Experiments should be conducted to measure the synthesis and salvage of fatty acids (e.g., by metabolic flux analysis), or the authors should acknowledge that both could be affected.

Response: We thank the reviewer very much for comments and suggestions. We partially agree with the comments that the depletion of transporters could affect lipids scavenged from the host cells, as endocytic vesicles are indeed derived from the parasite plasma membrane at the micropore and potentially from the host cell endo-membrane system, as demonstrated with the micropore endocytosis in our previous study (pmid: 36813769). Our latest study has addressed this by showing that the endocytic trafficking of GFP vesicles is regulated by prenylation of proteins (e.g. Rab1B and YKT6.1), depletion of which resulted in diffusion of GFP vesicles, but not disappearance of GFP vesicles in the parasites (pmid: 37548452), indicating that the vesicles (containing lipids) enter the parasites. In the current manuscript, the percentage of parasites containing GFP foci was significantly reduced in AMT1/AMT2-depleted parasites, and instead, parasites containing GFP diffusion appeared and the percentage was almost equal to the reduced level of parasites with GFP foci. These results suggested that endocytic vesicles (e.g. GFP vesicles) were continuously generated by the micropore in the parasites depleted with AMT1/AMT2, and that the vesicle trafficking was regulated by proteins modified by IPP derivatives that were derived from the apicoplast. Based on these observations, we considered that lipids in endocytic vesicles should not contribute to the reduced level of fatty acids and other lipids in parasites depleted with AMT1/AMT2. We have added in a short discussion concerning the fatty acids and lipids reduced in the parasites.

Reviewer #2 (Public Review):

In this study Hui Dong et al. identified and characterized two transporters of the monocarboxylate family, which they called Apcimplexan monocarboxylate 1 and 2 (AMC1/2) that the authors suggest are involved in the trafficking of metabolites in the non-photosynthetic plastid (apicoplast) of Toxoplasma gondii (the parasitic agent of human toxoplasmosis) to maintain parasite survival. To do so they first identified novel apicoplast transporters by conducting proximity-dependent protein labeling (TurboID), using the sole known apicoplast transporter (TgAPT) as a bait. They chose two out of the three MFS transporters identified by their screen based and protein sequence similarity and confirmed apicoplast localisation. They generated inducible knock down parasite strains for both AMC1 and AMC2, and confirmed that both transporters are essential for parasite intracellular survival, replication, and for the proper activity of key apicoplast pathways requiring pyruvate as carbon sources (FASII and MEP/DOXP). Then they show that deletion of each protein induces a loss of the apicoplast, more marked for AMC2 and affects its morphology both at its four surrounding membranes level and accumulation of material in the apicoplast stroma. This study is very timely, as the apicoplast holds several important metabolic functions (FASII, IPP, LPA, Heme, Fe-S clusters...), which have been revealed and studied in depth but no further respective transporter have been identified thus far. hence, new studies that could reveal how the apicoplast can acquire and deliver all the key metabolites it deals with, will have strong impact for the parasitology community as well as for the plastid evolution communities. The current study is well initiated with appropriate approaches to identify two new putatively important apicoplast transporters, and showing how essential those are for parasite intracellular development and survival. However, in its current state, this is all the study provides at this point (i.e. essential apicoplast transporters disrupting apicoplast integrity, and indirectly its major functions, FASII and IPP, as any essential apicoplast protein disruption does). The study fails to deliver further message or function regarding AMC1 and 2, and thus validate their study. Currently, the manuscript just describes how AMC1/2 deletion impacts parasite survival without answering the key question about them: what do they transport? The authors yet have to perform key experiments that would reveal their metabolic function. I would thus recommend the authors work further and determine the function of AMC1 and 2.

Response: We thank very much the reviewer for his/her positive evaluation of our work. To address the detailed function of the transporters, in the past three months, we have re-constructed plasmids (with codon-optimized DNA sequences of the genes) for expression of the transporters in a regular expression E. coli strain (BL21DE3) and in a pyruvate import knockout E. coli strain (a gift from Prof. Kirsten Jung), to examine the transport capability in vitro. And, we have re-constructed a new plasmid containing a new leading peptide for targeting the pyruvate sensor PyronicSF to the apicoplast in the parasite, to probe the possible substrate pyruvate. However, we were unable to successfully observe expression of the transporters in the above E. coli strains, and we were unable to target the sensor to the correct localization (the apicoplast) in the parasite. As a result, all these efforts have led the study to the current version of manuscript on the functional identification of transporters. We will keep working on this aspect, attempting to dissect out the exact transport function of the transporters in the near future. In this current manuscript, we have discussed the limitations of our study in the last part of the manuscript.

Reviewer #1 (Recommendations For The Authors):

Minor comments

Line 35: ...appears to have evolved...

Line 67: remove first comma

Line 105: thereafter or therefore?

Line 130: define ACP

Line 131: define TMD

Response: We thank very much the reviewer for the suggestions, and we have revised the points in the current manuscript.

Figure 1: more information on APT1 would be helpful for readers to interpret the results from turboID e.g., consider showing an illustration showing, according to Karnataki et al 2007 that APT1 likely occupies all 4 membranes of the apicoplast. Also, according to DeRocher et al 2012, APT1 N-term and C-term are both cytosolically exposed, at least in the outermost membrane. The orientation in the other membranes is not known.

Response: We thank very much the reviewer for the suggestions. We analyzed the localization information of APT1 in T. gondii, based on the studies as the reviewer proposed (Karnataki, et al., 2007; DeRocher et al., 2012). The HA tag at the C-terminus of APT1 was distributed at the four membranes of the apicoplast, indicating that the topology of APT1 might be difficult to be defined at the membranes. Considering this information, we felt hesitant to clearly describe the topology in a schematic diagram about the protein APT1. Nevertheless, the TurboID tagging at the C-terminus of APT1 was an excellent model for identification of potential transporters localized at membranes of the apicoplast. We have put more information about the topology of APT1 in the manuscript, thus providing a better understanding of the proteomic results.

Figure 2: add a space between "T." and "gondii"

Figure 2: remove period between "Fitness" and "scores"

Figure 2: different fonts are used within the figure. Consider using only one font such as arial. Same for Figure 4.

Figure 2: "Fitness scores" is not bold in panel A but is bold in panel B.

Response: We thank very much the reviewer for the suggestions. We have revised the points in the current version of the manuscript.

Line 187: superscript -7

Line 249: Caution should be used in interpreting two bands as being a precursor and mature product without additional experiments to establish such a relationship. Consider using the term "might" rather than "appear to". The presence of multiple bands could be due to phenomena other than proteolytic processing e.g., alternative splicing, alternative initiator codons, etc.

Response: We thank very much the reviewer for the suggestions. We have revised the sentences in the current version of manuscript.

Line 291: define IPP

Figure 3E. The data points for KD strains appear to be positioned above the zero value on the y-axis. Is this correct?

Response: We thank very much the reviewer for the suggestions. We have rechecked the figure and replaced it with the correct one.

Figure 3 G/H legend. Please describe what a single data point represents e.g., the average of one field of view, the average of a certain number of fields of view, or something else? Are the data combined from three experiments or from a representative experiment?

Response: We thank very much the reviewer for the suggestions. Three independent experiments were performed with at least three replicates. At least 150 vacuoles were scored in each replicate, thus resulting in at least 9 data points in total. The data points were shown with the results from each replicate.

Line 325: define MEP and explain how it is connected to IPP

Response: We thank very much the reviewer for the suggestions. We have provided the information in the current version of the manuscript.

Lines 351-355: The authors refer to Figure 4D to support this statement, but presumably they mean 4E. Also, the authors use the terms C14, C16, and C18. They should more precisely use the terms myristic acid, palmitoleic acid, and trans_oleic acid if this is what they are referring to. Finally, the authors should determine if there is a statistically significant difference between levels of these fatty acids between AMT1 KD and AMT2 KD. If not, they should suggest there is an overall trend toward lower levels of these fatty acids in AMT2 KD parasites compared to AMT1 KD parasites.

Response: We thank very much the reviewer for the suggestions. We have revised the information in the current version of the manuscript.

Lines 363-364: The basis of this comment is unclear. Please clarify.

Lines 369-370: the authors have not shown that the observed lower levels of fatty acids are due to synthesis, as noted above

Response: We thank very much the reviewer for the suggestions. We have accordingly revised the information in the current version of the manuscript.

Line 383: Should be Figure S6D

Line 386: An entire section of the results is used to describe data that are entirely in a supplemental figure. Consider moving this data to a main figure.

Response: We thank very much the reviewer for the suggestions. We have transferred the data to the main figure in the current version of the manuscript.

Line 391: Consider using the term virulence instead of growth since now experiments were performed to specifically assess parasite growth in the infected mice.

Response: We thank very much the reviewer for the suggestions. We have revised the terms in the Results section.

Line 427: Perhaps the authors mean "...strong growth defect..." or ...strong growth impairment..."

Line 460-461: This statement is unclear. Please explain how strong backgrounds in proteomics have made it difficult to identify apicoplast transporters. Because they are low abundance? Because they are membrane proteins?

Response: We thank very much the reviewer for the suggestions. We have revised the corresponding sentences in the current version. The strong backgrounds in the proteomics resulted from the high activity and nonspecific labeling of biotin ligase fused with the apicoplast proteins.

518-521: It would be helpful for non-specialists if the authors explained how pyruvate is connected to IPP biosynthesis.

523: delete period after "Escherichia"

548-549: "We observed similar decreases in level of the MEP biosynthesis activity upon depletion of AMT1 and AMT2..." Reword this since no experiments were done to measure MEP biosynthesis activity.

Response: We thank very much the reviewer for the suggestions. We have accordingly revised the relevant sentences in the manuscript.

Reviewer #2 (Recommendations For The Authors):

Major points:

• The metabolomic data on fatty acid synthesis and isoprenoid levels is relevant but cannot inform about the function of the transporter, since any protein causing loss of the apicoplast would behave in such a manner, i.e. block the apicoplast pathways.

Response: We thank very much the reviewer for the comment. We agree with this comment. We have thus discussed these points in a subsection in the Discussion, pointing out some of the limitations in the study.

• Currently, the manuscript fails to directly prove what AMC1 and AMC2 transports, potentially pyruvate as suggested to putatively fuel FASII and MEP/DOXP. Further experimental approaches using exogenous complementation and/or metabolomic analyses using stable isotope labelling (for example) should potentially bring light to the putative functions of AMC1/2.

Response: We thank very much the reviewer for the comments. As described above, we attempted several approaches to find out the substrates that the AMT1 and AMT2 transports. However, we could not successfully express the proteins in E. coli strains, and we did not generate a T. gondii strain that a pyruvate sensor was properly targeted to the apicoplast. At the end of the Discussion, we have a subsection that discusses the limitations of this study. We hope that our future approaches will be able to tackle these difficulties on the substrate identification.

Furthermore, the authors have not considered other pathways of interest, like heme or lysophosphatidic acid (LPA)n synthesis, which are two other key pathway, which may be related to AMC1/2 function. Those proposed experiments represent an important body of work, required to bring light to their metabolic functions.

Response: We thank very much the reviewer for the comments. We thought about that, but we finally decided to mainly discuss two of the pathways that the transporters might participate in, since the transporters contain specific domains on the proteins sequences that potentially are associated with pyruvate.

Further, the authors might have partially missed some referencing and data about the apicoplast in their introduction (and potentially to address other facets of the apicoplast metabolic functions/capacities in regards to AMC1/2 function): the introduction referencing and explanations are somehow not fully exact/precise for the part of the apicoplast and its pathway: references about the apicoplast, discovery and origin are not citing the original work (that should be Wilson et al. 1996, McFadden et al. 1996, Kohler et al. 1997,), same for the discovery of FASII and MEP./DOXP (Waller 1998, Jomaa et al...). The introduction (and the study?) lacks information about other key functions of the apicoplast: heme synthesis, lysophosphatidic acid synthesis (using FASII products). The explanations about the roles of FASII/DOXP are partial and not fully citing important references: Krishnan et al. 2020, and Amiar et al. 2020 are also key to understanding how the role of FASII is metabolically flexible depending on nutrient content. A whole part on the fact that FASII is not only dispensible but can also become essential under metabolic adaptations conditions, are missing (Botté et al. 2013, Amiar et al. 2020, Primo et al. 2021). These novel important facets of parasite biology should be mentioned as well as directly linked to the author's topic. This is more minor but could bring new ideas to the authors.

Response: We thank very much the reviewer for the suggestions. We have revised the relevant part in the introduction.

We are grateful for the suggestions to improve the manuscript.

2:40 sie holt sich ihre meinung vom mainstream, von leuten die die AFD hassen

auch lustig wie sie sagt "ich lese auch nachrichten über kontroverse themen" bei 1:23 also irgendwie ist ihr schon bewusst dass diese themen "kontrovers" sind...

aber gleichzeitig lässt sie sich blenden von "gewaltenteilung" und "vielfalt" während alle mainstream-kanäle komplett gleichgeschaltet sind

also die konsumiert den ganzen tag nur die "nachrichten" von einer seite aber fühlt sich "ausgewogen informiert"...

echte normies halt *facepalm

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This study presents potentially valuable results on glutamine-rich motifs in relation to protein expression and alternative genetic codes. The author's interpretation of the results is so far only supported by incomplete evidence, due to a lack of acknowledgment of alternative explanations, missing controls and statistical analysis and writing unclear to non experts in the field. These shortcomings could be at least partially overcome by additional experiments, thorough rewriting, or both.

We thank both the Reviewing Editor and Senior Editor for handling this manuscript.

Based on your suggestions, we have provided controls, performed statistical analysis, and rewrote our manuscript. The revised manuscript is significantly improved and more accessible to non-experts in the field.

Reviewer #1 (Public Review):

Summary

This work contains 3 sections. The first section describes how protein domains with SQ motifs can increase the abundance of a lacZ reporter in yeast. The authors call this phenomenon autonomous protein expression-enhancing activity, and this finding is well supported. The authors show evidence that this increase in protein abundance and enzymatic activity is not due to changes in plasmid copy number or mRNA abundance, and that this phenomenon is not affected by mutants in translational quality control. It was not completely clear whether the increased protein abundance is due to increased translation or to increased protein stability.

In section 2, the authors performed mutagenesis of three N-terminal domains to study how protein sequence changes protein stability and enzymatic activity of the fusions. These data are very interesting, but this section needs more interpretation. It is not clear if the effect is due to the number of S/T/Q/N amino acids or due to the number of phosphorylation sites.

In section 3, the authors undertake an extensive computational analysis of amino acid runs in 27 species. Many aspects of this section are fascinating to an expert reader. They identify regions with poly-X tracks. These data were not normalized correctly: I think that a null expectation for how often poly-X track occur should be built for each species based on the underlying prevalence of amino acids in that species. As a result, I believe that the claim is not well supported by the data.

Strengths

This work is about an interesting topic and contains stimulating bioinformatics analysis. The first two sections, where the authors investigate how S/T/Q/N abundance modulates protein expression level, is well supported by the data. The bioinformatics analysis of Q abundance in ciliate proteomes is fascinating. There are some ciliates that have repurposed stop codons to code for Q. The authors find that in these proteomes, Q-runs are greatly expanded. They offer interesting speculations on how this expansion might impact protein function.

Weakness

At this time, the manuscript is disorganized and difficult to read. An expert in the field, who will not be distracted by the disorganization, will find some very interesting results included. In particular, the order of the introduction does not match the rest of the paper.

In the first and second sections, where the authors investigate how S/T/Q/N abundance modulates protein expression levels, it is unclear if the effect is due to the number of phosphorylation sites or the number of S/T/Q/N residues.

There are three reasons why the number of phosphorylation sites in the Q-rich motifs is not relevant to their autonomous protein expression-enhancing (PEE) activities:

First, we have reported previously that phosphorylation-defective Rad51-NTD (Rad51-3SA) and wild-type Rad51-NTD exhibit similar autonomous PEE activity. Mec1/Tel1-dependent phosphorylation of Rad51-NTD antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min (1). (page 1, lines 11-14)

Second, in our preprint manuscript, we have already shown that phosphorylation-defective Rad53-SCD1 (Rad51-SCD1-5STA) also exhibits autonomous PEE activity similar to that of wild-type Rad53-SCD (Figure 2D, Figure 4A and Figure 4C). We have highlighted this point in our revised manuscript (page 9, lines 19-21).

Third, as revealed by the results of Figure 4, it is the percentages, and not the numbers, of S/T/Q/N residues that are correlated with the PEE activities of Q-rich motifs.

The authors also do not discuss if the N-end rule for protein stability applies to the lacZ reporter or the fusion proteins.

The autonomous PEE function of S/T/Q-rich NTDs is unlikely to be relevant to the N-end rule. The N-end rule links the in vivo half-life of a protein to the identity of its N-terminal residues. In S. cerevisiae, the N-end rule operates as part of the ubiquitin system and comprises two pathways. First, the Arg/N-end rule pathway, involving a single N-terminal amidohydrolase Nta1, mediates deamidation of N-terminal asparagine (N) and glutamine (Q) into aspartate (D) and glutamate (E), which in turn are arginylated by a single Ate1 R-transferase, generating the Arg/N degron. N-terminal R and other primary degrons are recognized by a single N-recognin Ubr1 in concert with ubiquitin-conjugating Ubc2/Rad6. Ubr1 can also recognize several other N-terminal residues, including lysine (K), histidine (H), phenylalanine (F), tryptophan (W), leucine (L) and isoleucine (I) (68-70). Second, the Ac/N-end rule pathway targets proteins containing N-terminally acetylated (Ac) residues. Prior to acetylation, the first amino acid methionine (M) is catalytically removed by Met-aminopeptidases (MetAPs), unless a residue at position 2 is non-permissive (too large) for MetAPs. If a retained N-terminal M or otherwise a valine (V), cysteine (C), alanine (A), serine (S) or threonine (T) residue is followed by residues that allow N-terminal acetylation, the proteins containing these AcN degrons are targeted for ubiquitylation and proteasome-mediated degradation by the Doa10 E3 ligase (71).

The PEE activities of these S/T/Q-rich domains are unlikely to arise from counteracting the N-end rule for two reasons. First, the first two amino acid residues of Rad51-NTD, Hop1-SCD, Rad53-SCD1, Sup35-PND, Rad51-ΔN, and LacZ-NVH are MS, ME, ME, MS, ME, and MI, respectively, where M is methionine, S is serine, E is glutamic acid and I is isoleucine. Second, Sml1-NTD behaves similarly to these N-terminal fusion tags, despite its methionine and glutamine (MQ) amino acid signature at the N-terminus. (Page 12, line 3 to page 13, line 2)

The most interesting part of the paper is an exploration of S/T/Q/N-rich regions and other repetitive AA runs in 27 proteomes, particularly ciliates. However, this analysis is missing a critical control that makes it nearly impossible to evaluate the importance of the findings. The authors find the abundance of different amino acid runs in various proteomes. They also report the background abundance of each amino acid. They do not use this background abundance to normalize the runs of amino acids to create a null expectation from each proteome. For example, it has been clear for some time (Ruff, 2017; Ruff et al., 2016) that Drosophila contains a very high background of Q's in the proteome and it is necessary to control for this background abundance when finding runs of Q's.

We apologize for not explaining sufficiently well the topic eliciting this reviewer’s concern in our preprint manuscript. In the second paragraph of page 14, we cite six references to highlight that SCDs are overrepresented in yeast and human proteins involved in several biological processes (5, 43) and that polyX prevalence differs among species (79-82).

We will cite a reference by Kiersten M. Ruff in our revised manuscript (38).

K. M. Ruff, J. B. Warner, A. Posey and P. S. Tan (2017) Polyglutamine length dependent structural properties and phase behavior of huntingtin exon1. Biophysical Journal 112, 511a.

The authors could easily address this problem with the data and analysis they have already collected. However, at this time, without this normalization, I am hesitant to trust the lists of proteins with long runs of amino acid and the ensuing GO enrichment analysis. Ruff KM. 2017. Washington University in St.

Ruff KM, Holehouse AS, Richardson MGO, Pappu RV. 2016. Proteomic and Biophysical Analysis of Polar Tracts. Biophys J 110:556a.

We thank Reviewer #1 for this helpful suggestion and now address this issue by means of a different approach described below.

Based on a previous study (43), we applied seven different thresholds to seek both short and long, as well as pure and impure, polyX strings in 20 different representative near-complete proteomes, including 4X (4/4), 5X (4/5-5/5), 6X (4/6-6/6), 7X (4/7-7/7), 8-10X (≥50%X), 11-10X (≥50%X) and ≥21X (≥50%X).

To normalize the runs of amino acids and create a null expectation from each proteome, we determined the ratios of the overall number of X residues for each of the seven polyX motifs relative to those in the entire proteome of each species, respectively. The results of four different polyX motifs are shown in our revised manuscript, i.e., polyQ (Figure 7), polyN (Figure 8), polyS (Figure 9) and polyT (Figure 10). Thus, polyX prevalence differs among species and the overall X contents of polyX motifs often but not always correlate with the X usage frequency in entire proteomes (43).

Most importantly, our results reveal that, compared to Stentor coeruleus or several non-ciliate eukaryotic organisms (e.g., Plasmodium falciparum, Caenorhabditis elegans, Danio rerio, Mus musculus and Homo sapiens), the five ciliates with reassigned TAAQ and TAGQ codons not only have higher Q usage frequencies, but also more polyQ motifs in their proteomes (Figure 7). In contrast, polyQ motifs prevail in Candida albicans, Candida tropicalis, Dictyostelium discoideum, Chlamydomonas reinhardtii, Drosophila melanogaster and Aedes aegypti, though the Q usage frequencies in their entire proteomes are not significantly higher than those of other eukaryotes (Figure 1). Due to their higher N usage frequencies, Dictyostelium discoideum, Plasmodium falciparum and Pseudocohnilembus persalinus have more polyN motifs than the other 23 eukaryotes we examined here (Figure 8). Generally speaking, all 26 eukaryotes we assessed have similar S usage frequencies and percentages of S contents in polyS motifs (Figure 9). Among these 26 eukaryotes, Dictyostelium discoideum possesses many more polyT motifs, though its T usage frequency is similar to that of the other 25 eukaryotes (Figure 10).

In conclusion, these new normalized results confirm that the reassignment of stop codons to Q indeed results in both higher Q usage frequencies and more polyQ motifs in ciliates.  

Reviewer #2 (Public Review):

Summary:

This study seeks to understand the connection between protein sequence and function in disordered regions enriched in polar amino acids (specifically Q, N, S and T). While the authors suggest that specific motifs facilitate protein-enhancing activities, their findings are correlative, and the evidence is incomplete. Similarly, the authors propose that the re-assignment of stop codons to glutamine-encoding codons underlies the greater user of glutamine in a subset of ciliates, but again, the conclusions here are, at best, correlative. The authors perform extensive bioinformatic analysis, with detailed (albeit somewhat ad hoc) discussion on a number of proteins. Overall, the results presented here are interesting, but are unable to exclude competing hypotheses.

Strengths:

Following up on previous work, the authors wish to uncover a mechanism associated with poly-Q and SCD motifs explaining proposed protein expression-enhancing activities. They note that these motifs often occur IDRs and hypothesize that structural plasticity could be capitalized upon as a mechanism of diversification in evolution. To investigate this further, they employ bioinformatics to investigate the sequence features of proteomes of 27 eukaryotes. They deepen their sequence space exploration uncovering sub-phylum-specific features associated with species in which a stop-codon substitution has occurred. The authors propose this stop-codon substitution underlies an expansion of ploy-Q repeats and increased glutamine distribution.

Weaknesses:

The preprint provides extensive, detailed, and entirely unnecessary background information throughout, hampering reading and making it difficult to understand the ideas being proposed.

The introduction provides a large amount of detailed background that appears entirely irrelevant for the paper. Many places detailed discussions on specific proteins that are likely of interest to the authors occur, yet without context, this does not enhance the paper for the reader.

The paper uses many unnecessary, new, or redefined acronyms which makes reading difficult. As examples:

1) Prion forming domains (PFDs). Do the authors mean prion-like domains (PLDs), an established term with an empirical definition from the PLAAC algorithm? If yes, they should say this. If not, they must define what a prion-forming domain is formally.

The N-terminal domain (1-123 amino acids) of S. cerevisiae Sup35 was already referred to as a “prion forming domain (PFD)” in 2006 (48). Since then, PFD has also been employed as an acronym in other yeast prion papers (Cox, B.S. et al. 2007; Toombs, T. et al. 2011).

B. S. Cox, L. Byrne, M. F., Tuite, Protein Stability. Prion 1, 170-178 (2007). J. A. Toombs, N. M. Liss, K. R. Cobble, Z. Ben-Musa, E. D. Ross, [PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain. PLoS One 6, e21953 (2011).

2) SCD is already an acronym in the IDP field (meaning sequence charge decoration) - the authors should avoid this as their chosen acronym for Serine(S) / threonine (T)-glutamine (Q) cluster domains. Moreover, do we really need another acronym here (we do not).

SCD was first used in 2005 as an acronym for the Serine (S)/threonine (T)-glutamine (Q) cluster domain in the DNA damage checkpoint field (4). Almost a decade later, SCD became an acronym for “sequence charge decoration” (Sawle, L. et al. 2015; Firman, T. et al. 2018).

L. Sawle and K, Ghosh, A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins. J. Chem Phys. 143, 085101(2015).

T. Firman and Ghosh, K. Sequence charge decoration dictates coil-globule transition in intrinsically disordered proteins. J. Chem Phys. 148, 123305 (2018).

3) Protein expression-enhancing (PEE) - just say expression-enhancing, there is no need for an acronym here.

Thank you. Since we have shown that the addition of Q-rich motifs to LacZ affects protein expression rather than transcription, we think it is better to use the “PEE” acronym.

The results suggest autonomous protein expression-enhancing activities of regions of multiple proteins containing Q-rich and SCD motifs. Their definition of expression-enhancing activities is vague and the evidence they provide to support the claim is weak. While their previous work may support their claim with more evidence, it should be explained in more detail. The assay they choose is a fusion reporter measuring beta-galactosidase activity and tracking expression levels. Given the presented data they have shown that they can drive the expression of their reporters and that beta gal remains active, in addition to the increase in expression of fusion reporter during the stress response. They have not detailed what their control and mock treatment is, which makes complete understanding of their experimental approach difficult. Furthermore, their nuclear localization signal on the tag could be influencing the degradation kinetics or sequestering the reporter, leading to its accumulation and the appearance of enhanced expression. Their evidence refuting ubiquitin-mediated degradation does not have a convincing control.

Although this reviewer’s concern regarding our use of a nuclear localization signal on the tag is understandable, we are confident that this signal does not bias our findings for two reasons. First, the negative control LacZ-NV also possesses the same nuclear localization signal (Figure 1A, lane 2). Second, another fusion target, Rad51-ΔN, does not harbor the NVH tag (Figure 1D, lanes 3-4). Compared to wild-type Rad51, Rad51-ΔN is highly labile. In our previous study, removal of the NTD from Rad51 reduced by ~97% the protein levels of corresponding Rad51-ΔN proteins relative to wild-type (1).

Based on the experimental results, the authors then go on to perform bioinformatic analysis of SCD proteins and polyX proteins. Unfortunately, there is no clear hypothesis for what is being tested; there is a vague sense of investigating polyX/SCD regions, but I did not find the connection between the first and section compelling (especially given polar-rich regions have been shown to engage in many different functions). As such, this bioinformatic analysis largely presents as many lists of percentages without any meaningful interpretation. The bioinformatics analysis lacks any kind of rigorous statistical tests, making it difficult to evaluate the conclusions drawn. The methods section is severely lacking. Specifically, many of the methods require the reader to read many other papers. While referencing prior work is of course, important, the authors should ensure the methods in this paper provide the details needed to allow a reader to evaluate the work being presented. As it stands, this is not the case.

Thank you. As described in detail below, we have now performed rigorous statistical testing using the GofuncR package (Figure 11, Figure 12 and DS7-DS32).

Overall, my major concern with this work is that the authors make two central claims in this paper (as per the Discussion). The authors claim that Q-rich motifs enhance protein expression. The implication here is that Q-rich motif IDRs are special, but this is not tested. As such, they cannot exclude the competing hypothesis ("N-terminal disordered regions enhance expression").

In fact, “N-terminal disordered regions enhance expression” exactly summarizes our hypothesis.

On pages 12-13 and Figure 4 of our preprint manuscript, we explained our hypothesis in the paragraph entitled “The relationship between PEE function, amino acid contents, and structural flexibility”.

The authors also do not explore the possibility that this effect is in part/entirely driven by mRNA-level effects (see Verma Na Comms 2019).

As pointed out by the first reviewer, we present evidence that the increase in protein abundance and enzymatic activity is not due to changes in plasmid copy number or mRNA abundance (Figure 2), and that this phenomenon is not affected in translational quality control mutants (Figure 3).

As such, while these observations are interesting, they feel preliminary and, in my opinion, cannot be used to draw hard conclusions on how N-terminal IDR sequence features influence protein expression. This does not mean the authors are necessarily wrong, but from the data presented here, I do not believe strong conclusions can be drawn. That re-assignment of stop codons to Q increases proteome-wide Q usage. I was unable to understand what result led the authors to this conclusion.

My reading of the results is that a subset of ciliates has re-assigned UAA and UAG from the stop codon to Q. Those ciliates have more polyQ-containing proteins. However, they also have more polyN-containing proteins and proteins enriched in S/T-Q clusters. Surely if this were a stop-codon-dependent effect, we'd ONLY see an enhancement in Q-richness, not a corresponding enhancement in all polar-rich IDR frequencies? It seems the better working hypothesis is that free-floating climate proteomes are enriched in polar amino acids compared to sessile ciliates.

We thank this reviewer for raising this point, however her/his comments are not supported by the results in Figure 7.

Regardless, the absence of any kind of statistical analysis makes it hard to draw strong conclusions here.

We apologize for not explaining more clearly the results of Tables 5-7 in our preprint manuscript.

To address the concerns about our GO enrichment analysis by both reviewers, we have now performed rigorous statistical testing for SCD and polyQ protein overrepresentation using the GOfuncR package (https://bioconductor.org/packages/release/bioc/html/GOfuncR.html). GOfuncR is an R package program that conducts standard candidate vs. background enrichment analysis by means of the hypergeometric test. We then adjusted the raw p-values according to the Family-wise error rate (FWER). The same method had been applied to GO enrichment analysis of human genomes (89).

The results presented in Figure 11 and Figure 12 (DS7-DS32) support our hypothesis that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition, Candida albicans filamentous growth, peptidyl-glutamic acid modification in ciliates with reassigned stop codons (TAAQ and TAGQ), Tetrahymena thermophila xylan catabolism, Dictyostelium discoideum sexual reproduction, Plasmodium falciparum infection, as well as the nervous systems of Drosophila melanogaster, Mus musculus, and Homo sapiens (78). In contrast, peptidyl-glutamic acid modification and microtubule-based movement are not overrepresented with Q-rich proteins in Stentor coeruleus, a ciliate with standard stop codons.

Recommendations for the authors:

Please note that you control which revisions to undertake from the public reviews and recommendations for the authors.

Reviewer #1 (Recommendations For The Authors):

The order of paragraphs in the introduction was very difficult to follow. Each paragraph was clear and easy to understand, but the order of paragraphs did not make sense to this reader. The order of events in the abstract matches the order of events in the results section. However, the order of paragraphs in the introduction is completely different and this was very confusing. This disordered list of facts might make sense to an expert reader but makes it hard for a non-expert reader to understand.

Apologies. We endeavored to improve the flow of our revised manuscript to make it more readable.

The section beginning on pg 12 focused on figures 4 and 5 was very interesting and highly promising. However, it was initially hard for me to tell from the main text what the experiment was. Please add to the text an explanation of the experiment, because it is hard to figure out what was going on from the figures alone. Figure 4 is fantastic, but would be improved by adding error bars and scaling the x-axis to be the same in panels B,C,D.

Thank you for this recommendation. We have now scaled both the x-axis and y-axis equivalently in panels B, C and D of Figure 4. Error bars are too small to be included.

It is hard to tell if the key variable is the number of S/T/Q/N residues or the number of phosphosites. I think a good control would be to add a regression against the number of putative phosphosites. The sequences are well designed. I loved this part but as a reader, I need more interpretation about why it matters and how it explains the PEE.

As described above, we have shown that the number of phosphorylation sites in the Q-rich motifs is not relevant to their autonomous protein expression-enhancing (PEE) activities.

I believe that the prevalence of polyX runs is not meaningful without normalizing for the background abundance of each amino acid. The proteome-wide abundance and the assumption that amino acids occur independently can be used to form a baseline expectation for which runs are longer than expected by chance. I think Figures 6 and 7 should go into the supplement and be replaced in the main text with a figure where Figure 6 is normalized by Figure 7. For example in P. falciparum, there are many N-runs (Figure 6), but the proteome has the highest fraction of N’s (Figure 7).

Thank you for these suggestions. The three figures in our preprint manuscript (Figures 6-8) have been moved into the supplementary information (Figures S1-S3). For normalization, we have provided four new figures (Figures 7-10) in our revised manuscript.

The analysis of ciliate proteomes was fascinating. I am particularly interested in the GO enrichment for “peptidyl-glutamic acid modification” (pg 20) because these enzymes might be modifying some of Q’s in the Q-runs. I might be wrong about this idea or confused about the chemistry. Do these ciliates live in Q-rich environments? Or nitrogen rich environments?

Polymeric modifications (polymodifications) are a hallmark of C-terminal tubulin tails, whereas secondary peptide chains of glutamic acids (polyglutamylation) and glycines (polyglycylation) are catalyzed from the γ-carboxyl group of primary chain glutamic acids. It is not clear if these enzymes can modify some of the Q’s in the Q-runs.

To our knowledge, ciliates are abundant in almost every liquid water environment, i.e., oceans/seas, marine sediments, lakes, ponds, and rivers, and even soils.

I think you should include more discussion about how the codons that code for Q’s are prone to slippage during DNA replication, and thus many Q-runs are unstable and expand (e.g. Huntington’s Disease). The end of pg 24 or pg 25 would be good places.

We thank the reviewer for these comments.

PolyQ motifs have a particular length-dependent codon usage that relates to strand slippage in CAG/CTG trinucleotide repeat regions during DNA replication. In most organisms having standard genetic codons, Q is encoded by CAGQ and CAAQ. Here, we have determined and compared proteome-wide Q contents, as well as the CAGQ usage frequencies (i.e., the ratio between CAGQ and the sum of CAGQ, CAGQ, TAAQ, and TAGQ).

Our results reveal that the likelihood of forming long CAG/CTG trinucleotide repeats are higher in five eukaryotes due to their higher CAGQ usage frequencies, including Drosophila melanogaster (86.6% Q), Danio rerio (74.0% Q), Mus musculus (74.0% Q), Homo sapiens (73.5% Q), and Chlamydomonas reinhardtii (87.3% Q) (orange background, Table 2). In contrast, another five eukaryotes that possess high numbers of polyQ motifs (i.e., Dictyostelium discoideum, Candida albicans, Candida tropicalis, Plasmodium falciparum and Stentor coeruleus) (Figure 1) utilize more CAAQ (96.2%, 84.6%, 84.5%, 86.7% and 75.7%) than CAAQ (3.8%, 15.4%, 15.5%, 13.3% and 24.3%), respectively, to avoid the formation of long CAG/CTG trinucleotide repeats (green background, Table 2). Similarly, all five ciliates with reassigned stop codons (TAAQ and TAGQ) have low CAGQ usage frequencies (i.e., from 3.8% Q in Pseudocohnilembus persalinus to 12.6% Q in Oxytricha trifallax) (red font, Table 2). Accordingly, the CAG-slippage mechanism might operate more frequently in Chlamydomonas reinhardtii, Drosophila melanogaster, Danio rerio, Mus musculus and Homo sapiens than in Dictyostelium discoideum, Candida albicans, Candida tropicalis, Plasmodium falciparum, Stentor coeruleus and the five ciliates with reassigned stop codons (TAAQ and TAGQ).

Author response table 1.

Usage frequencies of TAA, TAG, TAAQ, TAGQ, CAAQ and CAGQ codons in the entire proteomes of 20 different organisms.

Pg 7, paragraph 2 has no direction. Please add the conclusion of the paragraph to the first sentence.

This paragraph has been moved to the “Introduction” section” of the revised manuscript.

Pg 8, I suggest only mentioning the PFDs used in the experiments. The rest are distracting.

We have addressed this concern above.

Pg 12. Please revise the "The relationship...." text to explain the experiment.

We apologize for not explaining this topic sufficiently well in our preprint manuscript.

SCDs are often structurally flexible sequences (4) or even IDRs. Using IUPred2A (https://iupred2a.elte.hu/plot_new), a web-server for identifying disordered protein regions (88), we found that Rad51-NTD (1-66 a.a.) (1), Rad53-SCD1 (1-29 a.a.) and Sup35-NPD (1-39 a.a.) are highly structurally flexible. Since a high content of serine (S), threonine (T), glutamine (Q), asparanine (N) is a common feature of IDRs (17-20), we applied alanine scanning mutagenesis approach to reduce the percentages of S, T, Q or N in Rad51-NTD, Rad53-SCD1 or Sup35-NPD, respectively. As shown in Figure 4 and Figure 5, there is a very strong positive relationship between STQ and STQN amino acid percentages and β-galactosidase activities. (Page 13, lines 5-10)

Pg 13, first full paragraph, "Futionally, IDRs..." I think this paragraph belongs in the Discussion.

This paragraph is now in the “Introduction” section (Page 5, Lines 11-15).

Pg. 15, I think the order of paragraphs should be swapped.

These paragraphs have been removed or rewritten in the “Introduction section” of our revised manuscript.

Pg 17 (and other parts) I found the lists of numbers and percentages hard to read and I think you should refer readers to the tables.

Thank you. In the revised manuscript, we have avoided using lists of numbers and percentages, unless we feel they are absolutely essential.

Pg. 19 please add more interpretation to the last paragraph. It is very cool but I need help understanding the result. Are these proteins diverging rapidly? Perhaps this is a place to include the idea of codon slippage during DNA replication.

Thank you. The new results in Table 2 indicate that the CAG-slippage mechanism is unlikely to operate in ciliates with reassigned stop codons (TAAQ and TAGQ).

Pg 24. "Based on our findings from this study, we suggest that Q-rich motifs are useful toolkits for generating novel diversity during protein evolution, including by enabling greater protein expression, protein-protein interactions, posttranslational modifications, increased solubility, and tunable stability, among other important traits." This idea needs to be cited. Keith Dunker has written extensively about this idea as have others. Perhaps also discuss why Poly Q rich regions are different from other IDRs and different from other IDRs that phase-separate.

Agreed, we have cited two of Keith Dunker’s papers in our revised manuscript (73, 74).

Minor notes:

Please define Borg genomes (pg 25).

Borgs are long extrachromosomal DNA sequences in methane-oxidizing Methanoperedens archaea, which display the potential to augment methane oxidation (101). They are now described in our revised manuscript. (Page 15, lines 12-14)

Reviewer #2 (Recommendations For The Authors):

The authors dance around disorder but never really quantify or show data. This seems like a strange blindspot.

We apologize for not explaining this topic sufficiently well in our preprint manuscript. We have endeavored to do so in our revised manuscript.

The authors claim the expression enhancement is "autonomous," but they have not ruled things out that would make it not autonomous.

Evidence of the “autonomous” nature of expression enhancement is presented in Figure 1, Figure 4, and Figure 5 of the preprint manuscript.

Recommendations for improving the writing and presentation.

The title does not recapitulate the entire body of work. The first 5 figures are not represented by the title in any way, and indeed, I have serious misgivings as to whether the conclusion stated in the title is supported by the work. I would strongly suggest the authors change the title.

Figure 2 could be supplemental.

Thank you. We think it is important to keep Figure 2 in the text.

Figures 4 and 5 are not discussed much or particularly well.

This reviewer’s opinion of Figure 4 and Figure 5 is in stark contrast to those of the first reviewer.

The introduction, while very thorough, takes away from the main findings of the paper. It is more suited to a review and not a tailored set of minimal information necessary to set up the question and findings of the paper. The question that the authors are after is also not very clear.

Thank you. The entire “Introduction” section has been extensively rewritten in the revised manuscript.

Schematics of their fusion constructs and changes to the sequence would be nice, even if supplemental.

Schematics of the fusion constructs are provided in Figure 1A.

The methods section should be substantially expanded.

The method section in the revised manuscript has been rewritten and expanded. The six Javascript programs used in this work are listed in Table S4.

The text is not always suited to the general audience and readership of eLife.

We have now rewritten parts of our manuscript to make it more accessible to the broad readership of eLife.

In some cases, section headers really don't match what is presented, or there is no evidence to back the claim.

The section headers in the revised manuscript have been corrected.

A lot of the listed results in the back half of the paper could be a supplemental table, listing %s in a paragraph (several of them in a row) is never nice

Acknowledged. In the revised manuscript, we have removed almost all sentences listing %s.

Minor corrections to the text and figures.

There is a reference to table 1 multiple times, and it seems that there is a missing table. The current table 1 does not seem to be the same table referred to in some places throughout the text.

Apologies for this mistake, which we have now corrected in our revised manuscript.

In some places its not clear where new work is and where previous work is mentioned. It would help if the authors clearly stated "In previous work...."

Acknowledged. We have corrected this oversight in our revised manuscript.

Not all strains are listed in the strain table (KO's in figure 3 are not included)

Apologies, we have now corrected Table S2, as suggested by this reviewer.

Author response table 2.

S. cerevisiae strains used in this study

1. The tag above is infinite, it should not be so.
2. It should stop at the last tag name.
3. The corner black overlay need to be of the same colour as the background black #161616

The fact that people were paid such large amounts (especially for the time) for these animals is even more disturbing. So many animals were probably killed with the intention of being sold to this exhibit, but were majority were probably rejected because they were not up to standards.

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This important study reports jAspSnFR3, a biosensor that enables high spatiotemporal resolution of aspartate levels in living cells. To develop this sensor, the authors used a structurally guided amino acid substitution in a glutamate/aspartate periplasmic binding protein to switch its specificity towards aspartate. The in vitro and in cellulo functional characterization of the biosensor is convincing, but evidence of the sensor's effectiveness in detecting small perturbations of aspartate levels and information on its behavior in response to acute aspartate elevations in the cytosol are still lacking.

We thank the reviewers and editors for the detailed assessment of our work and for their constructive feedback. Most comments have now been experimentally addressed in the revised manuscript, which we feel is substantially improved from the initial draft.

Public Reviews:

Reviewer #1 (Public Review):

In this manuscript, Davidsen and coworkers describe the development of a novel aspartate biosensor jAspSNFR3. This collaborative work supports and complements what was reported in a recent preprint by Hellweg et al., (bioRxiv; doi: 10.1101/2023.05.04.537313). In both studies, the newly engineered aspartate sensor was developed from the same glutamate biosensor previously developed by the authors of this manuscript. This coincidence is not casual but is the result of the need to find tools capable of measuring aspartate levels in vivo. Therefore, it is undoubtedly a relevant and timely work carried out by groups experienced in aspartate metabolism and in the generation of metabolite biosensors.

Reviewer #2 (Public Review):

In this work the IGluSnFR3 sensor, recently developed by Marvin et al (2023) is mutated position S72, which was previously reported to switch the specificity from Glu to Asp. They made 3 mutations at this position, selected a S72P mutant, then made a second mutation at S27 to generate an Asp-specific version of the sensor. This was then characterized thoroughly and used on some test experiments, where it was shown to detect and allow visualization of aspartate concentration changes over time. It is an incremental advance on the iGluSnFR3 study, where 2 predictable mutations are used to generate a sensor that works on a close analog of Glu, Asp. It is shown to have utility and will be useful in the field of Asp-mediated biological effects.

Reviewer #3 (Public Review):

In this manuscript, Davidsen and collaborators introduce jAspSnFR3, a new version of aspartate biosensor derived from iGluSnFR3, that allows monitoring in real-time aspartate levels in cultured cells. A selective amino acids substitution was applied in a key region of the template to switch its specificity from glutamate to aspartate. The jAspSnFR3 does not respond to other tested metabolites and performs well, is not toxic for cultured cells, and is not affected by temperature ensuring the possibility of using this tool in tissues physiologically more relevant. The high affinity for aspartate (KD=50 uM) allowed the authors to measure fluctuations of this amino acid in the physiological range. Different strategies were used to bring aspartate to the minimal level. Finally, the authors used jAspSnFR3 to estimate the intracellular aspartate concentration. One of the highlights of the manuscript was a treatment with asparagine during glutamine starvation. Although didn't corroborate the essentiality of asparagine in glutamine depletion, the measurement of aspartate during this supplementation is a glimpse of how useful this sensor can be.

Reviewer #1 (Recommendations For The Authors):

The authors should evaluate the effectiveness of the sensor in detecting small perturbations of aspartate levels and its behavior in response to acute aspartate elevations in the cytosol. In vivo aspartate determinations were performed exclusively in conditions that cause aspartate depletion. By means the use of mitochondrial respiratory inhibitors or aspartate withdrawal, it was determined the reliability of the sensor performing readings during relatively long periods, until reaching a steady-state of aspartate-depletion 12-60 hours later. Although in Hellweg and coworkers, it has been demonstrated that a related aspartate sensor could detect increases in aspartate in cell overexpressing the aspartate-glutamate GLAST transporter, the differences reported here between both sensors advise testing whether this aspect is also improved, or not, using jAspSNFR3.

Similarly, Davidsen et al. did not test if the sensor can be able to detect transient variations in cytosolic aspartate levels. In proliferative cells aspartate synthesis is linked to NAD+ regeneration by ETC (Sullivan et al., 2015, Cell), indeed the authors deplete aspartate using CI or CIII inhibitors but do not analyze if those are recovered, and increased, after its removal. Furthermore, the sequential addition of oligomycin and uncouplers could generate measurable fluctuations of aspartate in the cytosol.

We agree with the reviewer that only including situations of aspartate depletion in our cell culture experiments provided an incomplete evaluation of the utility of this biosensor. In the revised manuscript we provide three additional experiments using secondary treatments that restore aspartate synthesis to conditions that initially caused aspartate depletion. First, we conducted experiments where cells expressing jAspSnFR3/NucRFP were changed into media without glutamine, inducing aspartate depletion, with glutamine being replenished at various time points to observe if GFP/RFP measurements recover. As expected, glutamine withdrawal caused a decay in the GFP/RFP signal and we found that restoring glutamine caused a subsequent restoration of the GFP/RFP signal at all time points, with each fully recovering the GFP/RFP signal over time (Revised Manuscript Figure 2E). Next, we conducted the experiment suggested by the reviewer, testing whether the published finding, that oligomycin induced aspartate limitation can be remedied by co-treatment with electron transport chain uncouplers, could be visualized using jAspSnFR3 measurements of GFP/RFP. Indeed, after 24 hours of oligomycin induced aspartate depletion, treatment with the ETC uncoupler BAM15 dose dependently restored GFP/RFP signal (Revised Manuscript Figure 2G). Finally, we also measured whether the ability of pyruvate to mitigate the decrease in aspartate upon co-treated with rotenone (Figure 2B) could also be detected in a sequential treatment protocol after aspartate depletion. Indeed, after 24 hours of aspartate depletion by rotenone treatment, the GFP/RFP signal was rapidly restored by additional treatment with pyruvate (Revised Manuscript Figure 2, figure supplement 1C). Collectively, these results provide support for the utility of jAspSnFR3 to measure transient changes in aspartate levels in diverse metabolic situations, including conditions that restore aspartate to cells that had been experiencing aspartate depletion.

Reviewer #2 (Recommendations For The Authors):

Weaknesses: Sensor basically identical to iGluSnFR3, but nevertheless useful and specific. The results support the conclusions, and the paper is very straightforward. I think the work will be useful to people working on the effects of free aspartate in biology and given it is basically iGluSnFR3, which is widely used, should be very reproducible and reliable.

We appreciate the reviewer’s comment that sensor is useful for specific detection of aspartate. We agree that the advance of the paper is primarily in demonstrating its utility to measure aspartate, rather than any fundamental innovation on the biosensor approach. We hope the fact that jAspSnFR3 derives from a well validated biosensor (iGluSnFR3) will support its adoption.

Reviewer #3 (Recommendations For The Authors):

Although this is a well-performed study, I have some comments for the authors to address:

1) A red tag version of the sensor (jAspSnFR3-mRuby3) was generated for normalization purposes, with this the authors plan to correct GFP signal from expression and movement artifacts. I naturally interpret "movement artifacts" as those generated by variations in cell volume and focal plane during time-lapse experiments. However, it was mentioned that jAspSnFR3-mRuby3 included a histidine tag that may induce a non-specific effect (responses to the treatment with some amino acids). This suggests that a version without the tag needs to be generated and that an alternative design needs to be set for normalization purposes. A nuclear-localized RFP was expressed in a second attempt to incorporate RFP as a normalization signal. Here the cell lines that express both signals (sensor and RFP) were generated by independent lentiviral transductions (insertions). Unless the number of insertions for each construct is known, this approach will not ensure an equimolar expression of both proteins (sensor and RFP). In this scenario is not clear how the nuclear expression of RFP will help the correction by expression or monitor changes in cell volume. The authors may be interested in attempting a bicistronic system to express both the sensor and RFP.

The reviewer noted several potential issues concerning the use of RFP for normalization, which will be separated into sections below:

Movement artifacts:

We are glad the reviewer raised this issue since we see how it was confusingly worded. We have deleted the text “and movement artefacts” from the sentence.

His-tag and non-specific responses to some amino acids:

We also found it concerning that non-specific responses to amino acids could potentially contribute to our RFP normalization signal, and so we conducted additional experiments to address whether this was likely to be an issue in intracellular measurements. We first tested whether the non-specific signal was related to the histidine tag, or was intrinsic to the mRuby3 protein itself, by comparing the fluorescence response to a titration of histidine (which showed the largest effect of red fluorescence), aspartate, and GABA (structurally related to glutamate and aspartate, but lacking a carboxylate group) across a group of mRuby containing variants, with or without histidine tags. We replicated the non-specific signal originally observed in jAspSnFR3-mRuby3-His and found that another biosensor with a histidine tagged on the C terminus of mRuby3 had a similar response (iGlucoSnFR2.mRuby3-His), as did mRuby3-His alone, indicating that the aspect of being fused with jAspSnFR3 or another binding protein was not required for this effect. Additionally, we also compared the fluorescence response of lysates expressing mRuby2 and mRuby3 without histidine tags and found that the non-specific signal was essentially absent (Revised Manuscript Figure 1, figure supplement 4B-D). Collectively. These data support our original hypothesis that the histidine tag was responsible for the non-specific signal, alleviating concerns about more substantial protein design issues or with using nuc-RFP for normalization. Since we also found that measuring aspartate signal using GFP/RFP ratios from cells with linked the jAspSnFR3-Ruby3-His agreed with measurements from cells separately expressing jAspSnFR3 and nucRFP (without a His tag), and the amino acid concentrations needed to significantly alter His tagged Ruby3 signal are above those typically found in cells, we conclude that this is unlikely to be a significant factor in cells. Nonetheless, we have added all the relevant data to the manuscript to allow readers to make their own decision about which construct would be best for their purposes.

Original text:

"Surprisingly, the mRuby3 component responds to some amino acids at high millimolar concentrations, indicating a non-specific effect, potentially interactions with the C-terminal histidine tag (Figure 1—figure Supplement 2, panel B). Notably, this increase in fluorescence is still an order of magnitude lower than the green fluorescence response and it occurs at amino acid concentrations that are unlikely to be achieved in most cell types."

Revised text:

"Surprisingly, the mRuby3 fluorescence of affinity-purified jAspSnFR3.mRuby3 responds to some amino acids at high millimolar concentrations, indicating a non-specific effect (Figure 1—figure Supplement 4, panel A). This was determined to be due to an unexpected interaction with the C-terminal histidine tag and could be reproduced with other proteins containing mRuby3 and purified via the same C-terminal histidine tag (Figure 1—figure Supplement 4, panel B and C). Interestingly, a structurally related, non-amino acid compound, GABA, does not elicit a change in red fluorescence; indicating, that only amino acids are interacting with the histidine tag (Figure 1—figure Supplement 4, panel D). Nevertheless, most of our cell culture experiments were performed with nuclear localized mRuby2, which lacks a C-terminal histidine tag, and these measurements correlated with those using the histidine tagged jAspSnFR3-mRuby3 construct (Figure 1—figure Supplement 1 panel D)."

Lentiviral transductions

We agree that splitting the two fluorescent proteins across two expression constructs and infections effectively guarantees that there will not be equimolar expression of jAspSnFR3 and RFP, however we do not think equimolar expression is necessary in this context. The primary goal of RFP measurements in these experiments (and in experiments using the jAspSnFR3-mRuby3 fused construct) is to control for global alterations in protein expression that might confound the interpretation that a change in GFP fluorescence corresponds to a change in aspartate levels. While a bicistronic system is arguably a better approach to improve the similarity of expression of jAspSnFR3 and nuc-RFP in a cell, we only require that the cells have consistent expression of both proteins across all cells in the population, not that the expression of one necessarily be a similar molarity to the other. We accomplish consistent expression of proteins by single cell cloning after expression of jAspSnFR3 and nucRFP (or jAspSnFR3-mRuby3), and screening for clones that have high enough expression of both proteins such that they are well detected by standard Incucyte conditions. Given that our data do not identify an obvious downside to separate expression of jASPSnFR3 and nuc-RFP compared to the fused jAspSnFR3-mRuby3 construct (where the fluorescent proteins are truly equimolar) (Figure 2, Figure Supplement 1C), we elected to prioritize the separate jAspSnFR3 and nuc-RFP combination, which provides additional opportunities to measure cell number in the same experiment (see below).

2) The authors were interested in establishing the temporal dynamics of aspartate depletion by genetics and pharmaceutical means. For the inhibition of mitochondrial complex I rotenone and metformin were used. Although the assays are clearly showing aspartate depletion the report of cell viability is missing. Considering that glutamine deprivation induces arrest in cell proliferation, I think will be important to know the conditions of the cell cultures after 60 hours of treatment with such inhibitors.

We agree that ensuring that cells are still viable in conditions where aspartate is depleted, as determined by GFP/RFP in jAspSnFR3 expressing cells, is an important goal. To this end, we added a new experiment investigating the restoration of glutamine on the GFP/RFP signal at different time points after glutamine depletion (Revised Manuscript Figure 2E, see response to reviewer 1). One advantage of using the nuclear RFP as a normalization marker is that it also enables measurements of nuclei counts, a surrogate measurement for cell number. In the same glutamine depletion experiment we therefore measured cell counts using nuclear RFP incidences and confluency as measurements of cell proliferation/growth. In both cases, the arrest in cell proliferation upon glutamine withdrawal was obvious, as was the restoration of cell proliferation following glutamine replenishment, with the amount of growth delay corresponding to the length of glutamine withdrawal (Revised Manuscript Figure 2, Figure Supplement 2A-B). Nonetheless, there was no obvious lasting defects in restarting cell proliferation even after 12 hours of glutamine withdrawal, indicating that cell viability is preserved. In the case of mitochondrial inhibitors, we also observe even that after 24 hours of treatment with oligomycin or rotenone, restoration of aspartate synthesis from BAM15 or pyruvate, respectively, can also restore GFP/RFP signal, supporting the conclusion that cellular metabolism is still active in these conditions (Revised Manuscript Figure 2G; Revised Manuscript Figure 2, figure supplement 1C).

3) The pH sensitivity was checked in vitro with jAspSnFR3-mRuby3 and the sensor reported suitable for measurements at physiological pH. It would be an opportunity to revisit the analysis for pH sensitivity in cultured cells using an untagged version of jAspSnFR3 coupled, for example, to a sensor for pH.

We thank the reviewer for the suggestion and agree that pH effects on sensor signal could be a confounding factor in some conditions. Unfortunately, measuring intracellular pH is not trivial and using multiple fluorescent sensors that change simultaneously would be complex to interpret, particularly in the absence of controls to unambiguously control intracellular pH and aspartate concentrations. Thus, we believe that proper investigation of the variable of pH is beyond the scope of this study. Nonetheless, we agree that measuring the contribution of pH to sensor signal is an important goal for future work, particularly if deploying it in conditions likely to cause substantial pH differences, such as comparing compartmentalized signal of jAspSnFR3 in the cytosol and mitochondria. We have added the following italicized text to the conclusions section to underscore this point:

“Another potential use for this sensor would be to dissect compartmentalized metabolism, with mitochondria being a critical target, although incorporating the influence of pH on sensor fluorescence will be an important consideration in this context.”

4) While the authors take an interesting approach to measuring intracellular aspartate concentration, it will be highly desirable if a calibration protocol can be designed for this sensor. Clearly, glutamine depletion grants a minimal ("zero") aspartate concentration. However, having a more dynamic way for calibration will facilitate the introduction of this tool for metabolism studies. This may be achieved by incorporating a cultured cell that already expresses the transporter or by ectopic expression in the cells that have already been used.

We appreciate the suggestion and would similarly desire a calibration protocol to serve as a quantitative readout of aspartate levels from fluorescence signal, if possible. While we do calibrate jAspSnFR3 fluorescence in purified settings, conducting an analogous experiment intracellularly is currently difficult, if not impossible. While we have several methods to constrain the production rate of aspartate (glutamine withdrawal, mitochondrial inhibitors, and genetic knockouts of GOT1 and GOT2), we cannot prevent cells from decreasing aspartate consumption and so cannot get a true intracellular zero to aid in calibration. Additionally, the impermeability of aspartate to cell membranes makes it challenging to specifically control intracellular concentrations using environmental aspartate, and the best-known aspartate transporter (SLC1A3) is concentrative and so has the reciprocal problem. Considering these issues, we are wary of implying to readers that any specific fluorescence measurement can be used to directly interpret aspartate concentration given the many variables that can impact its signal, both related to the biosensor system itself (expression of jAspSnFR3, expression of Nuc-RFP, sensitivity and settings of the fluorescence detector) and based on cell intrinsic variability (differences in basal ASP levels, different sensitivity to treatments, influence of pH, etc.). We maintain that jAspSnFR3 has utility to measure relative changes in aspartate within a cell line across treatment conditions and over time, but absolute quantitation of aspartate still will require complementary approaches, like mass spectrometry, enzymatic assays, or NMR.

5) jAspSnFR3 seems to have the potential to be incorporated easily for several research groups as a main tool. In general, a minor correction to replace F/F with ΔF/F in the text.

Thank you for catching this error, the text has been edited accordingly.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Davidsen and collaborators introduce jAspSnFR3, a new version of aspartate biosensor derived from iGluSnFR3, that allows to monitor in real-time aspartate levels in cultured cells. A selective amino acids substitution was applied in a key region of the template to switch its specificity from glutamate to aspartate. The jAspSnFR3 does not respond to other tested metabolites and performs well, is not toxic for cultured cells, and is not affected by temperature ensuring the possibility of using this tool in tissues physiologically more relevant. The high affinity for aspartate (KD=50 uM) allowed the authors to measure fluctuations of this amino acid in the physiological range. Different strategies were used to bring aspartate to the minimal level. Finally, the authors used jAspSnFR3 to estimate the intracellular aspartate concentration.

Strengths:<br /> One of the highlights of the manuscript was a treatment with asparagine during glutamine starvation. Although didn`t corroborate the essentiality of asparagine in glutamine depletion, the measurement of aspartate during this supplementation is a glimpse of how useful this sensor can be.

Weaknesses:<br /> Although this is a well-performed study, I have some comments for the authors to address:<br /> 1-A red tag version of the sensor (jAspSnFR3-mRuby3) was generated for normalization purposes, with this the authors plan to correct GFP signal from expression and movement artifacts. I naturally interpret "movement artifacts" as those generated by variations in cell volume and focal plane during time-lapse experiments. However, it was mentioned that jAspSnFR3-mRuby3 included a histidine tag that may induce a non-specific effect (responses to the treatment with some amino acids). This suggests that a version without the tag needs to be generated and that an alternative design needs to be set for normalization purposes. A nuclear-localized RFP was expressed in a second attempt to incorporate RFP as a normalization signal. Here the cell lines that express both signals (sensor and RFP) were generated by independent lentiviral transductions (insertions). Unless the number of insertions for each construct is known, this approach will not ensure an equimolar expression of both proteins (sensor and RFP). In this scenario is not clear how the nuclear expression of RFP will help the correction by expression or monitor changes in cell volume. The authors may be interested in attempting a bicistronic system to express both the sensor and RFP.<br /> 2-The authors were interested in establishing the temporal dynamics of aspartate depletion by genetics and pharmaceutical means. For the inhibition of mitochondrial complex I rotenone and metformin were used. Although the assays are clearly showing aspartate depletion the report of cell viability is missing. Considering that glutamine deprivation induces arrest in cell proliferation, I think will be important to know the conditions of the cell cultures after 60 hours of treatment with such inhibitors.<br /> 3-The pH sensitivity was checked in vitro with jAspSnFR3-mRuby3 and the sensor reported suitable for measurements at physiological pH. It would be an opportunity to revisit the analysis for pH sensitivity in cultured cells using an untagged version of jAspSnFR3 coupled, for example, to a sensor for pH.<br /> 4-While the authors take an interesting approach to measuring intracellular aspartate concentration, it will be highly desirable if a calibration protocol can be designed for this sensor. Clearly, glutamine depletion grants a minimal ("zero") aspartate concentration. However, having a more dynamic way for calibration will facilitate the introduction of this tool for metabolism studies. This may be achieved by incorporating a cultured cell that already expresses the transporter or by ectopic expression in the cells that have already been used.

Author Response

The following is the authors’ response to the original reviews.

First of all, we'd like to thank the three reviewers for their meticulous work that enable us to present now an improved manuscript and substantial changes were made to the article following reviewers' and editors' recommendations. We read all their comments and suggestions very carefully. Apart from a few misunderstandings, all comments were very pertinent. We responded positively to almost all the comments and suggestions, and as a result, we have made extensive changes to the document and the figures. This manuscript now contains 16 principal figures and 15 figure supplements.

The number of principal figures is now 16 (1 new figure), and additional panels have been added to certain figures. On the other hand, we have added 7 additional figures (supplement figures) to answer the reviewers' questions and/or comments.

Main figures

▪ Figures 1, 4, 5, 10, 11, 12, 13, 14: unchanged ▪ Figure 7 and 8 were switched.

▪ Figure 2: we added panel F in response to reviewer 3's and request for sperm defect statistics

▪ Figure 3: the contrast in panel B has been taken over to homogenize colors

▪ Figure 6: This figure was recomposed. The WB on testicular extract was suppressed and we present a new WB allowing to compare the presence of CCDC146 in the flagella fraction. Using an anti-HA Ab, we demonstrate that the protein is localized in the flagella in epididymal sperm. Request of the 3 reviewers.

▪ Figure 7 (old 8): to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. Moreover, the WB was removed and is now presented in figure 6 (improved as requested).

▪ Figure 8. Was old figure 7

▪ Figure 9: figure 9 was recomposed and improved for increased clarity as suggested by reviewer 2 and 3.

▪ Figure 16 was before appendix 11

Figure supplements and supplementary files

▪ Figure 1-Figure supplement 1 New. Sperm parameters of the 2 patients. requested by editor (remark #1) by the reviewer 1 (Note #3)

▪ Figure 2-Figure supplement 1 new. Sperm parameters of the line 2 (KO animals) requested by the reviewer 1 (Note #5)

▪ Figure 4-Figure supplement 1 New. Experiment to evaluate the specificity of the human CCDC146 antibody. Minimal revision request and reviewer 1 note #8

▪ Figure 6-Figure supplement 1 New. Figure recomposed; Asked by reviewer 2 note #4 and reviewer 3

▪ Figure 8-Figure supplement 1 New. We now provide new images to show the non-specific staining of the midpiece of human sperm by secondary Abs in ExM experiments; Asked by reviewer 2

▪ Figure 10-Figure supplement 1 New. We added new images to show the non-specific staining of the midpiece of mouse sperm by secondary Abs in IF (panel B). Rewiever 1 note #9 and reviewer 2 note #5

▪ Figure 12-Figure supplement 1 New. Control requested by reviewer 3 Note #23

▪ Figure 13-Figure supplement 1 New. We provide a graph and a statistical analysis demonstrating the increase of the length of the manchette in the Ccdc146 KO. Requested by editor and reviewer 3 Note 24

▪ Figure 15-Figure supplement 1 New. Control requested by reviewer 2. Minor comments

▪ Figure supplementary 1 New. Answer to question requested by reviewer 2 note #1

All the reviewers' and editors’ comments have been answered (see our point to point response) and we resubmit what we believe to be a significantly improved manuscript. We strongly hope that we meet all your expectations and that our manuscript will be suitable for publication in "eLife". We look forward to your feedback,

Point by point answer

Please note that there has been active discussion of the manuscript and the summarize points below is the minimal revision request that the reviewers think the authors should address even under this new review model system. It was the reviewers' consensus that the manuscript is prepared with a lot of oversights - please see all the minor points to improve your manuscript.

All minimal revision requests have been addressed

Minimal revision request

1) Clinical report/evaluation of the two patients should be given as it was not described even in their previous study as well as full description of CCDC146.

We provide now a new Figure 1-figure supplement 1 describing the patients sperm parameters

2) Antibody specificity should be provided, especially given two of the reviewers were not convinced that the mid piece signal is non-specific as the authors claim. As both KO and KI model in their hands, this should be straightforward.

To validate the specificity of the Antibody, we transfected HEK cells with a human DDK-tagged CCDC146 plasmid and performed a double immunostaining with a DDK antibody and the CCDC146 antibody. We show that both staining are superimposable, strongly suggesting that the CCDC146 Ab specifically target CCDC146. This experiment is now presented in Figure 4-Figure supplement 1. Next, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

3) The authors should improve statistical analysis to support their experimental results for the reader can make fair assessment. Combined with clear demonstration of ab specificity, this lack of statistical analysis with very few sample number is a major driver of dampening enthusiasm towards the current study.

Several statistical analyses were carried out and are now included:

1) distribution of the HA signal in mouse sperm cells (see point 2 Figure 7 panel B)

2) quantification and statistical analyses of the defect observed in Ccdc146 KO sperm (figure 2 panel E)

3) Quantification and statistical analyses of the length of the manchette in spermatids 13-15 steps (Figure 13-Figure supplement 1 new)

4) The authors need to clarify (peri-centriolar vs. centriole)

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein in somatic cells. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein, and this is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described, and the best example is maybe gamma-tubulin (PMID: 8749391).

or tone down (CCDC146 to be a MIP) of their claim/description.

Concerning its localization in sperm, we agree with the reviewer that our demonstration that CCDC146 is MIP would deserve more results. Because of that, we have toned down the MIP hypothesis throughout the manuscript. See lines 491495

Testis-specific expression of CCDC146 as it is not consistent with their data.

We have also modified our claim concerning the testis-expression of CCDC146. Line 176

Reviewer #1 (Recommendations For The Authors):

Major comments

1) As described in general comments, this study limits how the CCDC146 deficiency impairs abnormal centriole and manchette formation. The authors should explain their relationship in developing germ cells.

In fact, there are limited information about the relationship between the manchette and the centriole. However, few articles have highlighted that both organelles share molecular components. For instance, WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis (Commun Biol. 2021; 4: 645. doi: 10.1038/s42003-021-02171-5). Another study demonstrates that CCDC42 localizes to the manchette, the connecting piece and the tail (Front. Cell Dev. Biol. 2019 https://doi.org/10.3389/fcell.2019.00151). These articles underline that centrosomal proteins are involved in manchette formation and removal during spermiogenesis and support our results showing the impact of CCDC146 lack on centriole and manchette biogenesis. This information is now discussed. See lines 596-603

2) The authors generated knock-in mouse model. If then, are the transgene can rescue the MMAF phenotype in CCDC146-null mice? This reviewer strongly suggest to test this part to clearly support the pathogenicity by CCDC146.

We indeed wrote that we created a “transgenic mice”, which was misleading. We actually created a CCDC16 knock-in expressing a tagged-protein. The strain was actually made by CRISPR-Cas9 and a sequence coding for the HA-tag was inserted just before the first amino acid in exon 2, leading to the translation of an endogenous HA-tagged CCDC146 protein. We have removed the word transgenic from the text and made changes accordingly (see lines 250-253). We can therefore not use this strain to rescue the MMAF phenotype as suggested by the reviewer.

3) Although the authors cite the previous study (Coutton et al., 2019), the study does not describe any information for CCDC146 and clinical information for the patients. The authors must show the results for clinical analysis to clarify the attended patients are MMAF patients without other phenotypic defects.

We have now inserted a table, indicating all sperm parameters for the patients harboring a mutation in the CCDC146 gene (Figure 1-Figure supplement 1) and is now indicated lines 159-160

4) The authors describe CCDC146 expression is dominant in testes, However, the level in testis is only moderate in human (Supp Figure 1). Thus, this description is not suitable.

In Figure 1-figure supplement 2 (old FigS1), the median of expression in testis is around 12 in human, a value considered as high expression by the analysis software from Genevestigator. However, for mouse, it is true that the level of expression is medium. We assumed that reviewer’s comment concerned testis expression in mouse. To take into account this remark, we changed the text accordingly. See line 176.

5) Although the authors mentioned that two mice lines are generated, only one line information is provided. Authors must include information for another line and provide basic characterization results to support the shared phenotype within the lines.

We now provide a revised Figure 2-figure supplement 1CD, presenting the second line and the corresponding text in the main text is found lines 178-183.

6) In somatic cells, the CCDC146 localizes at both peri-centriole and microtubule but its intracellular localization in sperm is distinguished. The authors should explain this discrepancy.

The multi-localization of a centriolar protein is already discussed in detail in discussion lines 520-526. We have written:

“Despite its broad cellular distribution, the association of CCDC146 with tubulin-dependent structures is remarkable. However, centrosomal and axonemal localizations in somatic and germ cells, respectively, have also been reported for CFAP58 [37, 55], thus the re-use of centrosomal proteins in the sperm flagellar axoneme is not unheard of. In addition, 80% of all proteins identified as centrosomal are found in multiple localizations (https://www.proteinatlas.org/humanproteome/subcellular/centrosome). The ability of a protein to home to several locations depending on its cellular environment has been widely described, in particular for MAP. The different localizations are linked to the presence of distinct binding sites on the protein…. “

7) Authors mention CCDC146 is a centriolar protein in the title and results subtitle. However, the description in results part depicts CCDC146 is a peri-centriolar protein, which makes confusion. Do the authors claim CCDC146 is centrosomal protein?

In figure 4A, we have clearly shown that the protein colocalizes with centrin, a centriolar core protein. This colocalization strongly suggests that CCDC146 is therefore a centriolar protein in somatic cells, and is now clearly indicated lines 211-212. However, its localization is not restricted to the centrioles and a clear staining was also observed in the pericentriolar material (PCM). The presence of a protein in PCM and centriole was already described and the best example is maybe gamma-tubulin (PMID: 8749391).

8) Verification of the antibody against CCDC146 must be performed and shown to support the observed signal are correct. 2nd antibody only signal is not proper negative control.

It is a very important remark. The commercial antibody raised against human CCDC146 was validated in HEK293-cells expressing a DDK-tagged CCDC146 protein. Cells were co-marked with anti-DDK and anti-CCDC146 antibodies. We have a perfect colocalization of the staining. This experiment is now presented in Figure 4-figure supplement 1 and presented in the text (lines 206-208).

9) In human sperm, conventional immunostaining reveals CCDC146 is detected from acrosome head and midpiece. However, in ExM, the signal at acrosome is not detected. How is this discrepancy explained? The major concern for the ExM could be physical (dimension) and biochemical (properties) distortion of the sample. Without clear positive and negative control, current conclusion is not clearly understood. Furthermore, it is unclear why the authors conclude the midpiece signal is non-specific. The authors must provide experimental evidence.

Staining on acrosome should always be taken with caution in sperm. Indeed, numerous glycosylated proteins are present at the surface of the plasma membrane regarding the outer acrosomal membrane for sperm attachment and are responsible for numerous nonspecific staining. Moreover, this acrosomal staining was not observed in mouse sperm, strongly suggesting that it is not specific.

Concerning the staining in the midpiece observed in both conventional and Expansion microscopy, it also seems to be nonspecific and associated with secondary Abs.

For IF, we now provide new images showing clearly the nonspecific staining of the midpiece when secondary Ab were used alone (see Figure 10-figure supplement 1B).

For ExM, we provide new images in Figure 8-figure supplement 1B (POC5 staining) showing a staining of the midpiece (likely mitochondria), although POC5 was never described to be present in the midpiece. Both experiments (CCDC146 and POC5 staining by ExM) shared the same secondary Ab and the midpiece signal was likely due to it.

Moreover, we now provide new images (figure 7C) in ExM on mouse sperm showing no staining in the midpiece and demonstrating that the punctuated signal is present all along the flagellum. Finally, we would like to underline that we now provide new IF results, using an anti-HA conjugated with alexafluor 488 and confirming the ExM results.

These points are now discussed lines 498-502 for acrosome and lines 503-511 for midpiece staining.

10) For intracellular localization of the CCDC146 in mouse sperm, the authors should provide clear negative control using WT sperm which do not carry the transgene.

This experiment was performed.

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum.

11) Current imaging data do not clearly support the intracellular localization of the CCDC146. Although western blot imaging reveal that CCDC146 is detected from sperm flagella, this is crude approach. Thus, this reviewer highly recommends the authors provide more clear experimental evidence, such as immuno EM.

We provide now a WB comparing the presence of the protein in the flagellum and in the head fractions; see new figure 6. We show that CCDC146 is only present in the flagellum fraction; The detection of the band appeared very quickly at visualization and became very strong after few minutes, demonstrating that the protein is abundant in the flagella. It is important to note that epididymal sperm do not have centrioles and therefore this signal is not a centriolar signal. We also now provide new statistical analyses showing that the immuno-staining observed in the principal piece is very specific (Figure 7B). Altogether, these results demonstrate unequivocally the intracellular localization of CCDC146 in the flagellum. This point is now discussed lines 480-489

12) Although sarkosyl is known to dissociate tubulin, it is not well understood and accepted that the enhanced detection of CCDC146 by the detergent indicates its microtubule inner space. Sperm axoneme to carry microtubule is also wrapped peri-axonemal components with structural proteins, which are even not well solubilized by high concentration of the ionic detergent like SDS.

We agree with the reviewer that the solubilization of the protein by sarkozyl is not a proof of the presence of the protein inside microtubule. Taking into account this point, the MIP hypothesis was toned down and we now discuss alternative hypothesis concerning these results; See discussion lines 490-497

13) SEM image is not suitable to explain internal structure (line 317-323).

We agree with the reviewers and changes were made accordingly. See lines 354-357

Minor comments

1) In main text, supplementary figures are cited "Supp Figure". And the corresponding legends are written in "Appendix - Figure". Please unify them.

Done Labelled now “Figure X-figure supplement Y”

2) Line 159, "exon 9/19" is not clear.

We have written now exons 9 and indicated earlier that the gene contains 19 exons

3) Line 188, "positive cells" are vague.

Positive was changed by “fluorescent”

4) Representative TUNEL assay image for knockout testes were not shown in Supp Figure 3B.

It was a mistake now Figure 2-figure supplement 2C

5) Please provide full description for "IF" and "AB" when described first.

Done

6) Line 262, It is unclear what is "main piece".

Changed to principal piece

7) Line 340, Although the "stage" information might be applicable, this is information for "seminiferous tubule" rather than "spermatid". This reviewer suggests to provide step information rather than stage information.

We agree with the reviewer that there was a confusion between “stage” and “step”. We change to step spermatids

8) Line 342, Step 1 is not correct in here.

OK corrected. now steps 13-15 spermatids

9) Line 803, "C." is duplicated.

Removed

10) Figure 3A, it will be good to mark the defective nuclei which are described in figure legends.

These cells are now indicated by white arrow heads

11) Figure 5, Please provide what MT stands for.

Now explained in the legend of figure 5

12) Figure 6. Author requires clear blot images for C. In addition, Panel B information is not correct. If the blot was performed using HA antibody, then how "WT" lane shows bands rather than "HA" bands?

The reviewer is correct. It was a mistake; The figure was recomposed and improved.

Reviewer #2 (Recommendations For The Authors):

Overall, editing oversights are present throughout the manuscript, which has made the review process quite difficult. Some repetitive figures can be removed to streamline to grasp the overall story easier. Some claims are not fully supported by evidence that need to tone down. Some figures not referenced in the main text need to be mentioned at least once.

All figures are now referenced in the text

Major comments:

1) 163-164 - Please clarify the claim that there is going to be an absence of the protein or nonfunctional protein, especially for the patient with a deletion that could generate a truncated protein at two third size of the full-length protein. Similarly, 35% of the protein level is present for the patient with a nonsense mutation. Some in silico structural analysis or analysis of conserved domains would be beneficial to support these claims.

Both mutations are predicted to produce a premature stop codons: p.Arg362Ter and p.Arg704serfsTer7, leading either to the complete absence of the protein in case of non-sense mediated mRNA decay or to the production of a truncated protein missing almost two third or one fourth of the protein respectively. CCDC146 is very well conserved throughout evolution (Figure supplementary 1), including the 3’ end of the protein which contains a large coil-coil domain (Figure 1B). In view of the very high degree of conservation, it is most likely that the 3’ end of the protein, absent in both subjects, is critical for the CCDC146 function and hence that both mutations are deleterious. This explanation is now added to the discussion. see lines 439-448

2) 173, 423 - Please clearly state a rationale of your mouse model design (i.e., why a mouse model that recapitulate human mutation is not generated) as the truncations identified in human patients are located further towards the C-terminus, and it is not clear whether truncated proteins are present, and if so, they could still be functional. Basically, the current mouse model supports the causality of the human mutations.

This is an important question, which goes beyond the scope of this article, and raises the question of how to confirm the pathogenicity of mutations identified by high-throughput sequencing. The production of KO or KI animals is an important tool to help confirm one’ suspicions but the first element to take into consideration is the nature of the genetic data.

Here we had two patients with homozygous truncating variants. In human, it is well established that the presence of premature stop codons usually induces non-sense mediated mRNA decay (NMD), inducing the complete absence of the protein or a strong reduction in protein production. In the unlikely absence of NMD in our two patients, the identified variants would induce the production of proteins missing 60% and 30% of their C terminal part. Often (and it is particularly true for structural proteins) the production of abnormal proteins is more deleterious than the complete absence of the protein (and it is most likely the purpose of NMD, to limit the production of abnormal “toxic” proteins). For these reasons, to try to recapitulate the most likely consequences of the human variants, without risking obtaining an even more severe effect, we decided to introduce a stop codon in the first exon in order to remove the totality of the protein in the KO mice.

The second element is to interpret the phenotype of the KO animals. Here, the human sperm phenotype is perfectly recapitulated in the KO mice.

Overall, we have strong genetic arguments in human and the reproduction of the phenotype in KO mice confirming the pathogenicity of the variants identified in men.

This point is now discussed see lines 433-438

3) Figure 6A - the labelling is misleading as it seems to suggest that the specific cells were isolated from the testes for RT-PCR.

We have modified the labelling to avoid any confusion.

Figure 6B -Signal of HA-tag is shown in WT, not in transgenic. Please check the order of the labels. Figure 6C - This blot is NOT a publication-quality figure. The bands are very difficult to observe, especially in lane D18. Because it is one of the important data of this study, replacing this figure is a must.

The figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed.

4) Supplementary fig 6 is also not a publication-level figure, and the top part seems largely unnecessary (already in the figure legend).

The figure has been completely remade as well (now Figure 6-Figure Supplement 1).

5) 261/267- The conclusion that mitochondrial staining in the flagellum (in both mice and humans) is non-specific is not convincing. Supplementary fig 8 shows that the signal from secondary only IF possibly extends beyond the midpiece - but it is hard to determine as no mitochondrial-specific staining is present. Either need to tone down the conclusion or provide supporting experimental evidence.

First, to avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the flagellum. These experiments are now described lines 271-279

Second, we provide new images of the signal obtained with secondary Abs only that shows more clearly that the secondary Ab gave a non-specific staining (Figure 10-Figure supplement 1B). This point is discussed lines 503-511

6) Figure 9 A - Please relate the white line to Fig. 9B label in X-axis. The information from Fig 9A+D and 9E+F are redundant. The main text nor the figure legends indicate why these specific two sperm were chosen for quantification and demonstrating the outcomes. One of them could be moved to supplementary information or removed, or the two could be combined.

As suggested by the reviewer, we have combined the two sperm to demonstrate that CCDC146 staining is mostly located on microtubule doublets. Moreover, the figure was recomposed to make it clearer.

Minor comments:

All of the supplementary figures are referred to as Supp Fig X in the text, however, they are actually titled Appendix - Figure X. This needs to be consistent.

The figures are now referred as figure supplement x in both text and figures

Line 125 - edit spacing.

We think this issue (long internet link) will be curated later and more efficiently by the journal, during the step of formatting necessary for publication.

144 - With which to study  with which we studied?

We made the change as suggested.

151 - Supp Fig 1 - the text says that the gene is highly transcribed in human and mouse testes, but the information in the figure states that the level in mouse tissues is "medium"

We have corrected this mistake in the text; See line 176

165 - The two mutations are most likely deleterious. Please specifically mention what analyses done to predict the deleterious nature to support these claims.

Both variants, c.1084C>T and c.2112del, are extremely rare in the general population with a reported allele frequency of 6.5x10-5 and 6.5x10-06 respectively in gnomAD v3. Moreover, these variants are annotated with a high impact on the protein structure (MoBiDiC prioritization algorithm (MPA) score = 10, DOI: 10.1016/j.jmoldx.2018.03.009) and predicted to induce each a premature termination codon, p.(Arg362Ter) and p.(Arg704SerfsTer7) respectively, leading to the production of a truncated protein. This information is now given line 164-169

196-200/Figure 4 - As serum starved cells/basal body (B) are not mentioned in the main text, as is, Fig 4A would be sufficient/is relevant to the text. Please make the text reflect the contents of the whole figure, or re/move to supplement.

We agree with the reviewer that the full description of the figure should be in the text. We added two sentences to describe figure 4B see lines 217-218.

224 - spermatozoa (plural) fits better here, not spermatozoon

OK changed accordingly

236 - According to the figure legend, 6B is only showing data from the epididymal sperm, not postnatal time points; should be referencing 6C. Alignment of Marker label

As indicated above, the figure has been completely remade, including new results. See new figure 6. Figure 6C was suppressed. The corresponding text was changed accordingly see lines 249-266

255-256 - Referenced figure 7B3, however, 7B3 only shows tubulin staining, so no CCDC146 can be observed. Did authors mean to reference fig 7B as a whole?

Sorry for this mistake. We agree and the text is now figure 8B6 (figure 7 and 8 were switched)

305 - "of tubules" - I presume it is meant to be microtubules?

Yes; The text was changed as suggested

317-321 - a diagram of HTCA would be useful here

We have added a reference where HTCA diagram is available see line 363. Moreover, a TEM view of HTCA is presented figure 12A

322/Fig 11A - an arrow denoting the damage might be useful, as A1 and A3 look similar. The size of the marker bar is missing. Please update the information on figure legend.

Concerning, the comparison between A1 and A3, the take home message is that there is a great variability in the morphological damages. This point is now underlined in the corresponding text. We updated the size of the marker bar as suggested (200 nm). See line 365-367

323 - Please mark where capitulum is in the figure

Capitulum was changed for nucleus

Since Fig 11B2 is not referenced in the main text, it does not seem to add anything to the data, and could be removed/moved to supplement.

We added a sentence to describe figure 11B2 line 370

342-343 - manchette in step I is not seen clearly - the figure needs to be annotated better. However, DPY19L2 is absent in step I in the KO, but the main text does not reflect that - why is that?

We do not understand the remark of the reviewer “manchette in step I is not seen clearly”. The figure shows clearly the manchette (red signal) in both WT and KO (Figure 13 D1/D2).

For steps 13-15 WT spermatids, the size of the manchette decreases and become undetectable. In KO spermatids, the shrinkage of the manchette is hampered and in contrast continue to expand (Figure 13D2). We also provide a new Figure 13-figure supplement 1 for other illustrations of very long manchettes and a statistical analysis. In the meantime, the acrosome is strongly remodeled, as shown in figure 16-new, with detached acrosome (panel H). This morphological defect may induce a loss of the DPY19L2 staining (Figure 13 D2 stage I-III). This explanation is now inserted in the text line 396399

Figure 15B and 15C only show KO, corresponding images from the WT should be present for comparison.

WT images are now provided in Figure 1-figure supplement 1 new

Figure 12 - Figure 12 - JM?.

JM was removed. It does not mean anything

Figure 12C and Supplementary Fig 10 - structures need to be labelled, as it is unclear what is where

Done

338 - text mentions step III, but only sperm from step VII are shown in Figure 13

As suggested by reviewer 3, we changed stage by step. The text was modified to take into account this remark see lines 388-396

360 - This is likely supposed to say Supp Figure 11E-G, not 13??

Yes, it is a mistake. Corrected

388 Typo "in a in a".

Yes, it is a mistake. Corrected

820 - Fig 3 legend - in KO spermatid nuclei were elongated - could this be labelled by arrows? I am not convinced this phenotype is that different from the WT.

In fact, the nuclei of elongating KO spermatids are elongated and also very thin, a shape not observed in the WT; We have added arrow heads and modified the text to indicate this point line 200.

836 - Figure 5 legend says that in yellow is centrin, but that is not true for 5A, where the figure shows labelling for y-tubulin (presumably, according to the figure itself).

We have modified the text of the legend to take into account the remark

837- 5A supposedly corresponds to synchronized HEK293T cells, but the reasoning behind using synchronized cells is not mentioned at all in the main text; furthermore, how this synchronization is achieved is not explained in materials and methods (serum starvation? Thymidine block?).

Yes, figure 5A was obtained with synchronized cells. We have added one paragraph in the MM section. For cell synchronization experiments, cells underwent S-phase blockade with thymidine (5 mM, SigmaAldrich) for 17 h followed by incubation in a control culture medium for 5 h, then a second blockade at the G2-M transition with nocodazole (200 nM, Sigma-Aldrich) for 12 h. Cells were then fixed with cold methanol at different times for IF labelling. See line 224 for changes made in the result section and lines 700-704 for changes made in the MM section.

845- figure legend says that the RT-PCR was done on CCDC146-HA tagged mice, but the main text does not reflect that.

We made changes and the description of the KI is now presented before (line 240) the RT-PCR experiment (line 257).

949 - it is likely supposed to say A2, not B1 (B1 does not exist in Fig 15)

Yes, it is a mistake. Corrected

971 - Appendix Fig 3 legend - I believe that the description for B and C are swapped.

Yes, it is a mistake. Corrected

Furthermore, some questions to address in A would be: Which cross sections were from which animal/points? How many per animal? Were they always in the same location?

Yes, we have a protocol for arranging and orienting all testes in the same way during the paraffin embedding phase. The cross-sections are therefore not taken at random, and we can compare sections from the same part of the testis. The number of animals was already indicated in the figure legend (see line 1128)

Reviewer #3 (Recommendations For The Authors):

1) There are a number of grammatical and orthographical errors in the text. Careful proofreading should be performed.

We have sent the manuscript to a professional proofreader

2) The author should also check for redundancies between the introduction and the discussion.

The discussion has modified to take into account reviewers’ remarks. Nevertheless, we did our best to avoid redundancies between introduction and discussion.

3) Can the authors provide a rationale why they have chosen to tag their gene with an HA tag for localisation? One would rather think of fluorescent proteins or a Halo tag.

Because the functional domains of the protein are unknown, adding a fluorescent protein of 24 KDa may interfere with both the localization and the function of CCDC146. For this reason, we choose a small tag of only 1.1 KDa, to limit as such as possible the risk of interfering with the structure of the protein. This rational is now indicated in the manuscript lines 251-254. It is worth to note, that the tagged-strain shows no sperm defect, demonstrating that the HA-tag does not interfere with CCDC146 function.

4) In the abstract, line 53, "provide evidence" is not the right term for something that is just suggestive. The term "suggests" would be more appropriate.

The text was modified to take into account this remark

5) Line 74: "genetic deficiency" sounds strange here, do the authors mean simply "mutation"?

Infertility may be due to several genetic deficiency such as chromosomal defects (XXY (Klinefelter syndrome)), microdeletion of the Y chromosome or mutations in a single gene. Therefore, mutation is too restrictive. Nevertheless, we modified the sentence which is now “…or a genetic disorder including chromosomal or single gene deficiencies”

6) Lines 163-164: the authors describe the mutations (premature stop mutations) and say that they could either lead to complete absence of the gene product, or the expression of a truncated protein. Did they test this, for example, with some immuno blot analyses?

As stated above, unfortunately, we were unable to verify the presence of RNA-decay in these patients for lack of biological material.

7) Line 184 and Fig 2E: the sperm head morphologies should be quantitatively assessed.

We provide now a full statistical analysis of the observed defects: see new panel in Figure 2 F

8) Fig 3: The annotation should be more precise - KO certainly means CDCC146-KO. The colours of the IH panels is different, which attracts attention but is clearly a colour-adjustment artefact. Colours should be adjusted for the panels to look comparable. It would be also helpful to add arrowheads into the figure to point at the phenotypes that are highlighted in the text.

We have added Ccdc146 KO in all figures. We have added arrow heads to point out the spermatids showing a thin and elongated nucleus. Concerning adjustment of colors, we attempted to make images of panel B comparable. See new figure 3.

9) Fig 6A: the authors use RT PCR to determine expression dynamics of their gene of interested, and use actin (apparently) as control. However, actin and CDCC146 expression levels follow the same trend. How is the interpreted?

The reviewer did not understand the figure. The orange bars do not correspond to actin expression and the grey bars to Ccdc146 expression but both bars represent the mRNA expression levels of Ccdc146 relative to Actb (orange) and Hprt (grey) expression in CCDC146-HA mouse pups’ testes. We tested two housekeeping genes as reference to be sure that our results were not distorted by an unstable expression of a housekeeping gene. We did not see significant difference between both house keeping genes. Actin was not used.

10) In line 235, the authors suggest posttranslational modifications of their protein as potential cause for a slightly different migration in SDS PAGE as predicted from the theoretical molecular weight. This is not necessarily the case, some proteins do migrate just differently as predicted.

We have changed the text accordingly and now provide alternative explanation for the slightly different migration. See lines 258-259

11) The annotation of Fig 6 panels is problematic. First, why do the authors write "Laemmli" as description of the gel? It would be more helpful to write what is loaded on the gel, such as "sperm". Second, in panels B and C it would be helpful to add the antibodies used. It is not clear why there is a signal in the WT lane of panel B, but not in the HA lane (supposing an anti-HA antibody is used: why has WT a specific HA band?). In panel C, it is not clear why the blot that has so beautifully shown a single band in panel B suddenly gives such a bad labelling. Can the authors explain this? Also, they cut off the blot, likely because to too much background, but this is bad practice as full blots should be shown. In the current state, the panel C does not allow any clear conclusion. To make it conclusive, it must be repeated.

Several mistakes were present in this figure. This figure was recomposed. The WB on testicular extract was suppressed and we now present a new WB allowing to compare the presence of CCDC146 in the flagella and head fractions from WT and HA-CCDC146 sperm. Using an anti-HA Ab, we demonstrate that in epididymal sperm the protein is localized in the flagella only. See new figure 6. The corresponding text was changed accordingly.

12) The authors have raised an HA-knockin mouse for CDCC146, which they explained by the unavailability of specific antibodies. However, in Fig 7, they use a CDCC146 antibody. Can they clarify?

The commercial Ab work for HUMAN CCDC146 but not for MOUSE CCDC146. We have added few words to make the situation clearer, we have added the following information “the commercial Ab works for human CCDC146 only”. See line 240

13) In Fig 7A (line 258), the authors hypothesise that they stain mitochondria - why not test this directly by co-staining with mitochondria markers?

We chose another solution to resolve this question:

To avoid the issue of the non-specificity of secondary antibodies, we performed a new set of IF experiments using an HA Tag Alexa Fluor® 488-conjugated Antibody (anti-HA-AF488-C Ab) on WT and HA-CCDC146 sperm. These results are now presented in figure 7 panel A (new). The specificity of the signal obtained with the anti-HA-AF488-C Ab on mouse spermatozoa was evaluated by performing a statistical study of the density of dots in the principal piece of the flagellum from HA-CCDC146 and WT sperm. These results are now presented in figure 7 panel B (new). This study was carried out by analyzing 58 WT spermatozoa and 65 CCDC146 spermatozoa coming from 3 WT and 3 KI males. We found a highly significant difference, with a p-value <0.0001, showing that the signal obtained on spermatozoa expressing the tagged protein is highly specific. We have added a paragraph in the MM section to describe the process of image analysis. We finally present new images obtained by ExM showing no staining in the midpiece (figure 7C new). Altogether, these results demonstrate unequivocally the presence of the protein in the whole flagellum.

14) It seems that in both, Fig 7 and 8, the authors use expansion microscopy to localise CDCC146 in sperm tails. However, the staining differs substantially between the two figures. How is this explained?

In figure 8 we used the commercial Ab in human sperm, whereas in figure 7 we used the anti-HA Abs in mouse sperm. Because the antibodies do not target the same part of the CCDC146 protein (the tag is placed at the N-terminus of the protein, and the HPA020082 Ab targets the last 130 amino acids of the Cter), their accessibility to the antigenic site could be different. However, it is important to note that both antibodies target the flagellum. This explanation is now inserted see lines 304-312

15) Fig 8D and line 274: the authors do a fractionation, but only show the flagella fraction. Why?

Showing all fractions of their experiment would have underpinned the specific enrichment of CDCC146 in the flagella fraction, which is what they aim to show. Actually, given the absence of control proteins, the fact that the band in the flagellar fraction appears to be weaker than in total sperm, one could even conclude that there is more CDCC146 in another (not analysed) fraction of this experiment. Thus, the experiment as it stands is incomplete and does not, as the authors claim, confirm the flagellar localisation of the protein.

We agree with the reviewer’s remark. We provide now new results showing both flagella and nuclei fractions in new figure 6A. This experiment is presented lines 253-256

16) Line 283, Fig 9D,F: The description of the microtubules in this experiment is not easy to understand. Do the authors mean to say that the labelling shows that the protein is associated with doublet microtubules, but not with the two central microtubules? They should try to find a clearer way to explain their result.

As suggested by reviewer 2, we have changed the figure to make it clearer. The text was changed accordingly. See new figure 9 and new corresponding legend lines 1006.

17) Fig 9G - how often could the authors observe this? Why is the axoneme frayed? Does this happen randomly, or did the authors apply a specific treatment?

Yes, it happens randomly during the fixation process.

18) Line 300 and Fig 10A - the authors talk about the 90-kDa band, but do say anything about what they think this band is representing.

We have now added the following sentence lines 340-342: “This band may correspond to proteolytic fragment of CCDC146, the solubilization of microtubules by sarkosyl may have made CCDC146 more accessible to endogenous proteases.”

19) Fig 11A, lines 321-322: the authors write that the connecting piece is severely damaged. This is not obvious for somebody who does not work in sperm. Perhaps the authors could add some arrow heads to point out the defects, and briefly describe them in the text.

We realized from your remark that our message was not clear. In fact, there is a great variability in the morphological damages of the HTCA. For instance, the HTCA of Ccdc146 KO sperm presented in figure 10A2 is quite normal, whereas that in figure 10A4 is completely distorted. This point is now underlined in the corresponding text. See lines 367-369

We also added the size of the marker bar (200 nm), which were missing in the figure’s legend.

20) Line 323: it will be important to name which tubulin antibody has been used to identify centrioles, as they are heavily posttranslationally modified.

The different types of anti-tubulin Abs are described in the corresponding figure’s legend

21) Fig 11B - phenotypes must be quantified to make these observations meaningful.

We agree that a quantification would improve the message. However, testicular sperm are obtained by enzymatic separation of spermatogenic cells and the number of testicular sperm are very low. Moreover, not all sperm are stained. Taking these two points into account, it seems to us that quantification could be difficult to analyze. For this reason, the quantification was not done; however, it is important to note that these defects were not observed in WT sperm, demonstrating that these defects are cased by the lack of CCDC146. We have added a sentence to underline this point; See lines 374-375

22) Line 329: Figure 12AB - is this a typo - should it read Figure 12B?

We have split the panel A in A1 and A2 and changed the text accordingly. See line 378

23) Why are there not wildtype controls in Fig 12B, C?

We provide now as Figure 12-figure supplement 1, a control image for fig 12B. For figure 12C, the emergence of the flagellum from the distal centriole in WT is already shown in Fig 12A1

24) Fig 13: the authors write that the manchette is "clearly longer and wider than in WT cells" (lines 342-343). How can they claim this without quantitative data?

We now provide a statistical analysis of the length of the manchette. See figure 13-figure supplement 1A. We also provide a new a new image illustrating the length of the manchette in Ccdc146 KO spermatids; See Figure 13-figure supplement 1B.

Reviewer #3 (Public Review):

Male infertility is an important health problem. Among pathologies with multiple morphological abnormalities of the flagellum (MMAF), only 50% of the patients have no identified genetic causes. It is thus primordial to find novel genes that cause the MMAF syndrome. In the current work, the authors follow up the identification of two patients with MMAF carrying a mutation in the CCDC146 gene. To understand how mutations in CCDC146 lead to male infertility, the authors generated two mouse models: a CCDC146-knockout mouse, and a knockin mouse in which the CCDC146 locus is tagged with an HA tag. Male CCDC146-knockout mice are infertile, which proves the causative role of this gene in the observed MMAF cases. Strikingly, animals develop no other obvious pathologies, thus underpinning the specific role of CCDC146 in male fertility. The authors have carefully characterised the subcellular roles of CCDC146 by using a combination of expansion and electron microscopy. They demonstrate that all microtubule-based organelles, such as the sperm manchette, the centrioles, as well as the sperm axonemes are defective when CCDC146 is absent. Their data show that CCDC146 is a microtubule-associated protein, and indicate, but do not prove beyond any doubt, that it could be a microtubule-inner protein (MIP).

This is a solid work that defines CCDC146 as a novel cause of male infertility. The authors have performed comprehensive phenotypic analysis to define the defects in CCDC146 knockout mice. The manuscript text is well written and easy to follow also for non-specialists. The introduction and discussion chapters contain important background information that allow to put the current work into the greater context of fertility research. Overall, this manuscript provides convincing evidence for CCDC146 being essential for male fertility and illustrates this with a large panel of phenotypic observations. Together, the work provides important first insights into the role of a so-far unexplored proteins, CCDC146, in spermatogenesis, thereby broadening the spectrum of genes involved in male infertility.

Reviewer #2 (Public Review):

Summary:

The manuscript by Kaneda et al "FBXO24 ensures male fertility by preventing abnormal accumulation 2 of membraneless granules in sperm flagella" is a significant paper on the role of FBXO24 in murine male germ cell development and sperm ultrastructure and function. The body of experimental evidence that the authors present is extraordinarily strong in both breadth and depth. The authors investigate the protein's functions in male germ cells and sperm using a wide variety of approaches but focusing predominantly on their novel mouse model featuring deletion of the Fbxo24 gene and its product. Using this mouse, and a cross of it with another model that expresses reporters in the head and midpiece, they logically build from one experiment to the next. Together, their data show that this protein is involved in the regulation of membraneless electron-dense structures; loss of FBXO24 led to an accumulation of these materials and defects in the sperm flagellum and fertilizing ability. Interestingly, the authors found that several of the best-known components of electron-dense ribonucleoprotein granules that are found in the intermitochondrial cement and chromatoid body were not disrupted in the Fbxo24 knockout, suggesting that the electron-dense material and these structures are not all the same, and the biology is more complicated than some might have thought. They found evidence for the most changes in IPO5 and KPNB1, and biochemical evidence that FBXO24 and IPO5 could interact.

Strengths:

The authors are to be commended for the thoroughness of their experimental approaches and the extent to which they investigated impacts on sperm function and potential biochemical mechanisms. Very briefly, they start by showing that the Fbxo24 message is present in spermatids and that the protein can interact with SKP1, in a way that is dependent on its F-box domain. This points toward a potential function in protein degradation. To test this, they next made the knockout mouse, validated it, and found the males to be sterile, although capable of plugging a female. Looking at the sperm, they identified a number of ultrastructural and morphological abnormalities, which they looked at in high resolution using TEM. They also cross their model with RBGS mice so that they have reporters in both the acrosome and mitochondria. The authors test a variety of sperm functions, including motility parameters, ability to fertilize by IVF, cumulus-free IVF, zona-free-IVF, and ICSI. They found that ICSI could rescue the knockout but not other assisted reproductive technologies. Defects in male fertility likely resulted from motility disruption and failure to get through the utero-tubal junction but defects in acrosome exocytosis also were noted. The authors performed thorough investigations including both targeted and unbiased approaches such as mass spectrometry. These enabled them to show that although the loss of the FBXO24 protein led to more RNA and elevated levels of some proteins, it did not change others that were previously identified in the electron-dense RNP material.

The manuscript will be highly significant in the field because the exact functions of the electron-dense RNP materials have remained somewhat elusive for decades. Much progress has been made in the past 15 years but this work shows that the situation is more complex than previously recognized. The results show critical impacts of protein degradation in the differentiation process that enables sperm to change from non-descript round cells into highly polarized and compartmentalized mature sperm, with an equally highly compartmentalized flagellum. This manuscript also sets a high bar for the field in terms of how thorough it is, which reveals wide-ranging impacts on processes such as mitochondrial compaction and arrangement in the midpiece, the correct building of the major cytoskeletal elements in the flagellum, etc.

Weaknesses:

There are no real weaknesses in the manuscript that result from anything in the control of the authors. They attempted to rescue the knockout by expressing a FLAG-tagged Fbxo24 transgene, but that did not rescue the phenotype, either because of inappropriate levels/timing/location of expression, or because of interference by the tag. They also could not make anti-FBXO24 that worked for co-immunoprecipitation experiments, so relied on the FLAG epitope, an approach that successfully showed co-IP with IPO5 and SKP1.

Certainly! The way it works is when you create a new note and associate it with a user, you will typically provide the user's unique identifier (ID) as the value for the user field. In a typical scenario, this user ID would come from the "user" collection in your database.

Here's a brief example in code:

```javascript // Assume you have a User model and you have obtained a user's ID const userId = "someUserId"; // This would be the actual ID of the user in your database

// Creating a new note and associating it with the user by providing the user ID const newNote = new Notes({ user: userId, title: "Sample Note", description: "This is a sample note.", tag: "General", deadline: new Date(), deadlinetime: "12:00 PM", });

// Save the new note to the database newNote.save() .then((savedNote) => { console.log("Note saved successfully:", savedNote); }) .catch((error) => { console.error("Error saving note:", error); }); ```

In this example, the user field of the new note is set to the userId obtained from the "user" collection. When you save this note to the database, the user field will store the provided user ID. Later, when you retrieve this note, you can use this user ID to identify which user the note is associated with.

It's important to ensure that the user ID you provide for the user field actually corresponds to an existing user in the "user" collection. This way, you maintain the relationship between the "notes" and "user" collections in your MongoDB database.

Author Response

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Koumoundourou et al., identify a pathway downstream of Bcl11b that controls synapse morphology and plasticity of hippocampal mossy fiber synapses. Using an elegant combination of in vivo, ex vivo, and in vitro approaches, the authors build on their previous work that indicated C1ql2 as a functional target of Bcl11b (De Bruyckere et al., 2018). Here, they examine the functional implications of C1ql2 at MF synapses in Bcl11b cKO mice and following C1ql2 shRNA. The authors find that Bcl11b KO and shRNA against C1ql2 significantly reduces the recruitment of synaptic vesicles and impairs LTP at MF synapses. Importantly, the authors test a role for the previously identified C1ql2 binding partner, exon 25b-containing Nrxn3 (Matsuda et al., 2016), as relevant at MF synapses to maintain synaptic vesicle recruitment. To test this, the authors developed a K262E C1ql2 mutant that disrupts binding to Nrxn3. Curiously, while Bcl11b KO and C1ql2 KD largely phenocopy (reduced vesicle recruitment and impaired LTP), only vesicle recruitment is dependent on C1ql2-Nrxn3 interactions. These findings provide new insight into the functional role of C1ql2 at MF synapses. While the authors convincingly demonstrate a role for C1ql2-Nrxn3(25b+) interaction for vesicle recruitment and a Nrxn3(25b+)independent role for C1ql2 in LTP, the underlying mechanisms remain inconclusive. Additionally, a discussion of how these findings relate to previous work on C1ql2 at mossy fiber synapses and how the findings contribute to the biology of Nrxn3 would increase the interpretability of this work.

As suggested by reviewer #1, we extended our discussion of previous work on C1ql2 and additionally discussed the biology of Nrxn3 and how our work relates to it. Moreover, we extended our mechanistic analysis of how Bcl11b/C1ql2/Nrxn3 pathway controls synaptic vesicle recruitment as well as LTP (please see also response to reviewer #2 points 5 and 8 and reviewer #3 point 4 of public reviews below for detailed discussion).

Reviewer #2 (Public Review):

This manuscript describes experiments that further investigate the actions of the transcription factor Bcl11b in regulating mossy fiber (MF) synapses in the hippocampus. Prior work from the same group had demonstrated that loss of Bcl11b results in loss of MF synapses as well as a decrease in LTP. Here the authors focus on a target of Bcl11b a secreted synaptic organizer C1ql2 which is almost completely lost in Bcl11b KO. Viral reintroduction of C1ql2 rescues the synaptic phenotypes, whereas direct KD of C1ql2 recapitulates the Bcl1 phenotype. C1ql2 itself interacts directly with Nrxn3 and replacement with a binding deficient mutant C1q was not able to rescue the Bcl11b KO phenotype. Overall there are some interesting observations in the study, however there are also some concerns about the measures and interpretation of data.

The authors state that they used a differential transcriptomic analysis to screen for candidate targets of Bcl11b, yet they do not present any details of this screen. This should be included and at the very least a table of all DE genes included. It is likely that many other genes are also regulated by Bcl11b so it would be important to the reader to see the rationale for focusing attention on C1ql2 in this study.

The transcriptome analysis mentioned in our manuscript was published in detail in our previous study (De Bruyckere et al., 2018), including chromatin-immunoprecipitation that revealed C1ql2 as a direct transcriptional target of Bcl11b. Upon revision of the manuscript, we made sure that this was clearly stated within the main text module to avoid future confusion. In the same publication (De Bruyckere et al., 2018), we discuss in detail several identified candidate genes such as Sema5b, Ptgs2, Pdyn and Penk as putative effectors of Bcl11b in the structural and functional integrity of MFS. C1ql2 has been previously demonstrated to be almost exclusively expressed in DG neurons and localized to the MFS.

There it bridges the pre- and post-synaptic sides through interaction with Nrxn3 and KAR subunits, respectively, and regulates synaptic function (Matsuda et al., 2016). Taken together, C1ql2 was a very good candidate to study as a potential effector downstream of Bcl11b in the maintenance of MFS structure and function. However, as our data reveal, not all Bcl11b mutant phenotypes were rescued by C1ql2 (see supplementary figures 2d-f of revised manuscript). We expect additional candidate genes, identified in our transcriptomic screen, to act downstream of Bcl11b in the control of MFS.

All viral-mediated expression uses AAVs which are known to ablate neurogenesis in the DG (Johnston DOI: 10.7554/eLife.59291) through the ITR regions and leads to hyperexcitability of the dentate. While it is not clear how this would impact the measurements the authors make in MF-CA3 synapses, this should be acknowledged as a potential caveat in this study.

We agree with reviewer #2 and are aware that it has been demonstrated that AAV-mediated gene expression ablates neurogenesis in the DG. To avoid potential interference of the AAVs with the interpretability of our phenotypes, we made sure during the design of the study that all of our control groups were treated in the same way as our groups of interest, and were, thus, injected with control AAVs. Moreover, the observed phenotypes were first described in Bcl11b mutants that were not injected with AVVs (De Bruyckere et al., 2018). Finally, we thoroughly examined the individual components of the proposed mechanism (rescue of C1ql2 expression, over-expression of C1ql3 and introduction of mutant C1ql2 in Bcl11b cKOs, KD of C1ql2 in WT mice, and Nrxn123 cKO) and reached similar conclusions. Together, this strongly supports that the observed phenotypes occur as a result of the physiological function of the proteins involved in the described mechanism and not due to interference of the AAVs with these biological processes. We have now addressed this point in the main text module of the revised ms.

The authors claim that the viral re-introduction "restored C1ql2 protein expression to control levels. This is misleading given that the mean of the data is 2.5x the control (Figure 1d and also see Figure 6c). The low n and large variance are a problem for these data. Moreover, they are marked ns but the authors should report p values for these. At the least, this likely large overexpression and variability should be acknowledged. In addition, the use of clipped bands on Western blots should be avoided. Please show the complete protein gel in primary figures of supplemental information.

We agree with reviewer #2 that C1ql2 expression after its re-introduction in Bcl11b cKO mice was higher compared to controls and that this should be taken into consideration for proper interpretation of the data. To address this, based also on the suggestion of reviewer #3 point 1 below, we overexpressed C1ql2 in DG neurons of control animals. We found no changes in synaptic vesicle organization upon C1ql2 over-expression compared to controls. This further supports that the observed effect upon rescue of C1ql2 expression in Bcl11b cKOs is due to the physiological function of C1ql2 and not as result of the overexpression. These data are included in supplementary figure 2g-j and are described in detail in the results part of the revised manuscript.

Additionally, we looked at the effects of C1ql2 overexpression in Bcl11b cKO DGN on basal synaptic transmission. We plotted fEPSP slopes versus fiber volley amplitudes, measured in slices from rescue animals, as we had previously done for the control and Bcl11b cKO (Author response image 1a). Although regression analysis revealed a trend towards steeper slopes in the rescue mice (Author response image 1a and b), the observation did not prove to be statistically significant, indicating that C1ql2 overexpression in Bcl11b cKO animals does not strongly alter basal synaptic transmission at MFS. Overall, our previous and new findings support that the observed effects of the C1ql2 rescue are not caused by the artificially elevated levels of C1ql2, as compared to controls, but are rather a result of the physiological function of C1ql2.

Following the suggestion of reviewer #2 all western blot clipped bands were exchanged for images of the full blot. This includes figures 1c, 4c, 6b and supplementary figure 2g of the revised manuscript. P-value for Figure 1d has now been included.

Author response image 1.

C1ql2 reintroduction in Bcl11b cKO DGN does not significantly alter basal synaptic transmission at mossy fiber-CA3 synapses. a Input-output curves generated by plotting fEPSP slope against fiber volley amplitude at increasing stimulation intensities. b Quantification of regression line slopes for input-output curves for all three conditions. Control+EGFP, 35 slices from 16 mice; Bcl11b cKO+EGFP, 32 slices from 14 mice; Bcl11b cKO+EGFP-2A-C1ql2, 22 slices from 11 mice. The data are presented as means, error bars represent SEM. Kruskal-Wallis test (non-parametric ANOVA) followed by Dunn’s post hoc pairwise comparisons. p=0.106; ns, not significant.

Measurement of EM micrographs: As prior work suggested that MF synapse structure is disrupted the authors should report active zone length as this may itself affect "synapse score" defined by the number of vesicles docked. More concerning is that the example KO micrographs seem to have lost all the densely clustered synaptic vesicles that are away from the AZ in normal MF synapses e.g. compare control and KO terminals in Fig 2a or 6f or 7f. These terminals look aberrant and suggest that the important measure is not what is docked but what is present in the terminal cytoplasm that normally makes up the reserve pool. This needs to be addressed with further analysis and modifications to the manuscript.

As requested by reviewer #2 we analyzed and reported in the revised manuscript the active zone length. We found that the active zone length remained unchanged in all conditions (control/Bcl11b cKO/C1ql2 rescue, WT/C1ql2 KD, control/K262E and control/Nrxn123 cKO), strengthening our results that the described Bcl11b/C1ql2/Nrxn3 mechanism is involved in the recruitment of synaptic vesicles. These data have been included in supplementary figures 2c, 4h, 5f and 6g and are described in the results part of the revised manuscript.

We want to clarify that the synapse score is not defined by the number of docked vesicles to the plasma membrane. The synapse score, which is described in great detail in our materials and methods part and has been previously published (De Bruyckere et al., 2018), rates MFS based on the number of synaptic vesicles and their distance from the active zone and was designed according to previously described properties of the vesicle pools at the MFS. The EM micrographs refer to the general misdistribution of SV in the proximity of MFS. Upon revision of the manuscript, we made sure that this was clearly stated in the main text module to avoid further confusion.

The study also presents correlated changes in MF LTP in Bcl11b KO which are rescued by C1ql2 expression. It is not clear whether the structural and functional deficits are causally linked and this should be made clearer in the manuscript. It is also not apparent why this functional measure was chosen as it is unlikely that C1ql2 plays a direct role in presynaptic plasticity mechanisms that are through a cAMP/ PKA pathway and likely disrupted LTP is due to dysfunctional synapses rather than a specific LTP effect.

The inclusion of functional experiments in this and our previous study (de Bruyckere et al., 2018) was first and foremost intended to determine whether the structural alterations observed at MFB disrupt MFS signaling. From the signaling properties we tested, basal synaptic transmission (this study) and short-term potentiation (de Bruyckere et al., 2018) were unaltered by Bcl11b KO, whereas MF LTP was found to be abolished (de Bruyckere et al., 2018). Indeed, because MF LTP largely depends on presynaptic mechanisms, including the redistribution of the readily releasable pool and recruitment of new active zones (Orlando et al., 2021; Vandael et al., 2020), it appears to be particularly sensitive to the specific structural changes we observed. We therefore believe that it is valuable information that MF LTP is affected in Bcl11b cKO animals - it conveys a direct proof for the functional importance of the observed morphological alterations, while basic transmission remains largely normal. Furthermore, it subsequently provided a functional marker for testing whether the reintroduction of C1ql2 in Bcl11b cKO animals or the KD of C1ql2 in WT animals can functionally recapitulate the control or the Bcl11b KO phenotype, respectively.

We fully agree with the reviewer that C1ql2 is unlikely to directly participate in the cAMP/PKA pathway and that the ablation of C1ql2 likely disrupts MF LTP through an alternative mode of action. Our original wording in the paragraph describing the results of the forskolin-induced LTP experiment might have overstressed the importance of the cAMP pathway. We have now rephrased that paragraph to better describe the main idea behind the forskolin experiment, namely to circumvent the initial Ca2+ influx in order to test whether deficient presynaptic Ca2+ channel/KAR signaling might be responsible for the loss of LTP in Bcl11b cKO. The results are strongly indicative of a downstream mechanism and further investigation is needed to determine the specific mechanisms by which C1ql2 regulates MFLTP, especially in light of the result that C1ql2.K262E rescued LTP, while it was unable to rescue the SV recruitment at the MF presynapse. This raises the possibility that C1ql2 can influence MF-LTP through additional, yet uncharacterized mechanisms, independent of SV recruitment. As such, a causal link between the structural and functional deficits remains tentative and we have now emphasized that point by adding a respective sentence to the discussion of our revised manuscript. Nevertheless, we again want to stress that the main rationale behind the LTP experiments was to assess the functional significance of structural changes at MFS and not to elucidate the mechanisms by which MF LTP is established.

The authors should consider measures that might support the role of Bcl11b targets in SV recruitment during the depletion of synapses or measurements of the readily releasable pool size that would complement their findings in structural studies.

We fully agree that functional measurements of the readily releasable pool (RRP) size would be a valuable addition to the reported redistribution of SV in structural studies. We have, in fact, attempted to use high-frequency stimulus trains in both field and single-cell recordings (details on single-cell experiments are described in the response to point 8) to evaluate potential differences in RRP size between the control and Bcl11b KO (Figure for reviewers 2a and b). Under both recording conditions we see a trend towards lower values of the intersection between a regression line of late responses and the y-axis. This could be taken as an indication of slightly smaller RRP size in Bcl11b mutant animals compared to controls. However, due to several technical reasons we are extremely cautious about drawing such far-reaching conclusions based on these data. At most, they suffice to conclude that the availability of release-ready vesicles in the KO is likely not dramatically smaller than in the control.

The primary issue with using high-frequency stimulus trains for RRP measurements at MFS is the particularly low initial release probability (Pr) at these synapses. This means that a large number of stimulations is required to deplete the RRP. As the RRP is constantly replenished, it remains unclear when steady state responses are reached (reviewed by Kaeser and Regehr, 2017). This is clearly visible in our single-cell recordings (Author response image 2b), which were additionally complicated by prominent asynchronous release at later stages of the stimulus train and by a large variability in the shapes of cumulative amplitude curves between cells. In contrast, while the cumulative amplitude curves for field potential recordings do reach a steady state (Author response image 2a), field potential recordings in this context are not a reliable substitute for single cell or, in the case of MFB, singlebouton recordings. Postsynaptic cells in field potential recordings are not clamped, meaning that the massive release of glutamate due to continuous stimulation depolarizes the postsynaptic cells and reduces the driving force for Na+, irrespective of depletion of the RRP. This is supported by the fact that we consistently observed a recovery of fEPSP amplitudes later in the trains where RRP had presumably been maximally depleted. In summary, high-frequency stimulus trains at the field potential level are not a valid and established technique for estimating RRP size at MFS.

Specialized laboratories have used highly advanced techniques, such as paired recordings between individual MFB and postsynaptic CA3 pyramidal cells, to estimate the RRP size of MFB (Vandael et al., 2020). These approaches are outside the scope of our present study which, while elucidating functional changes following Bcl11b depletion and C1ql2 rescue, does not aim to provide a high-end biophysical analysis of the presynaptic mechanisms involved.

Author response image 2.

Estimation of RRP size using high-frequency stimulus trains at mossy fiber-CA3 synapses. a Results from field potential recordings. Cumulative fEPSP amplitude in response to a train of 40 stimuli at 100 Hz. All subsequent peak amplitudes were normalized to the amplitude of the first peak. Data points corresponding to putative steady state responses were fit with linear regression (RRP size is indirectly reflected by the intersection of the regression line with the yaxis). Control+EGFP, 6 slices from 5 mice; Bcl11b cKO+EGFP, 6 slices from 3 mice. b Results from single-cell recordings. Cumulative EPSC amplitude in response to a train of 15 stimuli at 50 Hz. The last four stimuli were fit with linear regression. Control, 5 cells from 4 mice; Bcl11b cKO, 3 cells from 3 mice. Note the shallow onset of response amplitudes and the subsequent frequency potentiation. Due to the resulting increase in slope at higher stimulus numbers, intersection with the y-axis occurs at negative values. The differences shown were not found to be statistically significant; unpaired t-test or Mann-Whitney U-test.

Bcl11b KO reduces the number of synapses, yet the I-O curve reported in Supp Fig 2 is not changed. How is that possible? This should be explained.

We agree with reviewer #2– this apparent discrepancy has indeed struck us as a counterintuitive result. It might be that synapses that are preferentially eliminated in Bcl11b cKO are predominantly silent or have weak coupling strength, such that their loss has only a minimal effect on basal synaptic transmission. Although perplexing, the result is fully supported by our single-cell data which shows no significant differences in MF EPSC amplitudes recorded from CA3 pyramidal cells between controls and Bcl11b mutants (Author response image 3; please see the response below for details and also our response to Reviewer #1 question 2).

Matsuda et al DOI: 10.1016/j.neuron.2016.04.001 previously reported that C1ql2 organizes MF synapses by aligning postsynaptic kainate receptors with presynaptic elements. As this may have consequences for the functional properties of MF synapses including their plasticity, the authors should report whether they see deficient postsynaptic glutamate receptor signaling in the Bcl11b KO and rescue in the C1ql2 re-expression.

We agree that the study by Matsuda et al. is of key importance for our present work. Although MF LTP is governed by presynaptic mechanisms and we previously did not see differences in short-term plasticity between the control and Bcl11b cKO (De Bruyckere et al., 2018), the clustering of postsynaptic kainate receptors by C1ql2 is indeed an important detail that could potentially alter synaptic signaling at MFS in Bcl11b KO. We, therefore, re-analyzed previously recorded single-cell data by performing a kinetic analysis on MF EPSCs recorded from CA3 pyramidal cells in control and Bcl11b cKO mice (Figure for reviewers 3a) to evaluate postsynaptic AMPA and kainate receptor responses in both conditions. We took advantage of the fact that AMPA receptors deactivate roughly 10 times faster than kainate receptors, allowing the contributions of the two receptors to mossy fiber EPSCs to be separated (Castillo et al., 1997 and reviewed by Lerma, 2003). We fit the decay phase of the second (larger) EPSC evoked by paired-pulse stimulation with a double exponential function, yielding a fast and a slow component, which roughly correspond to the fractional currents evoked by AMPA and kainate receptors, respectively. Analysis of both fast and slow time constants and the corresponding fractional amplitudes revealed no significant differences between controls and Bcl11b mutants (Figure for reviewers 3e-h), indicating that both AMPA and kainate receptor signaling is unaffected by the ablation of C1ql2 following Bcl11b KO.

Importantly, MF EPSC amplitudes evoked by the first and the second pulse (Author response image 3b), paired-pulse facilitation (Author response image 3c) and failure rates (Author response image 3d) were all comparable between controls and Bcl11b mutants. These results further corroborate our observations from field recordings that basal synaptic transmission at MFS is unaltered by Bcl11b KO.

We note that the results from single cell recordings regarding basal synaptic transmission merely confirm the observations from field potential recordings, and that the attempted measurement of RRP size at the single cell level was not successful. Thus, our single-cell data do not add new information about the mechanisms underlying the effects of Bcl11b-deficiency and we therefore decided not to report these data in the manuscript.

Author response image 3.

Basal synaptic transmission at mossy fiber-CA3 synapses is unaltered in Bcl11b cKO mice. a Representative average trace (20 sweeps) recorded from CA3 pyramidal cells in control and Bcl11b cKO mice at minimal stimulation conditions, showing EPSCs in response to paired-pulse stimulation (PPS) at an interstimulus interval of 40 ms. The signal is almost entirely blocked by the application of 2 μM DCG-IV (red). b Quantification of MF EPSC amplitudes in response to PPS for both the first and the second pulse. c Ratio between the amplitude of the second over the first EPSC. d Percentage of stimulation events resulting in no detectable EPSCs for the first pulse. Events <5 pA were considered as noise. e Fast decay time constant obtained by fitting the average second EPSC with the following double exponential function: I(t)=Afaste−t/τfast+Aslowe−t/τslow+C, where I is the recorded current amplitude after time t, Afast and Aslow represent fractional current amplitudes decaying with the fast (τfast) and slow (τslow) time constant, respectively, and C is the offset. Starting from the peak of the EPSC, the first 200 ms of the decaying trace were used for fitting. f Fractional current amplitude decaying with the fast time constant. g-h Slow decay time constant and fractional current amplitude decaying with the slow time constant. For all figures: Control, 8 cells from 4 mice; Bcl11b cKO, 8 cells from 6 mice. All data are presented as means, error bars indicate SEM. None of the differences shown were found to be statistically significant; Mann-Whitney U-test for nonnormally and unpaired t-test for normally distributed data.

Reviewer #3 (Public Review):

Overall, this is a strong manuscript that uses multiple current techniques to provide specific mechanistic insight into prior discoveries of the contributions of the Bcl11b transcription factor to mossy fiber synapses of dentate gyrus granule cells. The authors employ an adult deletion of Bcl11b via Tamoxifen-inducible Cre and use immunohistochemical, electron microscopy, and electrophysiological studies of synaptic plasticity, together with viral rescue of C1ql2, a direct transcriptional target of Bcl11b or Nrxn3, to construct a molecular cascade downstream of Bcl11b for DG mossy fiber synapse development. They find that C1ql2 re-expression in Bcl11b cKOs can rescue the synaptic vesicle docking phenotype and the impairments in MF-LTP of these mutants. They also show that C1ql2 knockdown in DG neurons can phenocopy the vesicle docking and plasticity phenotypes of the Bcl11b cKO. They also use artificial synapse formation assays to suggest that C1ql2 functions together with a specific Nrxn3 splice isoform in mediating MF axon development, extending these data with a C1ql2-K262E mutant that purports to specifically disrupt interactions with Nrxn3. All of the molecules involved in this cascade are disease-associated and this study provides an excellent blueprint for uncovering downstream mediators of transcription factor disruption. Together this makes this work of great interest to the field. Strengths are the sophisticated use of viral replacement and multi-level phenotypic analysis while weaknesses include the linkage of C1ql2 with a specific Nrxn3 splice variant in mediating these effects.

Here is an appraisal of the main claims and conclusions:

1) C1ql2 is a downstream target of Bcl11b which mediates the synaptic vesicle recruitment and synaptic plasticity phenotypes seen in these cKOs. This is supported by the clear rescue phenotypes of synapse anatomy (Fig.2) and MF synaptic plasticity (Fig.3). One weakness here is the absence of a control assessing over-expression phenotypes of C1ql2. It's clear from Fig.1D that viral rescue is often greater than WT expression (totally expected). In the case where you are trying to suppress a LoF phenotype, it is important to make sure that enhanced expression of C1ql2 in a WT background does not cause your rescue phenotype. A strong overexpression phenotype in WT would weaken the claim that C1ql2 is the main mediator of the Bcl11b phenotype for MF synapse phenotypes.

As suggested by reviewer #3, we carried out C1ql2 over-expression experiments in control animals. We show that the over-expression of C1ql2 in the DG of control animals had no effect on the synaptic vesicle organization in the proximity of MFS. This further supports that the observed effect upon rescue of C1ql2 expression in Bcl11b cKOs is due to the physiological function of C1ql2 and not a result of the artificial overexpression. These data are now included in supplementary figure 2g-j and are described in detail in the results part of the revised manuscript. Please also see response to point 3 of reviewer #2.

2) Knockdown of C1ql2 via 4 shRNAs is sufficient to produce the synaptic vesicle recruitment and MFLTP phenotypes. This is supported by clear effects in the shRNA-C1ql2 groups as compared to nonsense-EGFP controls. One concern (particularly given the use of 4 distinct shRNAs) is the potential for off-target effects, which is best controlled for by a rescue experiment with RNA insensitive C1ql2 cDNA as opposed to nonsense sequences, which may not elicit the same off-target effects.

We agree with reviewer #3 that the usage of shRNAs could potentially create unexpected off-target effects and that the introduction of a shRNA-insensitive C1ql2 in parallel to the expression on the shRNA cassette would be a very effective control experiment. However, the suggested experiment would require an additional 6 months (2 months for AAV production, 2-3 months from animal injection to sacrifice and 1-2 months for EM imaging/analysis and LTP measurements) and a high number of additional animals (minimum 8 for EM and 8 for LTP measurements). We note here, that before the production of the shRNA-C1ql2 and the shRNA-NS, the individual sequences were systematically checked for off-target bindings on the murine exome with up to two mismatches and presented with no other target except the proposed (C1ql2 for shRNA-C1ql2 and no target for shRNA-NS). Taking into consideration our in-silico analysis, we feel that the interpretation of our findings is valid without this (very reasonable) additional control experiment.

3) C1ql2 interacts with Nrxn3(25b+) to facilitate MF terminal SV clustering. This claim is theoretically supported by the HEK cell artificial synapse formation assay (Fig.5), the inability of the K262-C1ql2 mutation to rescue the Bcl11b phenotype (Fig.6), and the altered localization of C1ql2 in the Nrxn1-3 deletion mice (Fig.7). Each of these lines of experimental evidence has caveats that should be acknowledged and addressed. Given the hypothesis that C1ql2 and Nrxn3b(25b) are expressed in DG neurons and work together, the heterologous co-culture experiment seems strange. Up till now, the authors are looking at pre-synaptic function of C1ql2 since they are re-expressing it in DGNs. The phenotypes they are seeing are also pre-synaptic and/or consistent with pre-synaptic dysfunction. In Fig.5, they are testing whether C1ql2 can induce pre-synaptic differentiation in trans, i.e. theoretically being released from the 293 cells "post-synaptically". But the post-synaptic ligands (Nlgn1 and and GluKs) are not present in the 293 cells, so a heterologous synapse assay doesn't really make sense here. The effect that the authors are seeing likely reflects the fact that C1ql2 and Nrxn3 do bind to each other, so C1ql2 is acting as an artificial post-synaptic ligand, in that it can cluster Nrxn3 which in turn clusters synaptic vesicles. But this does not test the model that the authors propose (i.e. C1ql2 and Nrxn3 are both expressed in MF terminals). Perhaps a heterologous assay where GluK2 is put into HEK cells and the C1ql2 and Nrxn3 are simultaneously or individually manipulated in DG neurons?

C1ql2 is expressed by DG neurons and is then secreted in the MFS synaptic cleft, while Nrxn3, that is also expressed by DG neurons, is anchored at the presynaptic side. In our work we used the well established co-culture system assay and cultured HEK293 cells secreting C1ql2 (an IgK secretion sequence was inserted at the N-terminus of C1ql2) together with hippocampal neurons expressing Nrxn3(25b+). We used the HEK293 cells as a delivery system of secreted C1ql2 to the neurons to create regions of high concentration of C1ql2. By interfering with the C1ql2-Nrxn3 interaction in this system either by expression of the non-binding mutant C1ql2 variant in the HEK cells or by manipulating Nrxn expression in the neurons, we could show that C1ql2 binding to Nrxn3(25b+) is necessary for the accumulation of vGlut1. However, we did not examine and do not claim within our manuscript that the interaction between C1ql2 and Nrxn3(25b+) induces presynaptic differentiation. Our experiment only aimed to analyze the ability of C1ql2 to cluster SV through interaction with Nrxn3. Moreover, by not expressing potential postsynaptic interaction partners of C1ql2 in our system, we could show that C1ql2 controls SV recruitment through a purely presynaptic mechanism. Co-culturing GluK2-expressing HEK cells with simultaneous manipulation of C1ql2 and/or Nrxn3 in neurons would not allow us to appropriately answer our scientific question, but rather focus on the potential synaptogenic function of the Nrxn3/C1ql2/GluK2 complex and the role of the postsynaptic ligand in it. Thus, we feel that the proposed experiment, while very interesting in characterization of additional putative functions of C1ql2, may not provide additional information for the point we were addressing. In the revised manuscript we tried to make the aim and methodological approach of this set of experiments more clear.

4) K262-C1ql2 mutation blocks the normal rescue through a Nrxn3(25b) mechanism (Fig.6). The strength of this experiment rests upon the specificity of this mutation for disrupting Nrxn3b binding (presynaptic) as opposed to any of the known postsynaptic C1ql2 ligands such as GluK2. While this is not relevant for interpreting the heterologous assay (Fig.5), it is relevant for the in vivo phenotypes in Fig.6. Similar approaches as employed in this paper can test whether binding to other known postsynaptic targets is altered by this point mutation.

It has been previously shown that C1ql2 together with C1ql3 recruit postsynaptic GluK2 at the MFS. However, loss of just C1ql2 did not affect the recruitment of GluK2, which was disrupted only upon loss of both C1ql2 and C1ql3 (Matsuda et al., 2018). In our study we demonstrate a purely presynaptic function of C1ql2 through Nrxn3 in the synaptic vesicle recruitment. This function is independent of C1ql3, as C1ql3 expression is unchanged in all of our models and its over-expression did not compensate for C1ql2 functions (Fig. 2, 3a-c). Our in vitro experiments also reveal that C1ql2 can recruit both Nrxn3 and vGlut1 in the absence of any known postsynaptic C1ql2 partner (KARs and BAI3; Fig.5; please also see response above). Furthermore, we have now performed a kinetic analysis on single-cell data which we had previously collected to evaluate postsynaptic AMPA and kainate receptor responses in both the control and Bcl11b KO. Our analysis reveals no significant differences in postsynaptic current kinetics, making it unlikely that AMPA and kainate receptor signaling is altered upon the loss of C1ql2 following Bcl11b cKO (Author response image 3e-h; please also see our response to reviewer #2 point 8). Thus, we have no experimental evidence supporting the idea that a loss of interaction between C1ql2.K262E and GluK2 would interfere with the examined phenotype. However, to exclude that the K262E mutation disrupts interaction between C1ql2 and GluK2, we performed co-immunoprecipitation from protein lysate of HEK293 cells expressing GluK2myc-flag and GFP-C1ql2 or GluK2-myc-flag and GFP-K262E and could show that both C1ql2 and K262E had GluK2 bound when precipitated. These data are included in supplementary figure 5k of the revised manuscript.

5) Altered localization of C1ql2 in Nrxn1-3 cKOs. These data are presented to suggest that Nrx3(25b) is important for localizing C1ql2 to the SL of CA3. Weaknesses of this data include both the lack of Nrxn specificity in the triple a/b KOs as well as the profound effects of Nrxn LoF on the total levels of C1ql2 protein. Some measure that isn't biased by this large difference in C1ql2 levels should be attempted (something like in Fig.1F).

We acknowledge that the lack of specificity in the Nrxn123 model makes it difficult to interpret our data. We have now examined the mRNA levels of Nrxn1 and Nrxn2 upon stereotaxic injection of Cre in the DG of Nrxn123flox/flox animals and found that Nrxn1 was only mildly reduced. At the same time Nrxn2 showed a tendency for reduction that was not significant (data included in supplementary figure 6a of revised manuscript). Only Nrxn3 expression was strongly suppressed. Of course, this does not exclude that the mild reduction of Nrxn1 and Nrxn2 interferes with the C1ql2 localization at the MFS. We further examined the mRNA levels of C1ql2 in control and Nrxn123 mutants to ensure that the observed changes in C1ql2 protein levels at the MFS are not due to reduced mRNA expression and found no changes (data are included in supplementary figure 6b of the revised manuscript), suggesting that overall protein C1ql2 expression is normal.

The reduced C1ql2 fluorescence intensity at the MFS was first observed when non-binding C1ql2 variant K262E was introduced to Bcl11b cKO mice that lack endogenous C1ql2 (Fig.6). In these experiments, we found that despite the overall high protein levels of C1ql2.K262E in the hippocampus (Fig. 6c), its fluorescence intensity at the SL was significantly reduced compared to WT C1ql2 (Fig. 6d-e). The remaining signal of the C1ql2.K262E at the SL was equally distributed and in a punctate form, similar to WT C1ql2. Together, this suggests that loss of C1ql2-Nrxn3 interaction interferes with the localization of C1ql2 at the MFS, but not with the expression of C1ql2. Of course, this does not exclude that other mechanisms are involved in the synaptic localization of C1ql2, beyond the interaction with Nrxn3, as both the mutant C1ql2 in Bcl11b cKO and the endogenous C1ql2 in Nrxn123 cKOs show residual immunofluorescence at the SL. Further studies are required to determine how C1ql2-Nrxn3 interaction regulates C1ql2 localization at the MFS.

Reviewer #1 (Recommendations For The Authors):

In addition to addressing the comments below, this study would benefit significantly from providing insight and discussion into the relevant potential postsynaptic signaling components controlled exclusively by C1ql2 (postsynaptic kainate receptors and the BAI family of proteins).

We have now performed a kinetic analysis on single-cell data that we had previously collected to evaluate postsynaptic AMPA and kainate receptor responses in both the control and Bcl11b cKO. Our analysis reveals no significant differences in postsynaptic current kinetics, making it unlikely that AMPA and kainate receptor signaling differ between controls and upon the loss of C1ql2 following Bcl11b cKO (Author response image 3e-h; please also see our response to Reviewer #2 point 8). This agrees with previous findings that C1ql2 regulates postsynaptic GluK2 recruitment together with C1ql3 and only loss of both C1ql2 and C1ql3 results in a disruption of KAR signaling (Matsuda et al., 2018). In our study we demonstrate a purely presynaptic function of C1ql2 through Nrxn3 in the synaptic vesicle recruitment. This function is independent of C1ql3, as C1ql3 expression is unchanged in all of our models and its over-expression did not compensate for C1ql2 functions (Fig. 2, 3a-c). Our in vitro experiments also reveal that C1ql2 can recruit both Nrxn3 and vGlut1 in the absence of any known postsynaptic C1ql2 partner (KARs and BAI3; Fig.5; please also see our response to reviewer #3 point 4 above). We believe that further studies are needed to fully understand both the pre- and the postsynaptic functions of C1ql2. Because the focus of this manuscript was on the role of the C1ql2-Nrxn3 interaction and our investigation on postsynaptic functions of C1ql2 was incomplete, we did not include our findings on postsynaptic current kinetics in our revised manuscript. However, we increased the discussion on the known postsynaptic partners of C1ql2 in the revised manuscript to increase the interpretability of our results.

Major Comments:

The authors demonstrate that the ultrastructural properties of presynaptic boutons are altered after Bcl11b KO and C1ql2 KD. However, whether C1ql2 functions as part of a tripartite complex and the identity of the postsynaptic receptor (BAI, KAR) should be examined.

Matsuda and colleagues have nicely demonstrated in their 2016 (Neuron) study that C1ql2 is part of a tripartite complex with presynaptic Nrxn3 and postsynaptic KARs. Moreover, they demonstrated that C1ql2, together with C1ql3, recruit postsynaptic KARs at the MFS, while the KO of just C1ql2 did not affect the KAR localization. In our study we demonstrate a purely presynaptic function of C1ql2 through Nrxn3 in the synaptic vesicle recruitment. This function is independent of C1ql3, as C1ql3 expression is unchanged in all of our models and its over-expression did not compensate for C1ql2 functions (Fig. 2, 3a-c). Our in vitro experiments also reveal that C1ql2 is able to recruit both Nrxn3 and vGlut1 in the absence of any known postsynaptic C1ql2 partner (Fig. 5; please also see our response to reviewer #3 point 4 above). Moreover, we were able to show that the SV recruitment depends on C1ql2 interaction with Nrxn3 through the expression of a non-binding C1ql2 (Fig. 6) that retains the ability to interact with GluK2 (supplementary figure 5k of revised manuscript) or by KO of Nrxns (Fig. 7). Furthermore, we have now performed a kinetic analysis on single-cell data which we had previously collected to evaluate postsynaptic AMPA and kainate receptor responses in both the control and Bcl11b cKO. Our analysis reveals no significant differences in postsynaptic current kinetics, making it unlikely that AMPA and kainate receptor signaling differ between controls and Bcl11b mutants (Author response image 3e-h; please also see our response to Reviewer #2 question 8). Together, we have no experimental evidence so far that would support that the postsynaptic partners of C1ql2 are involved in the observed phenotype. While it would be very interesting to characterize the postsynaptic partners of C1ql2 in depth, we feel this would be beyond the scope of the present study.

Figure 1f: For a more comprehensive understanding of the Bcl11b KO phenotype and the potential role for C1ql2 on MF synapse number, a complete quantification of vGlut1 and Homer1 for all conditions (Supplement Figure 2e) should be included in the main text.

In our study we focused on the role of C1ql2 in the structural and functional integrity of the MFS downstream of Bcl11b. Bcl11b ablation leads to several phenotypes in the MFS that have been thoroughly described in our previous study (De Bruyckere et al., 2018). As expected, re-expression of C1ql2 only partially rescued these phenotypes, with full recovery of the SV recruitment (Fig. 2) and of the LTP (Fig. 3), but had no effect on the reduced numbers of MFS nor the structural complexity of the MFB created by the Bcl11b KO (supplementary figure 2d-f of revised manuscript). We understand that including the quantification of vGlut1 and Homer1 co-localization in the main figures would help with a better understanding of the Bcl11b mutant phenotype. However, in our manuscript we investigate C1ql2 as an effector of Bcl11b and thus we focus on its functions in SV recruitment and LTP. As we did not find a link between C1ql2 and the number of MFS/MFB upon re-expression of C1ql2 in Bcl11b cKO or now also in C1ql2 KD (see response to comment #4 below), we believe it is more suitable to present these data in the supplement.

Figure 3/4: Given the striking reduction in the numbers of synapses (Supplement Figure 2e) and docked vesicles (Figure 2d) in the Bcl11b KO and C1ql2 KD (Figure 4e-f), it is extremely surprising that basal synaptic transmission is unaffected (Supplement Figure 2g). The authors should determine the EPSP input-output relationship following C1ql2 KD and measure EPSPs following trains of stimuli at various high frequencies.

We fully acknowledge that this is an unexpected result. It is, however, well feasible that the modest displacement of SV fails to noticeably influence basal synaptic transmission. This would be the case, for example, if only a low number of vesicles are released by single stimuli, in line with the very low initial Pr at MFS. In contrast, the reduction in synapse numbers in the Bcl11b mutant might indeed be expected to reflect in the input-output relationship. It is possible, however, that synapses that are preferentially eliminated in Bcl11b cKO are predominantly silent or have weak coupling strength, such that their loss has only a minimal effect on basal synaptic transmission. Finally, we cannot exclude compensatory mechanisms (homeostatic plasticity) at the remaining synapses. A detailed analysis of these potential mechanisms would be a whole project in its own right.

As additional information, we can say that the largely unchanged input-output-relation in Bcl11b cKO is also present in the single-cell level data (Author response image 3; details on single-cell experiments are described in the response to Reviewer #2 point 8).

As suggested by the reviewer, we have now additionally analyzed the input-output relationship following C1ql2 KD and again did not observe any significant difference between control and KD animals. We have incorporated the respective input-output curves into the revised manuscript under Supplementary figure 3c-d.

Figure 4: Does C1ql2 shRNA also reduce the number of MFBs? This should be tested to further identify C1ql2-dependent and independent functions.

As requested by reviewer #1 we quantified the number of MFBs upon C1ql2 KD. We show that C1ql2 KD in WT animals does not alter the number of MFBs. The data are presented in supplementary figure 4d of the revised manuscript. Re-expression of C1ql2 in Bcl11b cKO did not rescue the loss of MFS created by the Bcl11b mutation. Moreover, C1ql2 re-expression did not rescue the complexity of the MFB ultrastructure perturbed by the Bcl11b ablation. Together, this suggests that Bcl11b regulates MFs maintenance through additional C1ql2-independent pathways. In our previously published work (De Bruyckere et al., 2018) we identified and discussed in detail several candidate genes such as Sema5b, Ptgs2, Pdyn and Penk as putative effectors of Bcl11b in the structural and functional integrity of MFS (please also see response to reviewer #2- point 1 of public reviews).

Figure 5: Clarification is required regarding the experimental design of the HEK/Neuron co-culture: 1. C1ql2 is a secreted soluble protein - how is the protein anchored to the HEK cell membrane to recruit Nrxn3(25b+) binding and, subsequently, vGlut1?

C1ql2 was secreted by the HEK293 cells through an IgK signaling peptide at the N-terminus of C1ql2. The high concentration of C1ql2 close to the secretion site together with the sparse coculturing of the HEK293 cells on the neurons allows for the quantification of accumulation of neuronal proteins. We have now described the experimental conditions in greater detail in the main text module of the revised manuscript

2) Why are the neurons transfected and not infected? Transfection efficiency of neurons with lipofectamine is usually poor (1-5%; Karra et al., 2010), while infection of neurons with lentiviruses or AAVs encoding cDNAs routinely are >90% efficient. Thus, interpretation of the recruitment assays may be influenced by the density of neurons transfected near a HEK cell.

We agree with reviewer #1 that viral infection of the neurons would have been a more effective way of expressing our constructs. However, due to safety allowances in the used facility and time limitation at the time of conception of this set of experiments, a lipofectamine transfection was chosen.

However, as all of our examined groups were handled in the same way and multiple cells from three independent experiments were examined for each experimental set, we believe that possible biases introduced by the transfection efficiency have been eliminated and thus have trust in our interpretation of these results.

3) Surface labeling of HEK cells for wild-type C1ql2 and K262 C1ql2 would be helpful to assess the trafficking of the mutant.

We recognize that potential changes to the trafficking of C1ql2 caused by the K262E mutation would be important to characterize, in light of the reduced localization of the mutant protein at the SL in the in vivo experiments (Fig. 6e). In our culture system, C1ql2 and K262E were secreted by the HEK cells through insertion of an IgK signaling peptide at the N-terminus of the myc-tagged C1ql2/K262E. Thus, trafficking analysis on this system would not be informative, as the system is highly artificial compared to the in vivo model. Further studies are needed to characterize C1ql2 trafficking in neurons to understand how C1ql2-Nrxn3 interaction regulates the localization of C1ql2. However, labeling of the myc-tag in C1ql2 or K262E expressing HEK cells of the co-culture model reveals a similar signal for the two proteins (Fig. 5a,c). Nrxn-null mutation in neurons co-cultured with C1ql2-expressing HEK cells disrupted C1ql2 mediated vGlut1 accumulation in the neurons. Selective expression of Nrxn3(25b) in the Nrxn-null neurons restored vGlut1 clustering was (Fig. 5e-f). Together, these data suggest that it is the interaction between C1ql2 and Nrxn3 that drives the accumulation of vGlut1.

Figure 6: Bcl11b KO should also be included in 6f-h.

As suggested by reviewer #1, we included the Bcl11b cKO in figures 6f-h and in corresponding supplementary figures 5c-j.

Figure 7b: What is the abundance of mRNA for Nrxn1 and Nrxn2 as well as the abundance of Nrxns after EGFP-Cre injection into DG?

We addressed this point raised by reviewer #1 by quantifying the relative mRNA levels of Nrxn1 and Nrxn2 via qPCR upon Nrxn123 mutation induction with EGFP-Cre injection. We have now examined the mRNA levels of Nrxn1 and Nrxn2 upon stereotaxic injection of Cre in the DG of Nrxn123flox/flox animals and found that Nrxn1 was only mildly reduced. At the same time Nrxn2 showed a tendency for reduction that was not significant. The data are presented in supplementary figure 6a of the revised maunscript.

Minor Comments for readability:

Synapse score is referred to frequently in the text and should be defined within the text for clarification.

'n' numbers should be better defined in the figure legends. For example, for protein expression analysis in 1c, n=3. Is this a biological or technical triplicate? For electrophysiology (e.g. 3c), does "n=7" reflect the number of animals or the number of slices? n/N (slices/animals) should be presented.

Figure 7a: Should the diagrams of the cre viruses be EGFP-Inactive or active Cre and not CRE-EGFP as shown in the diagram?

Figure 7b: the region used for the inset should be identified in the larger image.

All minor points have been fixed in the revised manuscript according to the suggestions.

Reviewer #3 (Recommendations For The Authors):

-Please describe the 'synapse score' somewhere in the text - it is too prominently featured to not have a clear description of what it is.

The description of the synapse score has been included in the main text module of the revised manuscript.

-The claim that Bcl11b controls SV recruitment "specifically" through C1ql2 is a bit stronger than is warranted by the data. Particularly given that C1ql2 is expressed at 2.5X control levels in their rescue experiments. See pt.2

Please see response to reviewer #3 point 1 of public reviews. To address this, we over-expressed C1ql2 in control animals and found no changes in the synaptic vesicle distribution (supplementary figure 2g-j of revised manuscript). This supports that the observed rescue of synaptic vesicle recruitment by re-expression of C1ql2 is due to its physiological function and not due to the artificially elevated protein levels. Of course, we cannot exclude the possibility that other, C1ql2-independent, mechanisms also contribute to the SV recruitment downstream of Bcl11b. Our data from the C1ql2 rescue, C1ql2 KD, the in vitro experiments and the interruption of C1ql2-Nrxn3 in vivo, strongly suggest C1ql2 to be an important regulator of SV recruitment.

-Does Bcl11b regulate Nrxn3 expression? Considering the apparent loss of C1ql2 expression in the Nrxn KO mice, this is an important detail.

We agree with reviewer #3 that this is an important point. We have previously done differential transcriptomics from DG neurons of Bcl11b cKOs compared to controls and did not find Nrxn3 among the differentially expressed genes. To further validate this, we now quantified the Nrxn3 mRNA levels via qPCR in Bcl11b cKOs compared to controls and found no differences. These data are included in supplementary figure 5a of the revised manuscript.

-It appears that C1ql2 expression is much lower in the Nrxn123 KO mice. Since the authors are trying to test whether Nrxn3 is required for the correct targeting of C1ql2, this is a confounding factor. We can't really tell if what we are seeing is a "mistargeting" of C1ql2, loss of expression, or both. If the authors did a similar analysis to what they did in Figure 1 where they looked at the synaptic localization of C1ql2 (and quantified it) that could provide more evidence to support or refute the "mistargeting" claim.

Please also see response to reviewer #3 point 5 of public reviews. To exclude that reduction of fluorescence intensity of C1ql2 at the SL in Nrxn123 KO mice is due to loss of C1ql2 expression, we examined the mRNA levels of C1ql2 in control and Nrxn123 mutants and found no changes (data are included in supplementary figure 6b of the revised manuscript), suggesting that C1ql2 gene expression is normal. The reduced C1ql2 fluorescence intensity at the MFS was first observed when non-binding C1ql2 variant K262E was introduced to Bcl11b cKO mice that lack endogenous C1ql2 (Fig.6). In these experiments, we found that despite the overall high protein levels of C1ql2.K262E in the hippocampus (Fig. 6c), its fluorescence intensity at the SL was significantly reduced compared to WT C1ql2 (Fig. 6d-e). The remaining C1ql2.K262E signal in the SL was equally distributed and in a punctate form, similar to WT C1ql2. Together, this indicates that the loss of C1ql2-Nrxn3 interaction interferes with the localization of C1ql2 along the MFS, but not with expression of C1ql2. Of course, this does not exclude that additional mechanisms regulate C1ql2 localization at the synapse, as both the mutant C1ql2 in Bcl11b cKO and the endogenous C1ql2 in Nrxn123 cKO show residual immunofluorescence at the SL.

We note here that we have not previously quantified the co-localization of C1ql2 with individual synapses. C1ql2 is a secreted molecule that localizes at the MFS synaptic cleft. However, not much is known about the number of MFS that are positive for C1ql2 nor about the mechanisms regulating C1ql2 targeting, transport, and secretion to the MFS. Whether C1ql2 interaction with Nrxn3 is necessary for the protection of C1ql2 from degradation, its surface presentation and transport or stabilization to the synapse is currently unclear. Upon revision of our manuscript, we realized that we might have overstated this particular finding and have now rephrased the specific parts within the results to appropriately describe the observation and have also included a sentence in the discussion referring to the lack of understanding of the mechanism behind this observation.

-Title of Figure S5 is "Nrxn KO perturbs C1ql2 localization and SV recruitment at the MFS", but there is no data on C1ql2 localization.

This issue has been fixed in the revised manusript.

-S5 should be labeled more clearly than just Cre+/-

This issue has been fixed in the revised manuscript.

References

Castillo, P.E., Malenka, R.C., Nicoll, R.A., 1997. Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons. Nature 388, 182–186. https://doi.org/10.1038/40645

De Bruyckere, E., Simon, R., Nestel, S., Heimrich, B., Kätzel, D., Egorov, A.V., Liu, P., Jenkins, N.A., Copeland, N.G., Schwegler, H., Draguhn, A., Britsch, S., 2018. Stability and Function of Hippocampal Mossy Fiber Synapses Depend on Bcl11b/Ctip2. Front. Mol. Neurosci. 11. https://doi.org/10.3389/fnmol.2018.00103

Kaeser, P.S., Regehr, W.G., 2017. The readily releasable pool of synaptic vesicles. Curr. Opin. Neurobiol. 43, 63–70. https://doi.org/10.1016/j.conb.2016.12.012

Lerma, J., 2003. Roles and rules of kainate receptors in synaptic transmission. Nat. Rev. Neurosci. 4, 481–495. https://doi.org/10.1038/nrn1118

Orlando, M., Dvorzhak, A., Bruentgens, F., Maglione, M., Rost, B.R., Sigrist, S.J., Breustedt, J., Schmitz, D., 2021. Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons. PLoS Biol. 19, e3001149. https://doi.org/10.1371/journal.pbio.3001149

Vandael, D., Borges-Merjane, C., Zhang, X., Jonas, P., 2020. Short-Term Plasticity at Hippocampal Mossy Fiber Synapses Is Induced by Natural Activity Patterns and Associated with Vesicle Pool Engram Formation. Neuron 107, 509-521.e7. https://doi.org/10.1016/j.neuron.2020.05.013

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

It is somewhat speculative that the structure represents the EIIa-bound regulatory state. There's a strong enough case that it should be analyzed in the discussion, but I don't think it is firmly established. Therefore, the title of the paper should be changed.

Our answer: Thank you for the comment. We have changed the title to “Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter”

Reading through the manuscript, it was challenging to distinguish what is new in the current manuscript and what has been done previously. There were a lot of parts where it was hard for me to identify the main point of the current study among all the details of previous studies. It would also benefit from shortening. For example:

-Page 6: Nb725 binding has already been characterized extensively in the very nice JBC paper earlier this year. It's important to test 725-4 for binding, but since it doesn't change the binding interaction, and probably wouldn't be expected to, the entire section could be written more succinctly. The main point, which is that 725-4 behaves like 725, is lost among all the details

Our answer: Thanks for this instructive suggestion. We have shortened the description in this section.

-Page 9-10. I don't understand what summarizing all of the results from the previous D59C studies adds to the current story. It's important because it provides an indication of the substrate binding site, but its mechanism of action does not seem relevant to the current work.

Our answer: We have shortened the description of the sugar-binding site and moved the previous Fig. 3b to supplementary figure sFig. 11. According to your comment about showing the location of the binding sites, which is also suggested by Reviewer #2, we modified Fig. 3 and added two panels to map the location of the bound Na+ in the inward-facing structure and the bound sugar in the outward-facing structure.

The sugar-binding site identified in the published structure is critical to construct the mobile barrier mechanism. The sugar-binding residues identified in the published structure provided essential data to support the conclusion that the sugar-binding pocket is broken in the inward-facing structure. Thus, this published structure is mechanistically relevant to the current study.

-Page 12. Too much summary of the previous outward structure. Since this is already part of the literature, it would be more efficient to reference the previous data when it is important to interpret the new data (or show as a figure).

Our answer: The introduction of the previous sugar-binding sit is important for the detailed comparison between the two states as discussed above, but we agree with this reviewer and have significantly shortened the paragraph by moving the detailed description into the legend to the sFig. 11.

-Instead of providing the PDB ID in figures of the current structure, just say "current work" or similar. Then it is obvious you are not citing a previous structure.

Our answer: To distinguish clearly the new data and published results, the citation of the cryoEM structure [PDP ID 8T60] has been completely removed from the main text but kept in sTable 1.

-An entire panel of Figure 3 is dedicated to ligand binding in a previous outward-facing structure.

Showing it in the overlay would be sufficient.

Our answer: It is the first time for us to show a structure with a bound-Na+. Fig. 3 also illustrates the spatial relationship between the sugar-binding pocket and the cation-binding pocket since both binding sites are determined now. As stated above, according to two reviewers’ comments, we have modified the Figures and the Fig. 3d is the overlay.

Please increase the size of the font in all figures. It should be 6-8 point when printed on a standard sheet of paper. Labels in Figure 3, distances in Figure 4, and everything in Figure 5 is hard to see.

Our answer: Thank you for the comments and the enlargement of the figure size and label font in all figures have been made.

Figure 2: would be helpful to show Figure S8 in the main text, orienting the reader to the approximate location of substrate binding. What is known about the EIIA-Glc binding interface? Has anyone probed this by mutagenesis? Where are these residues on the overall structure, and are they somewhere other than the nanobody interface?

Our answer: Thank you for this comment. We have added a panel for orienting the readers about the substrate location in MelB in Figure 3c. The sFig. 8 actually focuses on the details of Nb interactions with MelB. Our current data strongly supported the notion that the Nb-bound MelBSt structure mimics the EIIAGlc-bound MelB but is not structurally resolved, so we have tuned down our statement on EIIAGlc. There is one study suggesting the C-terminal tail helix may be involved in the EIIAGlc binding, which has been added to the discussion.

Can Figure 5 be split into 2 figures and simplified?

Our answer: thanks for the suggestion. We have split it into Figs. 5b and 6 and also moved the peptide mapping to the Fig 5a.

What is the difference between cartoon and ribbon rendering?

Our answer: Ribbon: illustrating the structure; cartoon: highlighting the positions with statistically significant protection or deprotection. The statistically significant changes are implied by the ribbon representation; Sphere: not covered by labeled peptides.

Can the panels showing the kinetic data be enlarged? I don't think they need to surround the molecule. An array underneath would be fine.

Our answer: We have enlarged all figures and labels. The placement of selected plots around the model could clearly show the difference in deuterium uptake rates between the transmembrane domain and extra-membrane regions. We will maintain this arrangement.

Do colors in panel A correspond with colors in panel B?

Our answer: The color usage in both are different. Now the two panels have been separated.

Do I understand correctly that in the HDX experiments, negative values indicate positions that exchange more quickly in the nanobody-free protein relative to the nanobody-bound protein?

Our answer: Your understanding is correct.

I assume some of this is due to the protein changing conformation, but some of it might be due to burial at the nanobody-binding interface. Can those peptides be indicated?

Our answer: Thank you for this comment. We have marked the peptide carrying the Nb-binding residues on uptake plots in Figs.6 and Extended Fig. 1. There are only three Nb-binding residues covered by many overlapping peptides. Most are not covered, either not carried by the labeled peptides (Tyr205, Ser206, and Ser207) or with insignificant changes (Pro132 and Thr133), except for Asp137, Lys138, and Arg141 which are presented in 8 labeled peptides.

Few buried positions in the outward-facing state are expected to be solvent in the inward-facing state; unfortunately, inward-facing state they are buried by Nb binding.

Make figure legends easier to interpret by removing non-essential methods details (like buffer conditions).

Our answer: We removed the detailed method descriptions in most figure legends. Thank you.

Check throughout for typos.

ie page 9 Lue Leu

Page 9 like likely

Our answer: We have corrected them. Thank you!

Reviewer #2 (Recommendations For The Authors):

I have mostly minor questions/remarks.

• Why not do the hdx-ms experiments in the presence of sugar? That would give a proper distinction between two conformational states, instead of an ensemble of states vs one state.

Our answer: MelB conformation induced by sugar is also multiple states, and likely most are outward-facing states and occluded intermediate states. This is also supported by the new finding of an inward state with low sugar affinity. The ideal design should be one inward and one outward to understand the inward-outward transition. We have not identified an outward-facing mutant while we can obtain the inward by the Nb. WT MelBSt with bound Na+ favors the outward-facing state. Although our design is not ideal, we do have one state vs a predominant outward-facing WT with bound Na+.

Minor comments:

• Fig 5 is misleading as the peptide number does not match with the amino acid sequence. I would suggest putting a heat map with coverage on top. Or showing deuterium uptake per peptide. See examples below.

Our answer: The peptide number should not match with sequence number. We have 155 overlapping peptides that cover the entire amino acid sequence including the 10-His tag, and there are 60 residues with no data because they are not covered by a labeled peptide. The residue positions that are covered by peptides are estimated by bars on the top. The cylinder length does not correspond to the length of the transmembrane helix, just for mapping purposes.

• Can the authors explain how they found that the Nbs bind to the cytoplasmic side (before obtaining the structure)?

Our answer: Our in vivo two-hybrid assay between the Nb and MelBSt indicated their interaction on the cytoplasmic surface of MelBSt, which is further confirmed by the melibiose fermentation and transport assay, where the transport activities were completely inhibited by intracellularly coexpressed Nb and MelBSt. Thanks for raising this question.

• The authors use the word "substrate" indifferently for sugar and Na+ binding, which is a bit confusing. Technically, only sugar is the substrate and Na+ is a ligand, or cotransported-ion, that powers the reaction of transport. This might sound like nit-picking but it can lead to misunderstandings (at some point I thought two sugars were transported, and then I was looking for the second Na+ binding site).

Our answer: We used to call the sugar and Na as co-substrate but we agree with this comment.

We have changed by using substrate for the cargo sugar and coupling cation for the driving cation.

• Abstract "only the inner barrier" - the is missing.

Thanks. We have corrected this.

• p.3 intro "and identified that the positive cooperativity of cation and melibiose, " something is missing.

Thanks again. We missed the “as the core symport mechanism”.

• P.6 Nb275_4 instead of Nb725_4

Thank you very much for your careful reading.

• P.7. Also, affinity affinities

Thank you very much. We changed to “; and also, the -NPG affinity decreased by 21~32-fold for both Nbs”

• P.8 " contains 417 MelBSt residues (positions 2-210, 219-355, and 364-432). This does not sum up to 417 residues.

Thanks for your critical reading. We changed 364-432 to 262-432.

• p.9 Lue 54

We have corrected it to Leu54.

• I find fig.3 hard to read. Can the authors show the Na+ binding pockets and sugar binding pockets within the structure? Especially figure 3b. why are the residues in different colors?

Our answer: We have moved Fig 3b into sFig. 11. We colored the residues in the previous Fig 3B to match the hosting helices. We have added two panels to show the location of both sugar and Na in the molecular. Thank you for your comments.

• Fig4 bcef. Colored circles at the end of the helices. What are they for?

Our answer: We revised the legend. “The paired helices involved in either barrier formation were highlighted in the same colored circles.”

• 86% coverage includes the his-tag - it would be good to clarify that.

Our answer: Yes, it includes the 10-His tag.

• Fig.7 - anti clockwise cycle of transport is counter-intuitive.

Our answer: We have re-arranged. Our model was constructed originally to explain efflux due to limited information at the earlier state. Now more data are available allowing us to explain inflow and active transport.

• Where are all the uptake plots per peptide for the HDX-MS data?

Our answer: We have added the course raw data and prepared all uptake plots for all 71 peptides with statistically significant changes as an Extended Fig. 1.

• P.22 protein was concentrated to 50 mg/mL. Really? That is a lot.

This is correct. We can even concentrate MelBSt protein to greater than 50 mg/ml.

• Have the authors looked into the potential role of lipids in regulating the conformational transition? Since the structure was obtained in nanodiscs, have they observed some unexplained densities? The role of lipid-protein interactions in regulating such transitions was observed for several transporters including MFS (Gupta K, et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature. 2017 10.1038/nature20820. Martens C, et al. Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat Commun. 2018 10.1038/s41467-018-06704-1.). Furthermore, I see the authors have already observed lipid specific functional regulation of MelB (ref: Hariharan, P., et al BMC Biol 16, 85 (2018). https://doi.org/10.1186/s12915-018-0553-0). A few words about this previous work, and even commenting on the absence of lipid-protein interactions in this current work is worthwhile.

Our answer: Thanks for this very relevant comment. We paid attention to the unmodelled densities. There is one with potential but it is challenging to model it. We have added a sentence “There is no unexplained density that can be clearly modeled by lipids.” in the method to address this concern.

Reviewer #3 (Recommendations For The Authors):

1) In the following sentence, the authors report high errors for the Kd value. The anti-Fab Nb binding to NabFab was two-fold poorer than Nb725_4 at a Kd value of 0.11 {plus minus} 0.16 μM. The figure however indicates that the error value is 0.016 µM. Pls correct.

Our answer: Thank you. You are correct. The error has been corrected. 0.16 ± 0.02 uM. In this revised manuscript, we present the data in nM units.

2) Is the stoichiometry of the MelB:Na+ symport clearly known in this transporter. It can be mentioned in the discussion with appropriate references.

Our answer: Yes, the stoichiometry of unity has been clearly determined, which was included in the second paragraph of the previous version.

3) In the last section of results, the authors seem to suggest a greater movement within their Cterminal helical bundle compared to N-terminal helices. Is there evidence to suggest an asymmetry in the rocker switch between the two states of the transporter?

Our answer: Our structural data revealed that the C-terminal bundle is more dynamic compared with the N-terminal bundle where hosts the residues for specific binding of galactoside and Na+. The HDX data showed that the most dynamic regions are the structurally unresolved C-terminal tail by either method, the conserved tail helix and the middle-loop helix. transmembrane helices are relatively less dynamic with similar distributions on both transmembrane bundles. Since the most dynamic regions are peripheral element associated with the C-terminal domain, it might give a wrong impression. With regard to the symmetric or asymmetric movement, which will certainly affect the dynamic interactions between the transporter and the lipids, we favor the notion that MelBSt performs symmetric movement during the rocker switch between inward and outward states at the least cost for the protein-lipids interaction.

4) Figure 1. Are the thermograms exothermic or endothermic? clarify

Our answer: In our thermograms, all positive peaks are exothermic due to the direct detection of the heat release by the TA instrument. We clarified this in Method and now we stress this in figure legends to avoid confusion.

5) Figure 4a,d. Please put in a membrane bilayer and depict cytosolic and extracellular compartments for clarity.

Thank you. We have added a bilayer and labeled the sidedness in this figure and other related figures.

6) Fig 7. Melibiose symport cannot be referred to as Melibiose efflux transport in the legend as the latter refers to antiport. Pls rectify.

Our answer: Influx and efflux are conventionally used to describe the direction of movement of a substrate. The use of symport and antiport indicates the directions of the coupling reaction for the cargo and cation. For the symporter MelB, melibiose efflux means that sugar with the coupled cation moves out, which is driven by the melibiose concentration. During the steady state of melibiose active transport, efflux rate = influx rate.

7) Page 11 "A common feature of carrier transporters". The authors can use either carriers or transporters. Need not use both simultaneously.

Sorry for overlooking this. We have deleted carriers. Thank you very much for your time.

8) Several typos were noticed in this manuscript. some are listed below. pls correct.

Page 4- last paragraph "Furthermore"

We have corrected it. Thank you again!

Page 7 - second para one repharse "affinity reduced by 21~32 fold/units.." pls clarify

Added 21~32 fold.

Page 9 - "so it is highly likely that inward-open conformation" pls correct.

We have corrected to “likely”.

Fig. S9c - correct the spelling "Distance".

We have corrected to “Distance”

https://youtu.be/3Lru4k9Q55k?si=2TsGAi8iMEMunKii&t=15

I opened the image in Youtube and then under share link I choose the start at time ootion. Please note the three tags I added. Note, once you have created the required tag of physical-computing it will auotcomplete. Also, you need to hit enter after you type in each tag, be sure to check the tags got added, as you are being graded on your ability to tag web content.

Certainly! Let's break it down:

1. What is root.unmount()?

2. root.unmount() is a function in React that you can use to clean up and remove a rendered tree inside a React root. It's like saying, "Hey React, I'm done with this part, please clean it up."

3. When do you need it?

4. You might need it when the DOM node where your React app lives is going to be removed by some other code. For example, if you have a tab panel made with jQuery that removes inactive tabs, calling root.unmount() helps React know that it should stop managing the components inside the removed tab.

5. What does it do?

6. When you call root.unmount(), it removes all the React components in that part of the app and disconnects React from the corresponding DOM node. This cleanup includes removing event handlers and state.

7. How to use it?

8. If your entire app is built with React, you typically create one root at the beginning, and you don't need to call root.unmount(). You set it up once, and React takes care of everything. javascript const root = createRoot(document.getElementById('root')); root.render(<App />);

9. Cautions and Restrictions:

10. After calling root.unmount(), you can't call root.render() again on the same root. You would need to create a new root for the same DOM node.

11. If you're using server rendering, use hydrateRoot instead of createRoot for the initial setup.

12. Troubleshooting Tips:

13. If nothing is displayed, make sure you actually called root.render(<App />);.
14. If you get a "Target container is not a DOM element" error, check if the DOM node you're passing to createRoot is valid.
15. If you get a "Functions are not valid as a React child" error, ensure you're passing a React component, not just a function.

In simple terms, root.unmount() is like telling React, "I'm done with this part of the app, clean it up," and you typically use it in specific situations where parts of your app might be removed dynamically.

Certainly! Let's break down the information in a simpler way with examples:

1. createElement Function:
2. What it does: createElement is a function in React that allows you to create a React element. It's an alternative to using JSX.

3. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello');

4. Parameters:

5. type: Specifies the type of element you want to create. It can be a tag name string (e.g., 'div', 'span') or a React component.
6. props: An object that holds the properties (attributes) for the element.
7. ...children: Optional. Represents child elements or content.

8. Example: javascript createElement('h1', { className: 'greeting' }, 'Hello', createElement('i', null, 'World'));

9. Returns:

10. The function returns a React element object with properties like type, props, ref, and key.

11. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello'); console.log(element.type); // 'h1' console.log(element.props); // { className: 'greeting' }

12. Usage Caveats:

13. React elements and their props should be treated as immutable. They should not be changed after creation.
14. JSX tags should start with a capital letter for custom components.
15. When using JSX, dynamic children should be passed as an array to ensure React warns about missing keys for dynamic lists.

16. Example: javascript createElement('ul', {}, listItems); // Dynamic children as an array

17. Creating an Element Without JSX:

18. If you're not using JSX, you can use createElement to create elements.

19. Example: javascript function Greeting({ name }) { return createElement('h1', { className: 'greeting' }, 'Hello ', createElement('i', null, name), '. Welcome!'); }

20. Comparison with JSX:

21. JSX is a more concise and readable way to create elements compared to using createElement.

22. Example: javascript function Greeting({ name }) { return <h1 className="greeting">Hello <i>{name}</i>. Welcome!</h1>; }

23. Rendering Your Own Component:

24. You can use createElement to render your own React components.
25. Example: javascript export default function App() { return createElement(Greeting, { name: 'Taylor' }); }
26. With JSX: javascript export default function App() { return <Greeting name="Taylor" />;

In summary, createElement is a foundational function in React for creating elements, and it is often used behind the scenes when JSX is transpiled. While it's useful, JSX provides a more readable syntax for creating React elements. You can choose the style that best fits your project.

PE is the field I am going into. After reading this article I have learned a couple new things. Responsibility, respectfulness, and gratefulness are very important in any classrroom. I like the things talked about, and even the videos of the frozen tag game he explained.

20% der Schneemasse auf der Nordhalbkugel bedecken Gebiete, die im Winter meist wärmer sind als 8°. In diesen Gebieten hat die Schneedecke in den letzten Jahrzehnten bereits deutlich abgenommen. Für ihre Zukunft ist jedes Zehntelgrad mehr oder weniger Erhitzung entscheidend. Der Verlust der Schneedecke führt zu Problemen bei der Wasserversorgung etwa der Donau und des Mississippi. https://www.liberation.fr/environnement/y-aura-t-il-encore-de-la-neige-en-2050-20240117_UPOQVWROIZEBVDQRD5JBRA4EH4/

Mehr zur selben Studie: https://hypothes.is/search?q=tag%3A%22Evidence%20of%20human%20influence%20on%20Northern%20Hemisphere%20snow%20loss%22

Die OPEC geht davon aus, dass sich die Nachfrage nach Öl in diesem Jahr um 2,25 Millionen Barrel pro Tag erhöhen wird. Für das kommende Jahr erwartet die OPEC eine Steigerung um 1,85 Millionen Barrel am Tag. Die Prognosen der OPEC liegen deutlich höher als die der IEA. Die USA haben in der zweiten Januarwoche mit mit 13,3 Millionen Barrel pro Tag einen neuen Rekord in der Ölproduktion aufgestellt. https://www.reuters.com/business/energy/oil-prices-edge-higher-opec-demand-estimate-while-cold-hits-us-output-2024-01-18/

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

In this study, the authors attempt to describe alterations in gene expression, protein expression, and protein phosphorylation as a consequence of chronic adenylyl cyclase 8 overexpression in a mouse model. This model is claimed to have resilience to cardiac stress.

Major strengths of the study include 1) the large dataset generated which will have utility for further scientific inquiry for the authors and others in the field, 2) the innovative approach of using cross-analyses linking transcriptomic data to proteomic and phosphoproteomic data. One weakness is the lack of a focused question and clear relevance to human disease. These are all critical biological pathways that the authors are studying and essentially, they have compiled a database that could be surveyed to generate and test future hypotheses.

Thank you for your efforts to review our manuscript, we are delighted to learn that you found our approach to link transcriptomic, proteomic and phosphoproteome data in our analysis to be innovative. Your comment that we have not focused on a question with clear relevance to human disease is “right on point!”

During chronic pathophysiologic states e.g., chronic heart failure (CHF) in humans, AC/cAMP/PKA/Ca2+ signaling increases progressively the degree of heart failure progresses, leading to cardiac inflammation, mediated in part, by cyclic-AMP- induced up- regulation of renin-angiotensin system (RAS) signaling. Standard therapies for CHF include β-adrenoreceptor blockers and RAS inhibitors, which although effective, are suboptimal in amelioration of heart failure progression. One strategy to devise novel and better therapies for heart failure, would be to uncover the full spectrum of concentric cardio- protective adaptations that becomes activated in response to severe, chronic AC/cAMP/PKA/Ca2+ -induced cardiac stress.

We employed unbiased omics analyses, in our prior study (https://elifesciences.org/articles/80949v1) of the mouse harboring cardiac specific overexpression of adenylyl cyclase type 8 (TGAC8), and identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, that differed in TGAC8 left ventricle compared to WT. These bioinformatic analyses revealed that marked overexpression of AC8 engages complex, concentric adaptation "circuity" that has evolved in mammalian cells to confer resilience to stressors that threaten health or life. The main human disease category identified in these analyses was Organismal Injury and Abnormalities, suggesting that defenses against stress were activated as would be expected, in response to cardiac stress. Specific concentric signaling pathways that were enriched and activated within the TGAC8 protection circuitry included cell survival initiation, protection from apoptosis, proliferation, prevention of cardiac-myocyte hypertrophy, increased protein synthesis and quality control, increased inflammatory and immune responses, facilitation of tissue damage repair and regeneration and increased aerobic energetics. These TGAC8 stress response circuits resemble many adaptive mechanisms that occur in response to the stress of disease states and may be of biological significance to allow for proper healing in disease states such as myocardial infarction or failure of the heart. The main human cardiac diseases identified in bioinformatic analyses were multiple types cardiomyopathies, again suggesting that mechanisms that confer resilience to the stress of chronic increased AC-PKA-Ca2+ signaling are activated in the absence of heart failure in the super-performing TGAC8 heart at 3-months of age.

In the present study, we performed a comprehensive in silico analysis of transcription, translation, and post-translational patterns, seeking to discover whether the coordinated transcriptome and proteome regulation of the adaptive protective circuitry within the AC8 heart that is common to many types of cardiac disease states identified in our previous study (https://elifesciences.org/articles/80949v1) extends to the phosphoproteome.

Reviewer #2 (Public Review):

In this study, the investigators describe an unbiased phosphoproteomic analysis of cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) mice that was then integrated with transcriptomic and proteomic data. The phosphoproteomic analysis was performed using tandem mass tag-labeling mass spectrometry of left ventricular (LV) tissue in TGAC8 and wild-type mice. The initial principal component analysis showed differences between the TGAC8 and WT groups. The integrated analysis demonstrated that many stress-response, immune, and metabolic signaling pathways were activated at transcriptional, translational, and/or post-translational levels.

The authors are to be commended for a well-conducted study with quality control steps described for the various analyses. The rationale for following up on prior transcriptomic and proteomic analyses is described. The analysis appears thorough and well-integrated with the group's prior work. Confirmational data using Western blot is provided to support their conclusions. Their findings have the potential of identifying novel pathways involved in cardiac performance and cardioprotection.

Thank you for your efforts to review our manuscript, we are delighted to learn that you found our approach to link transcriptomic, proteomic and phosphoproteome data in our analysis. We are delighted that you found our work to be well-conducted, to have been well performed, and that our analysis was thorough and well-integrated with our prior work in this arena and that are findings have the potential of identifying novel pathways involved in cardiac performance and cardioprotection.

Reviewer #1 (Recommendations For The Authors):

I humbly suggest that the authors reconsider the title, as it could be more clear as to what they are studying. Are the authors trying to highlight pathways related to cardiac resilience? Resilience might be a clearer word than "performance and protection circuitry".

Thank you for this important comment. We have revised the title accordingly: Reprogramming of cardiac phosphoproteome, proteome and transcriptome confers resilience to chronic adenylyl cyclase-driven stress.

Perhaps the text can be reviewed in detail by a copy-editor, as there are many grammatically 'awkward' elements (for example, line 56: "mammalians" instead of mammals), inappropriate colloquialisms (for example, line 73: "port-of-call"), and stylistic unevenness that make it difficult to read.

We have reviewed the text in detail, with the assistance of a copy editor, in order to identify and correct awkward elements and to search for other colloquialisms. Finally, although “stylistic unevenness” to which you refer may be difficult for us to identify during our re-edits, we have tried our best to identify and revise them.

The best-written sections are the first few paragraphs of the discussion section, which finally clarify why the TGAC8 mouse is important in understanding cardiac resilience to stress and how the present study leverages this model to disentangle the biological processes underlying the resilience. I wish this had been presented in this manner earlier in the paper, (in the abstract and introduction) so I could have had a clearer context in which to interpret the data. It would also be helpful to point out whether the TGAC8 mouse has any correlates with human disease.

Thank you for this very important comment. Well put! In addition to recasting the title to include the concept of resilience, we have revised both the abstract and introduction to feature what you consider to be important to the understanding of cardiac resilience to stress, and how the present study leverages this model to disentangle the biological processes underlying the resilience.

Reviewer #2 (Recommendations For The Authors):

1. How were the cutoffs determined to distinguish between upregulated/downregulated phosphoproteins and phosphopeptides?

Thank you for this important question. We used the same criteria to distinguish differences between TGAC8 and WT for unnormalized and normalized phosphoproteins, -log10(p-value) > 1.3, and log2FoldChange <= -0.4 (down) or log2FoldChange >= 0.4 (up), as stated in the methods section, main text and figure legend. The results were consistent across all analyses and selectively verified by experiments.

1. Were other models assessed for correlation between transcriptome and phosphoproteome other than a linear relationship of log2 fold change?

Thank you for this comment. In addition to a linear relationship of log2 fold change of molecule expression, we also compared protein activities, e.g., Fig 4F, and pathways enriched from different omics, e.g., Fig 3D, 5J, 6B and 6F.

1. Figures 1A and 5G seem to show outliers. How many biological and technical replicates would be needed to minimize error?

Thank you for the question. Figures 1A and 5G were PCA plots which, as expected, manifested some genetic variability among the same genotypes. The PCA plots, however, are useful in determining how the identified items separated, both within and among genotypes. For bioinformatics analysis such as ours, 4-5 samples are sufficient to accomplish this, as demonstrated by separation, by genotype, of samples in PCA. Thus, in addition to discovery of true heterogeneity among the samples, our results are still able to robustly discover the true differences between the genotypes.

1. Were the up/downregulated genes more likely to be lowly expressed (which would lead to larger log2 changes identified)?

In response to your query, we calculated the average expression of phosphorylation levels across all samples to observe whether they were expressed in low abundance in all samples. We also generated the MA plots, an application of a Bland–Altman plot, to create a visual representation of omics data. The MA plots in Author response image 1 illustrate that the target molecules with significantly changed phosphorylation levels did not aggregate within the very low abundance. To confirm this conclusion, we adopted two sets of cutoffs: (1) change: -log10(p-value) > 1.3, and log2FoldChange < 0 (down) or log2FoldChange > 0 (up); and (2) change_2: -log10(p-value) > 1.3, and log2FoldChange <= -0.4 (down) or log2FoldChange >= 0.4 (up).

Author response image 1.

1. "We verified some results through wet lab experiments" in the abstract is vague.

Thank you for the good suggestion. What we meant to indicate here was that identified genotypic differences in selected proteins, phosphoproteins and RNAs discovered in omics were verified by western blots, protein synthesis detection, proteosome activity detection, and protein soluble and insoluble fractions detection. However, we have deleted the reference to the wet lab experiments in the revised manuscript.

1. There are minor syntactical errors throughout the text.

Thank you very much for the suggestion. As noted in our response, we have edited and revised those errors throughout the text.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

1. General Statements [optional]

We are thankful to the reviewers for the time and effort invested in assessing our manuscript and for their suggestions to improve it. We have now considered the points raised by them, carried out additional experiments, and modified the text and figures to address them. We feel that the new experiments and modifications have been able to solve all concerns raised by the reviewers and have improved the manuscript substantially, strengthening and extending our conclusions.

The main modifications include:

• We have extended the analysis of the overexpression strains to highly stringent conditions, which revealed a mild acidification defect for the strain overexpressing Oxr1. In addition, we have included in our analysis a strain in which both proteins are overexpressed, which resulted in a further growth defect.
• We have analyzed the recruitment of Rtc5 to the vacuole under additional conditions: deletion of the main subunit of the RAVE complex RAV1, medium containing galactose as the sole carbon source and pharmacological inhibition of the V-ATPase. These experiments allowed us to strengthen and extend our conclusions regarding the requirements for Rtc5 targeting to the vacuole.
• We have analyzed V-ATPase disassembly in intact cells, by addressing the localization to the vacuole of subunit C (Vma5) in glucose and galactose-containing medium. The results strengthen our conclusion that both Rtc5 and Oxr1 promote an in vivo state of lower V-ATPase assembly.
• We have extended our analyses of V-ATPase function to medium containing galactose as a carbon source, since glucose availability is one of the main regulators of V-ATPase function in vivo. The results are consistent with what we observed in glucose-containing medium.
• We have included a diagram of the structure of the V-ATPase for reference.
• We have included a diagram and a paragraph describing Oxr1 and Rtc5 regarding protein length and domain architecture and comparing them to other TLDc domain-containing proteins.
• We have made changes to the text and figures to improve clarity and accuracy, including a methods section that was missing. We include below a point-by-point response to the reviewers´ comments.

2. Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

__ __Suggestions:

1. The authors observed that knockout of Rtc5p or Oxr1p does not affect vacuolar pH. If Rtc5p and Oxr1p both cooperate to dissociate V-ATPase, the authors may wish to characterize the effect of a ∆Rtc5p∆Oxr1p double knockout on vacuolar pH. The double mutant ∆rtc5∆oxr1 was already included in the original manuscript (the growth test is shown in Figure 5 B and the BCECF staining is shown in Figure 5C). This strain behaved like wt in both of these assays. Of note, what we observe for the deletion strains is increased assembly (Figure 5 D - G), so we expect that it would be hard to observe a difference in vacuole acidity or growth in the presence of metals.

Therefore, we have now also included a strain with the double overexpression of Oxr1 and Rtc5. Since overexpression of the proteins results in decreased assembly, it is more likely that this strain will show impaired growth under conditions that strongly rely on V-ATPase activity. Indeed, we observed that the overexpression of Oxr1 alone resulted in a slight growth defect in media containing high concentrations of ZnCl2 and the double overexpression strain showed an even further defect (Figure 6 A and C).

The manuscript would benefit from a well-labelled diagram showing the subunits of V-ATPase (e.g. in Figure 2D).

We agree with the reviewer and we have now added a diagram of the structure of the V-ATPase labeling the different subunits in Figure 2B.

The images of structures, especially in Figure 1-Supplement 1B, are not particularly clear and could be improved (e.g. by removing shadows or using transparency).

We are thankful to the reviewer for this suggestion. To improve the clarity of the structures in Figure 1 C and Figure 1 – Supplement 1A, we are now presenting the different subunits in the structures with different shades of blue and grey.

The authors should clearly describe the differences between Rtc5p and Oxr1p in terms of protein length, sequence identity, domain structure, etc.

We are thankful for this suggestion and we have now included a diagram of the domain architecture and protein length of Rtc5 and Oxr1, comparing with two human proteins containing a TLDc domain in Figure 5A. In addition, we have added the following paragraph describing the features of the proteins.

“Rtc5 is a 567 residue-long protein. Analysis of the protein using HHPred (Zimmermann et al., 2018), finds homology to the structure of porcine Meak7 (PDB ID: 7U8O, (Zi Tan et al., 2022)) over the whole protein sequence (residues 37-559). For both yeast Rtc5 and human Meak7 (Uniprot ID: Q6P9B6), HHPred detects homology of the C-terminal region to other TLDc domain containing proteins like yeast Oxr1 (PDBID: 7FDE), Drosophila melanogaster Skywalker (PDB ID: 6R82), and human NCOA7 (PDB ID: 7OBP), while the N-terminus has similarity to EF-hand domain calcium-binding proteins (PDB IDs: 1EG3, 2CT9, 1S6C6, Figure 5A). HHPred analysis of the 273 residue long Saccharomyces cerevisiae Oxr1, on the other hand, only detects similarity to TLDc domain containing proteins (PDB IDs: 7U80, 6R82, 7OBP), which spans the majority of the sequence of the protein (residues 71-273). The overall sequence identity between Oxr1 and Rtc5 is 24% according to a ClustalOmega alignment within Uniprot. The Alphafold model that we generated for Rtc5 is in good agreement with the available partial structure of Oxr1 (7FDE) (root mean square deviation (RMSD) of 3.509Å) (Figure 5 - S1 A), indicating they are structurally very similar, in the region of the TLDc domain. Taken together, these analyses suggest that Oxr1 belongs to a group of TLDc domain-containing proteins consisting mainly of just this domain like the splice variants Oxr1-C or NCOA7-B in humans (NP_001185464 and NP_001186551, respectively), while Rtc5 belongs to a group containing an additional N-terminal EF-hand-like domain and a N-myristoylation sequence, like human Meak7 (Finelli & Oliver, 2017) (Figure 5 A).”

Minor:

1. The "O" in VO should be capitalized. This has been corrected.

In Figure 4 supplement 1, the labels "I", "S", and "P" should be defined.

This has been clarified in the figure legend.

Please clarify what is meant by "switched labelling"

This refers to the SILAC vacuole proteomics experiments, for which yeast strains are grown in medium containing either L-Lysine or 13C6;15N2- L-Lysine to produce normal (‘light’) or heavy isotope-labeled (‘heavy’) proteins. This allows comparing two conditions. To increase the robustness of the comparisons, the experiments are done twice with both possible labeling schemes (condition A – light, condition B – heavy + condition A – heavy + condition B – light), which is commonly described as switched labeling or label switching.

We have exchanged the original sentence in the manuscript for:

“Performing the same experiments but switching which strain was labeled with heavy and light amino acids gave consistent results.”

The meaning of the sentence "Indeed, this was the case for both of them" is ambiguous.

We have now replaced this sentence with the following:

“Indeed, overexpression of either Rtc5 or Oxr1 resulted in increased growth defects in the context of Stv1 deletion (Figure 7 H and I).”

For Figure 1-Supplement 1B it is hard to see the crosslink distances.

We have updated this figure to improve the visibility of the cross-links. In addition, we now include a supplemental table (supplemental table 5) with a list of the Cα- Cα distances measured for all the crosslinks we mapped onto high-resolution structures.

The statement "The effects of Oxr1 are greater than those caused by Rtc5" requires more context. Is there a way of quantifying this effect for the reader?

We agree that this sentence was too general and vague. The effects caused by one or the other protein depend on the condition and the assay. We have thus deleted this sentence, and we think it is better to refer to the description of the individual assays performed.

The phrase "negative genetic interaction" should be clarified.

We have included in the text the following explanation of genetic interactions:

“A genetic interaction occurs when the combination of two mutations results in a different phenotype from that expected from the addition of the phenotypes of the individual mutations. For example, deletion of OXR1 or RTC5 has no impact on growth in neutral pH media containing zinc in a control background but improves the growth of RAV1 deletion strains (Figure 7 E and F), so this is a positive genetic interaction. On the other hand, overexpression of either Rtc5 or Oxr1 results in a growth defect in a background lacking Rav1 in neutral media containing zinc, a negative genetic interaction.”

* * In the sentence "Isogenic strains with the indicated modifications in the genome where spotted as serial dilutions in media with pH=5.5, pH=7.5 or pH=7.5 and containing 3 mM ZnCl2", "where" should be "were".

This has been corrected.

Figure 2D: the authors should consider re-coloring these models, as it is challenging to distinguish Rtc5p from the V-ATPase.

We have changed the coloring of this structure and added a diagram of the V-ATPase structure with the same coloring scheme to improve clarity.

Reviewer #1 (Significance (Required)):

The vacuolar protein interaction map alone from this manuscript is a nice contribution to the literature. Experiments establishing colocalization of Rtc5p to the vacuole are convincing, as is dependence of this association on the presence of assembled V-ATPase. Similarly, experiments related to myristoylation are convincing. The observed mislocalization of V-ATPases that contain Stv1p to the vacuole (which is also known to occur when Vph1p has been knocked out) upon knockout of Oxr1p is also extremely interesting. Overall, this is an interesting manuscript that contributes to our understand of TLDc proteins.

We are thankful to the reviewer for their appreciation of the significance of our work, including the interactome map of the vacuole as a resource and the advances on the understanding of the regulation of the V-ATPase by TLDc domain-containing proteins.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Major points:

1. The evidence of Oxr1 and Rtc5 as V-ATPase disassembly factors is circumstantial. The authors base their interpretation primarily on increased V1 (but not Vo) at purified vacuoles from Oxr1- or Rtc5-deleted strains, which does not directly address disassembly. Of course, the results regarding Oxr1 confirm detailed disassembly experiments with the purified protein complex (PMID 34918374), but on their own are open to other interpretations, e.g. suppression of V-ATPase assembly. Of note, the authors emphasize that they provide first evidence of the in vivo role of Oxr1, but monitor V1 recruitment with purified vacuoles and do not follow V-ATPase assembly in intact cells. We are thankful to the reviewer for pointing this out. We did not want to express that the molecular activity of the proteins is the disassembly of the complex, as our analyses include in vivo and ex vivo experiments and do not directly address this. We rather meant that both proteins promote an in vivo state of lower assembly of the V-ATPase. We have modified the wording throughout the manuscript to be clearer about this.

In addition, we have added new experiments to monitor V-ATPase assembly in intact cells, as suggested by the reviewer. Previous work has shown that in yeast, only subunit C leaves the vacuole membrane under conditions that promote disassembly, while the other subunits remain at the vacuole membrane (Tabke et al 2014). Our own experiments agree with what was published (Figure 3 D). We have thus monitored Vma5 localization to the vacuole under glucose or after shift to galactose containing media in cells lacking or overexpressing Rtc5 or Oxr1. We observed that cells overexpressing either TLDc domain protein show lower levels of Vma5 recruitment to the vacuole in glucose (Figure 6 D and E). Additionally cells lacking either Rtc5 or Oxr1 contain higher levels of Vma5 at the vacuole after 20 minutes in galactose medium (Figure 5 F and G). Thus, these results re-inforce our conclusions that Rtc5 and Oxr1 promote states of lower assembly.

Oxr1 and Rtc5 have very low sequence similarity. It would be helpful if the authors provided more detail on the predicted structure of the putative TLDc domain of Rtc5 and its relationship to the V-ATPase - Oxr1 structure. Is Rtc5 more closely related to established TLDc domain proteins in other organisms?

We have now included a diagram of the domain architecture of Rtc5 and Oxr1, and comparison to the features of other TLDc domain containing proteins in Figure 5 A, as well as a paragraph describing them:

“Rtc5 is a 567 residue-long protein. Analysis of the protein using HHPred (Zimmermann et al., 2018), finds homology to the structure of porcine Meak7 (PDB ID: 7U8O, (Zi Tan et al., 2022)) over the whole protein sequence (residues 37-559). For both yeast Rtc5 and human Meak7 (Uniprot ID: Q6P9B6), HHPred detects homology of the C-terminal region to other TLDc domain containing proteins like yeast Oxr1 (PDBID: 7FDE), Drosophila melanogaster Skywalker (PDB ID: 6R82), and human NCOA7 (PDB ID: 7OBP), while the N-terminus has similarity to EF-hand domain calcium-binding proteins (PDB IDs: 1EG3, 2CT9, 1S6C6, Figure 5A). HHPred analysis of the 273 residue long Saccharomyces cerevisiae Oxr1, on the other hand, only detects similarity to TLDc domain containing proteins (PDB IDs: 7U80, 6R82, 7OBP), which spans the majority of the sequence of the protein (residues 71-273). The overall sequence identity between Oxr1 and Rtc5 is 24% according to a ClustalOmega alignment within Uniprot. The Alphafold model that we generated for Rtc5 is in good agreement with the available partial structure of Oxr1 (7FDE) (root mean square deviation (RMSD) of 3.509Å) (Figure 5 - S1 A), indicating they are structurally very similar, in the region of the TLDc domain. Taken together, these analyses suggest that Oxr1 belongs to a subfamily of TLDc domain-containing proteins consisting mainly of just this domain like the splice variants Oxr1-C or NCOA7-B in humans (NP_001185464 and NP_001186551, respectively) , while Rtc5 belongs to a subfamily containing an additional N-terminal EF-hand-like domain and a N-myristoylation sequence, like human Meak7 (Finelli & Oliver, 2017) (Figure 5 A).”

The authors conclude vacuolar recruitment of Rtc5 depends on the assembled V-ATPase, based on deletion of different V1 and Vo domain subunits. However, these genetic manipulations likely cause a strong perturbation of vacuolar acidification; indeed, the images show drastically altered vacuolar morphology. To strengthen their conclusion, it would be helpful to show that Rtc5 recruitment is not blocked by inhibition of vacuolar acidification, and that conversely it is blocked by deletion of rav1.

We are thankful to the reviewer for this insightful suggestion and we have now performed both experiments suggested. The experiment regarding rav1Δ is now Figure 3C, and we observed that this mutation also disrupts Rtc5 localization to the vacuole. In addition, we decided to include an experiment showing the subcellular localization of Rtc5 after shifting the cells to galactose containing medium for 20 minutes, as a physiologically relevant condition that results in disassembly of the complex (Figure 3D). We observed that under these conditions Rtc5 re-localizes to the cytosol. This result is particularly interesting given that in yeast only subunit C (but not other V1 subunits) re-localizes to the cytosol under these conditions. In addition, the experiment using Bafilomycin A to inhibit the V-ATPase shows that Rtc5 is still localized at the vacuole membrane under conditions of V-ATPase inhibition (Figure 3 F). Taken together these results allowed us to strengthen our original interpretation that Rtc5 requires an assembled V-ATPase for its localization and extend it to the fact that the V-ATPase does not need to be active.

Reviewer #2 (Significance (Required)):

This is an interesting paper that confirms and extends previous findings on TLDc domain proteins as a novel class of proteins that interact with and regulate the V-ATPase in eukaryotes. The title seems to exaggerate the findings a bit, as the authors do not investigate V-ATPase (dis)assembly directly and only phenotypically describe altered subcellular localization of the Golgi V-ATPase in Oxr1-deleted cells. A recent structural and biochemical characterization of Oxr1 as a V-ATPase disassembly factor (PMID 34918374) somewhat limits the novelty of the results, but the function of Oxr1 in regulating subcellular V-ATPase localization and the identification of a second potential TLDc domain protein in yeast provide relevant insights into V-ATPase regulation. This paper will be of interest to cell biologists and biochemists working on lysosomal biology, organelle proteomics and V-ATPase regulation.

We thank the reviewer for the assessment of our work, and for recognizing the novel insights that we provide. Regarding the previous biochemical work on Oxr1 and the V-ATPase, we have clearly cited this work in the manuscript. In our opinion, our results complement and extend this article, showing that the function in disassembly is relevant in vivo. Additionally, this is only one of five major points of the article, the other four being

• The interactome map of the vacuole as a resource
• The identification of Rtc5 as a second yeast TLDc domain containing protein and interactor of the V-ATPase.
• The identification of the role of Rtc5 in V-ATPase assembly.
• The identification of the role of Oxr1 in Stv1 subcellular localization. We believe these additional points add important insights to researchers interested in lysosomes, the V-ATPase, intracellular trafficking and TLDc-domain containing proteins.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Major comments

__1) Re: A cross-linking mass spectrometry map of vacuolar protein interactions (results) __ While XL-MS is a very powerful method, it is a high-throughput approach and there should be some kind of negative control in these experiments. In cross-linking experiments, non-cross-linked samples are usually used as negative controls. What was the negative control in cross-linking mass-spectrometry experiments here? If there was no negative control, how the specificity of interactions was evaluated? Maybe the authors analyzed the dataset for highly improbable interactions and found very few of them?

We fully agree that it is crucial to ensure the specificity of the interactions detected by XL-MS. To achieve this, one needs to control (1) the specificity of the data analysis (i.e. that the recorded mass spectrometry data are correctly matched to cross-linked peptides from the sequence database) and (2) the biological specificity (i.e. that the cross-linking captured natively occurring interactions).

To ascertain that criterion (1) is met, cross-link identifications are filtered to a pre-defined false-discovery rate (FDR) – an approach that the XL-MS field adopted from mass spectrometry-based proteomics. As a result, low-confidence identifications (e.g. cross-linked peptides that are only supported by a few signals in a given mass spectrum) are removed from the dataset. FDR filtering in XL-MS is a rather complex matter as it can be done at different points during data analysis and the optimal FDR cut-off depends on the specific scientific question at hand (for more details see for example Fischer and Rappsilber, Anal Chem, 2017). Generally speaking, an overly restrictive FDR cut-off would remove a lot of correct identifications, thereby greatly limiting the sensitivity of the analysis. On the other hand, a too relaxed FDR cut-off would dilute the correct identifications with a high number of false-positives, which would impair the robustness and specificity of the dataset. While many XL-MS study control the FDR on the level of individual spectrum matches, we opted for a 2% FDR cut-off on the level of unique residue pairs, which is more stringent (see Fischer and Rappsilber, Anal Chem, 2017). Our FDR parameters are described in the Methods section (Cross-linking mass spectrometry of isolated vacuoles - Data analysis). Of note, we have made all raw mass spectrometry data publicly available through the PRIDE repository (https://www.ebi.ac.uk/pride/ ; accession code PXD046792; login details during peer review: Username = reviewer_pxd046792@ebi.ac.uk, Password = q1645lTP). This will allow other researchers to re-analyze our data with the data analysis settings of their choice in the future.

To ascertain that criterion (2) is met, we mapped the identified cross-links onto existing high-resolution structures of vacuolar protein complexes. Taking into account the length of our cross-linking reagent, the side-chain length of the cross-linkable amino acids (i.e. lysines), and a certain degree of in-solution flexibility, cross-links can reasonably occur between lysines with a mutual Cα-Cα distance of up to 35 Å. Using this cut-off, the lysine-lysine pairs in the high-resolution structures we studied can be split into possible cross-linking partners (Cα-Cα distance 35 Å). Of all cross-links we could map onto high-resolution structures, 95.2% occurred between possible cross-linking partners. In addition, our cross-links reflect numerous known vacuolar protein interactions that have not yet been structurally characterized. These lines of evidence increase our confidence that our XL-MS approach captured genuine, natively occurring interactions. These analyses are described in more detail in the first Results sub-section (“A cross-linking mass spectrometry map of vacuolar protein interactions”).

In addition, the high purity of vacuole preparation is critical. How was it assessed by the authors?

We disagree that the purity of the vacuole preparation is critical for this analysis to be valid. The accuracy of the protein-protein interactions detected will depend on their preservation during sample preparation until the sample encounters the cross-linker, and the data analysis, as described above. The experiment would have been equally valid if performed on whole cell lysates without any enrichment of vacuoles, but the coverage of vacuolar proteins would have likely been very low. For this reason, we decided to use the vacuole isolation procedure to obtain better coverage of the proteins of this particular organelle. The use of the Ficoll gradient protocol (Haas, 1995) was based on that it is a protocol that yields strong enrichment of proteins annotated with the GO Term “vacuole” (Eising et al, 2019) and that it preserves the functionality of the organelle, as evidenced by its use for multiple functional assays (vacuole-vacuole fusion (Haas, 1995), autophagosome-vacuole fusion (Gao et al, 2018), polyphosphate synthesis by the VTC complex (Desfougéres et al, 2016), among others).

2) Re: Rtc5 and Oxr1 counteract the function of the RAVE complex (results)

Taken together, data, presented in this section of the manuscript, provide strong evidence that Rtc5 and Oxr1 negatively regulate V-ATPase activity, counteracting the V-ATPase assembly, facilitated by the activity of the RAVE complex. However, the complete deletion of the major RAVE subunit Rav1p was required to observe this effect in vivo in yeast. The other way to induce V-ATPase disassembly in yeast is glucose deprivation. It will be interesting to study if there is a synergistic effect between glucose deprivation and RTC5/OXR1 deletion on V-ATPase assembly, vacuolar pH, and growth of single oxr1Δ, rtc5Δ or double oxr1Δrtc5Δ mutants (OPTIONAL). Glucose deprivation is a more physiologically relevant condition than a deletion of an entire gene.

We would like to point out that an effect on assembly is observed without deleting the RAVE complex: deletions of Oxr1 or Rtc5 resulted in increased V-ATPase assembly in vivo in the presence of glucose and of the RAVE complex (Figures 5 D and E). We have now also added the experiments showing that the overexpression strains have a mild growth defect under conditions that force cells to strongly rely on V-ATPase activity (Figures 6 A and C).

Nevertheless, we agree that addressing the effect of changing the levels of Oxr1 and Rtc5 under low-glucose conditions is an interesting physiologically relevant question. We have now included growth assays and BCECF staining in medium containing galactose as the carbon source (Figures 5 – Supplement 1 B and C, and Figure 6 C and Figure 6- Supplement 1A). In addition, we have addressed the vacuolar localization of Vma5 in medium containing glucose or after shifting to medium containing galactose for 20 minutes, as a proxy for V-ATPase disassembly in intact cells (Figure 5 F and G, Figure 6 D and E). Taken together, these analyses reinforce our conclusions that both Rtc5 and Oxr1 promote an in vivo state of lower V-ATPase assembly, based on the following observations:

• Higher localization of Vma5 to the vacuole after 20 mins in galactose in cells lacking Oxr1 or Rtc5 (Figure 5 F and G).
• Lower localization of Vma5 to the vacuole in medium containing glucose in cells overexpressing Oxr1 or Rtc5 (Figure 6 D and E).
• Growth defect of the strain overexpressing Oxr1 in medium containing galactose with pH = 7.5 and zinc chloride, with a further growth defect caused by additional overexpression of Rtc5 (Figure 6 C). 3) Re: Figure 6 - supplement 1. The title is relevant to panel D only, it should be renamed to reflect the results of the disassembly of V-ATPase in rav1Δ mutant strains, while results about the stv1Δ-based strains (Panel D) should be shown together with similar experiments in Figure 7 - supplement 2 for clarity.

We have shifted the Panel D from the original Figure 6 – Supplement 1 to the main Figure (now Figure 7 – H and I). Regarding the title of the Figure, whether Supplemental Figures have titles or not will depend on the journal where the manuscript is published. For now, we have removed all titles from supplemental figures, as they are conceived to complement the main Figures.

4) Re: Figure 7 - supplement 1, Panel A. The proper assay to show that Stv1-mNeonGreen is functional is to express it in double mutant vph1Δstv1Δ to see if the growth defect is reversed. In addition, the vph1Δ growth defect is not changed (improved or worsened) in the presence of Stv1-mNeonGreen, so it means that the expression of Stv1-mNeonGreen does not further compromise the V-ATPase function, but it does not mean that it improves its function.

It is clear from the experiment suggested by the reviewer that they think that we have expressed Stv1-mNeonGreen from a plasmid. This was not the case, Stv1 was C-terminally tagged with mNeonGreen in the genome. It is thus the only expressed version in the strain. The experiment we have performed is thus equivalent to the one suggested by the reviewer, but for genomically expressed variants. For reference, the genotypes of all the strains used can be found in Supplemental Table 1.

5) Re: Figure 7 - supplement 2. This figure should be combined with Fig. 6- suppl 1, panel D as also mentioned above. The figure seems to lack some labels, and conclusions are not accurate as discussed below. However, this data provides important additional information about relationships between isoform-specific subunits of V-ATPase Vph1 and Stv1 and both Rtc5 and Oxr1 and should be repeated if it is not done yet to have a better idea about these relationships.

Panel B: Based on this picture, deletion of RTC5 has a negative genetic interaction with the deletion of VPH1, since double deletion mutant vph1Δ rtc5Δ grows worse than each individual mutant. Although it also means that there is no positive interaction, it is not the same.

Indeed, there is a negative genetic interaction between the deletion of RTC5 and VPH1. We have replaced the growth tests in this figure (Figure 8 – Supplement 2 A in the new manuscript) to show this negative genetic interaction better. This effect is reproducible, as shown in the repetitions of the experiments.

Panel C: Same as for panel B. Based on this picture, the deletion of OXR1 has a weak negative genetic interaction with the deletion of STV1, since double deletion mutant stv1Δ oxr1Δ grows worse than each individual mutant at 6 mM ZnCl2.

Panel D: Same as for panels B and C. Based on this picture, deletion of RTC5 has a negative genetic interaction with the deletion of STV1, since double deletion mutant stv1Δ rtc5Δ grows worse than each individual mutant at 6 mM ZnCl2. There is no label in the middle panel (growth conditions) and no growth assay data in the presence of CaCl2.

However, these results will be then in contradiction with the results from Figure 6 - Supplement 1, panel D, showing negative genetic interaction between the overexpression of Rtc5 or Oxr1 and deletion of Stv1, since both deletion and overexpression of Rtc5 or Oxr1 would have negative genetic interactions with Stv1.

For both Panels C and D (Now Figure 8 - Supplement 2 B and C). The effect pointed out by the reviewer (slightly stronger growth defect for the double mutants than for the single mutants) is very mild. We have attempted to make it more evident by assessing growth in medium with higher and lower concentrations of zinc and this was not possible. This is in contrast with the very clear positive genetic interaction that we observe between the deletion of OXR1 and VPH1 (Now Figure 8 H). This is the reason that we decided to report the lack of a positive genetic interaction instead of the presence of a negative one, as we do not want to draw conclusions based on results that are borderline detectable.

In addition, there is no label for the media in the middle panel, is it just YPAD pH=7.5, without the addition of any metals?

Indeed, the media is YPAD pH=7.5, without the addition of any metals. The line drawn above several images based on this media indicated this. Since this form of labeling appears to be confusing, we have now replaced it and placed the label directly above the image.

Why there is no growth assay in the presence of CaCl2, like in panels A and B?

Every growth test shown in the manuscript was performed including growth in YPD pH=5,5 as a control of a permissive condition for lack of V-ATPase activity, and then in YPD pH=7,5 including a broad range of Zinc Chloride and Calcium chloride concentrations. From all these pictures, the conditions where the differences among strains were clearly visible were chosen to assemble the figures. Conditions that did not provide any information for that particular experiment were not included in the figure to avoid making them unnecessarily large and crowded.

Re: Figure 7 - supplement 2, continued. How many times all these experiments were repeated? These experiments should be repeated at least 3 times, which is especially necessary for the experiments in panel C, because the effects are borderline. If results are reproducible and statistically significant, although small, the conclusion should be changed from "no positive genetic interactions" to "negative genetic interactions", which is more precise and informative.

All growth tests shown in the manuscript were repeated at least three times for the conditions shown. We are thankful to the reviewer for pointing out that this was not mentioned, and we have added this to the methods section. We have assembled a file with all repetitions of the shown growth tests and added it at the end of this file. In doing so, these are already available for the public. These repetitions show that all effects reported are reproducible. We will then discuss with the editors of the journal where this manuscript is published about the necessity of including it with the final article.

Regarding reporting the lack of a positive genetic interaction vs. a negative one, we have discussed this above. Shortly, for Panel B (Figure 8 – Supplement 2 A in the new manuscript) we have changed the conclusion to “negative genetic interaction” as adjusting the zinc chloride concentration allowed us to show this clearly and reproducibly, as shown by the repetitions of the experiments. For panels C and D (Now Figure 8 - Supplement 2 B and C), the effect is really mild and barely detectable, even when we tried a wide range of zinc chloride concentrations. For this reason, we would prefer to maintain the “no positive genetic interaction” conclusion.

Re: Methods. There is no description of yeast serial dilution growth assay at all. In addition, why the specific media (neutral pH, in the presence of high concentrations of calcium or zinc) was used is not explained either in the results or methods. Appropriate references should be included, for example, PMID: 2139726, PMID: 1491236.

We apologize for the oversight of the missing methods section, which we have now included.

Regarding the explanation of the media used, the following section was already a part of the results section, before the description of the first growth test:

“The V-ATPase is not essential for viability in yeast cells, and mutants lacking subunits of this complex grow similarly to a wt strain in acidic media. However, when cells grow at near-neutral pH or in the presence of divalent cations such as calcium and zinc, the mutants lacking V-ATPase function show a strong growth impairment (Kane et al, 2006).”

We have now replaced this with the following, more complete version:

“As a first approach for addressing the role of these proteins, we tested growth phenotypes related to V-ATPase function in strains lacking or overexpressing them. The V-ATPase is not essential for viability in yeast cells, and mutants lacking subunits of this complex grow similarly to a wt strain in acidic media, but display a growth defect at near-neutral pH the mutants (Nelson & Nelson, 1990). In addition, the proton gradient across the vacuole membrane generated by the V-ATPase energizes the pumping of metals into the vacuole, as a mechanism of detoxification. Thus, increasing concentrations of divalent cations such as calcium and zinc, generate conditions in which growth is increasingly reliant on V-ATPase activity (Förster & Kane, 2000; MacDiarmid et al, 2002; Kane, 2006).”

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MINOR COMMENTS

Yeast proteins are named with "p" at the end, such as "Rtc5p".

This nomenclature rule is falling into disuse during the last decades, as the use of capitals vs lowercase and italics allows to distinguish between genes proteins and strains (OXR1 = gene, Oxr1 = protein, oxr1Δ = strain). As an example, I include a list of the latest papers by some of the major yeast labs around the world, all of which use the same nomenclature as we do (in alphabetical order). This list even includes some work in the field of the V-ATPase.

• Alexey Merz, USA. PMID: 33225520
• Benoit Kornmann, UK. PMID: 35654841
• Christian Ungermann, Germany. PMID: 37463208
• Claudio de Virgilio, Switzerland. PMID: 36749016
• Daniel E. Gottschling, USA. PMID: 37640943
• David Teis, Austria. PMID: 32744498
• Elizabeth Conibear, Canada. PMID: 35938928
• Fulvio Reggiori, Denmark. PMID: 37060997
• J Christopher Fromme, USA. PMID: 37672345
• Maya Schuldiner, Israel. PMID: 37073826
• Patricia Kane, USA. PMID: 36598799
• Scott Emr, USA. PMID: 35770973
• W Mike Henne, USA. PMID: 37889293
• Yoshinori Ohsumi, Japan. PMID: 37917025 In addition, we would prefer to keep the nomenclature that we already use, to keep consistency with other published articles from our lab.

Re: Introduction. In the introduction it should be indicated that Rtc5 was originally discovered as a "restriction of telomere capping 5", using screening of temperature-sensitive cdc13-1 mutants combined with the yeast gene deletion collection [PMID: 18845848]. A couple of sentences should be written about the RAVE complex and its role in V-ATPase assembly.

We are thankful for this suggestion and we have now included both pieces of information in the introduction.

*“The re-assembly of the V1 onto the VO complex when glucose becomes again available, is aided by a dedicated chaperone complex known as the RAVE complex, which also likely has a general role in V-ATPase assembly (Seol et al, 2001; Smardon et al, 2002, 2014).” *

“In our cross-linking mass spectrometry interactome map of isolated vacuoles we found that the only other TLDc-domain containing protein of yeast, Rtc5, is a novel interactor of the V-ATPase. Rtc5 is a protein of unknown function, originally described in a genetic screen for genes related to telomere capping (Addinall et al, 2008)”

Re: The TLDc domain-containing protein of unknown function Rtc5 is a novel interactor of the vacuolar V-ATPase (results)

1) It is important to understand, that Oxr1 was co-purified before with the V1 domain of V-ATPase from a certain mutant strain, not wild-type yeast [PMID: 34918374]. It may explain why the authors did not identify it in their original protein-protein interactions screen here.

The structural work on the V1 domain bound to Oxr1 (Khan et al, 2022) showed that the binding of Oxr1 prevented V1 from assembling onto the Vo. Since our experiments rely on the purification of vacuoles, they should contain mainly only V1 assembled onto the VO, and not the free soluble V1. This is likely the reason that we do not detect Oxr1, in addition to it being less abundant. We have clarified this now in the manuscript and added the fact that Oxr1 was co-purified with a V1 containing a mutant version of the H subunit.

“In a previous study, Oxr1 was co-purified with a V1 domain containing a mutant version of the H subunit, and its presence prevented the in vitro assembly of this V1 domain onto the VO domain and promoted disassembly of the holocomplex (Khan et al., 2022). This is likely the reason why we do not detect Oxr1 in our experiments, which rely on isolated vacuoles and thus would only include V1 domains that are assembled onto the membrane. In addition, Oxr1 is less abundant in yeast cells than Rtc5 according to the protein abundance database PaxDb (Wang et al, 2015).”

2) It is a wrong conclusion that because Rtc5 was co-purified with both V1 and V0 domain subunits it interacts with the assembled V-ATPase, this does not exclude a possibility that Rtc5 also interacts with separate V1 sector or separate V0 sector of V-ATPase.

We agree with the reviewer that the co-purification of Rtc5 with both V1 and VO domain subunits does not necessarily mean that it interacts with the assembled V-ATPase. Thus, we have modified the text in this part to:

“The fact that we can co-enrich Rtc5 both with Vma2 and with Vph1 indicates that it can interact either with both the VO and V1 domains or with the assembled V-ATPase.”

However, other results throughout the manuscript can be taken into account to strengthen this idea:

1. Rtc5 requires an assembled V-ATPase to localize to the vacuole membrane, and thus seems not to interact with free VO domains, which would be available when we delete V1 subunits or in medium containing galactose.
2. Rtc5 becomes cytosolic in galactose-containing media. This would indicate that it also does not interact with free V1 domains, which are still localized to the vacuole membrane under these conditions. Taken together with the pull-downs, these results suggest that Rtc5 interacts with the assembled V1-VO V-ATPase. Thus, we have included the following sentence after Figure 3, which shows the subcellular localization experiments.

*“Taking into account that Rtc5 is co-enriched with subunits of both the VO and V1 domain, and that it localizes at the vacuole membrane dependent on an assembled V-ATPase, we suggest that Rtc5 interacts with the assembled V-ATPase complex.” *

Re: Figure 1, Panel C. Is it possible to show individual proteins in different colors for clarity?

Panel D. How were cross-link distances measured? It is not obvious if you are not an expert in the field and it is not described in the methods.

We have modified Figure 1 C and Figure 1 – Supplement 1B (now Figure 1 – Supplement 1 A) to present the different subunits in the structures with different shades of blue and grey.

Furthermore, we have clarified the distance measurement approach in the methods section and in the legend of Fig 1D: “Ca-Ca distances were determined using the measuring function in Pymol v.2.5.2 (Schrodinger LLC).”

__Re: Figure 1 - Supplement 1, __

Panel A. What scientific information are we getting from this picture?

This panel was just a visual representation of the complexity of the protein network we obtained. Indeed, there was no specific scientific message, so we have decided to remove this panel from the revised manuscript.

Panel B. Why are these complexes shown separately from the complexes in Figure 1, panel C? Also, can individual proteins be colored differently here as well?

We did not want to overload Fig 1C, so we decided to show some of the protein complexes in Fig 1 – Supplement 1B. The most important information is the histogram showing that 95% of the mapped cross-links fall within the expected length range, and this is shown in the main Figure (Figure 1D). As stated above, we have adjusted the subunit coloring in Figure 1 C to improve clarity.

Re: Figure 3. It will be nice to show the localization of the untagged protein as well if antibodies are available (OPTIONAL).

Unfortunately, there are no available antibodies for either Rtc5 or Oxr1. This hinders us from detecting the endogenous untagged proteins. We would like to point out that we have been very careful in showing which tagged proteins are functional (C-terminally tagged Rtc5) and which are not (C-terminally tagged Oxr1), so that the reader can know how to interpret the localization data.

Re: Figure 4. Why different tags were used in panels A (GFP), C (msGFP2) and D

(mNeonGreen)?

In general, we prefer to use mNeonGreen as a tag for microscopy experiments because it is brighter and more stable, and msGFP2 as a tag for experiments involving Western blots because we have better antibodies available. There was a mistake in the labeling, and actually, all constructs labeled as GFP were msGFP2. We have now corrected this. Of note, we have tested the functionality of both tagged version (mNeonGreen and msGFP2).

Panels B and C. Were Rtc5 fusions detected using anti-GFP antibodies?

Indeed, Rtc5-msGFP2 was detected with an anti-GFP antibody. We have now indicated next to each Western blot membrane the primary antibody used. In addition, all antibodies are detailed in Supplemental Figure 3.

The authors should have full-size Western blots available, not just cut-out bands, as some journals and reviewers require them for publication.

For all western blots, we always showed a good portion of the membrane and not cut-out bands. The cropping was performed to avoid making figures unnecessarily large. The whole membranes are of course available and will be included in an “extended data file” if required by the journal.

Re: Figure 4 - Supplement 1, Panel A. Does "-" and "+" mean -/+ Azido-Myr?

Indeed. We have now added this label to the figure.

Panel B. There is no blot with a membrane protein marker (Vam3 or Vac8), it should be included.

We have replaced this western blot for a different repetition of this experiment in which a membrane protein marker was included. Of note, the two other repetitions of the experiment shown (Figure 4 – Supplement 1 panel C and Figure 4 panel C) also include both a membrane protein marker and a soluble protein marker.

Re: Figure 5. The title does not describe all results in this figure and should be modified accordingly.

The original data from Figure 5 is now separated into Figures 5 and 6 because of the additional experiments included during revisions. We have modified the Figure titles to be descriptive of the overall message of the Figures.

Panel C. Statistical significance value for *** should be indicated in the legend.

This has been indicated in the Figure legend.

It is not clear how many times yeast growth assays were repeated. Usually, all experiments should be done in triplicates or more.

All shown growth tests were performed at least three times for the conditions shown. We have now indicated this in the materials and methods section. In addition, we now provide in this response a file with all repetitions of growth tests, which will be appended to the article if deemed necessary by the editors.

Re: Figure 5 - supplement 1. No title

Re: Figure 5 - supplement 2. No title

Whether the supplemental Figures should have a title or not will depend on the style of the journal where the manuscript is finally published. The current idea of the supplemental Figures is that they complement the corresponding main Figure. For this reason, we have removed all titles from supplemental Figures.

Re: Figure 6. There is a typo on the second lane in the legend: "...the genome were", not "...the genome where".

This has been corrected.

Panel C. Why the analysis of BCECF vacuole staining of double mutants oxr1Δrav1Δ and rtc5Δrav1Δ is not shown? Was it done at all?

We had not included this piece of data, as we thought that the genetic interaction of RTC5 and OXR1 and rav1Δ was sufficiently well supported with the included data (growth tests in combination with the deletion, growth tests in combination with the overexpression, vacuole proteomics in combination with overexpression, and BCECF staining in combination with the overexpression). Because of the request of the reviewer, we have now included this experiment as Figure 7 G.

Re: Figure 6 - Supplement 2. Why were two different tags (2xmNG and msGFP2) used?

We tried both tags to see if one of them would be functional. Unfortunately, they both resulted in non-functional proteins, as shown by the corresponding growth tests.

Did the authors study N-terminally tagged Oxr1? Was it functional?

We have tagged Oxr1 N-terminally, and this unfortunately resulted in a protein that was not completely functional. We show below the localization of N-terminally mNeon-tagged Oxr1, under the control of the TEF1 promoter. The protein appears cytosolic (Panel A) but is not completely functional (Panel B). The localization of Oxr1 had already been misreported by using a tagged version that we now show to be non-functional. For this reason, we preferred not to include this data in the manuscript, to avoid again including in the literature subcellular localizations that correspond to non-functional or partially functional proteins.

Panel B. Results for the untagged TEF1pr-Oxr1 overexpression are not shown, thus tagged and untagged proteins can't be compared. Are they available? What is the promoter for the expression of 2xmNG fusion constructs?

Oxr1-2xmNG was C-terminally tagged in the genome, which means that the promoter is the endogenous one, it was not modified. For this reason, the correct controls are a strain expressing Oxr1 at endogenous levels (the wt strain) and a strain lacking Oxr1. Both controls were included in the Figure, and in all repetitions made of this experiment. For reference, all the genotypes of the strains used are found in Supplemental Table 1.

Re: Methods. Were vacuoles prepared differently for XL-MS and SILAC-based vacuole proteomics (there are different references) and why? Methods for XL-MS and quantitative SILAC-based proteomics can be placed together for clarity.

The basis for the method of vacuole purification is the same, from (Haas, 1995). This reference was included in both protocols that include vacuole purifications. However, modifications of this method were performed to fit the crosslinking method (higher pH, no primary amines) or to fit the SILAC labeling (combination of two differentially labeled samples in one purification). The reference for the vacuole proteomics (Eising et al 2022) corresponds to a paper in which the SILAC-based comparison of vacuoles from different mutant strains was optimized, and includes not only the vacuole purification but the growth conditions and downstream processing of the vacuoles.

Since both the SILAC-based vacuole proteomics and the XL-MS are multi-step methods, containing numerous parameters including the sample preparation, processing for MS, MS run and data analysis, we would prefer to keep them separate. We think this would allow a person attempting to reproduce these methods to go through them step by step.

What is CMAC dye? Why was it used to stain the vacuolar lumen?

We apologize for this oversight, we have included the definition of CMAC as 7-Amino-4-Chlormethylcumarin. It is a standard-used organelle marker for the lumen of the vacuole.

Some abbreviations (TEAB, ACN) are not explained.

We apologize for this oversight. We have now replaced these abbreviations with the full names of the compounds in the article.

What is 0% Ficoll?

We used the term 0% Ficoll, because this is the name given to the buffer in the original Haas 1995 paper on vacuole purifications. However, we agree that the term is misleading and we have now added the composition of the buffer (10 mM PIPES/KOH pH=6.8, 0.2 M Sorbitol).

Reviewer #3 (Significance (Required)):

The vacuolar-type proton ATPase, V-ATPase, is the key proton pump, that hydrolases ATP and uses this energy to pump protons across membranes. Amazingly, this proton pump and its function are conserved in eukaryotes from yeast to mammals. While V-ATPase structure and function have been studied for more than 30 years in various organisms, its regulation is not completely understood. The very recent discoveries of two new V-ATPase interacting proteins in yeast, first Oxr1 (OXidative Resistance 1), and now Rtc5 (Restriction of Telomere Capping 5), both the only two members of TLDc (The Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic) proteins in yeast, provide new insights in V-ATPase regulation in yeast, and because the interaction is conserved in mammals its relevance to mammalian V-ATPases regulation as well.

TLDc proteins are best known for their role in protection from oxidative stress, in particular in yeast and in the nervous system in mammals. The discovery of the novel Rtc5-V-ATPase interaction points to the role of V-ATPase not only in protection from oxidative stress but also in restriction of telomere capping in yeast and most likely higher species. The studies of other species also highlight the possible conserved role of V-ATPase in lifespan determination and Torc1 signaling, mediated through these interactions. Thus, the discovery of this new functionally important interaction between the second TLDc family member in yeast, Rtc5, and V-ATPase will shed light on the molecular mechanisms of all these essential biological processes and pathways.

In addition, because the authors performed a comprehensive proteomics protein-protein interaction study of the purified yeast vacuole it provides a valuable resource for all researchers who study vacuoles and/or related to them lysosomes.

The follow-up functional studies using the rav1Δ strain clearly demonstrated that Rtc5 and Oxr1 disassemble V-ATPase and counteract the function of V-ATPase assembly RAVE complex in vivo in yeast. Thus, they are essentially the first discovered endogenous eukaryotic protein inhibitors of V-ATPase. Moreover, because the authors obtained the evidence that Oxr1 is the regulator of the specific subunit isoform of V-ATPase Stv1p in vivo in yeast, it suggests that different TLDc proteins may regulate different specific V-ATPase subunit isoforms in cell- and tissue-specific manner in higher eukaryotes. The mechanism of this isoform-specific regulation in yeast and other species needs further investigation in the future.

Because of the conservation of the TLDc-V-ATPase interactions, all this information can be extrapolated to higher species, all the way to humans, in whom genetic mutations in various TLDc proteins are known to cause devastating diseases and syndromes.

We are thankful to the reviewer for their positive comments about the significance of our work.

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

The biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria is still not fully understood, particularly substrate recognition and insertion by beta-assembly machinery (BAM). In the studies, the authors present their studies that in addition to recognition by the last strand of an OMP, sometimes referred to as the beta-signal, an additional signal upstream of the last strand is also important for OMP biogenesis.

Strengths:

1. Overall the manuscript is well organized and written, and addresses an important question in the field. The idea that BAM recognizes multiple signals on OMPs has been presented previously, however, it was not fully tested.

2. The authors here re-address this idea and propose that it is a more general mechanism used by BAM for OMP biogenesis.

3. The notion that additional signals assist in biogenesis is an important concept that indeed needs fully tested in OMP biogenesis.

4. A significant study was performed with extensive experiments reported in an attempt to address this important question in the field.

5. The identification of important crosslinks and regions of substrates and Bam proteins that interact during biogenesis is an important contribution that gives clues to the path substrates take en route to the membrane.

Weaknesses:

Major critiques (in no particular order):

1. The title indicates 'simultaneous recognition', however no experiments were presented that test the order of interactions during OMP biogenesis.

We have replaced the word “Simultaneous” with “Dual” so as not to reflect on the timing of the recognition events for the distinct C-terminal signal and -5 signal.

1. Aspects of the study focus on the peptides that appear to inhibit OmpC assembly, but should also include an analysis of the peptides that do not to determine this the motif(s) present still or not.

We thank the reviewer for this comment. Our study focuses on the peptides which exhibited an inhibitory effect in order to elucidate further interactions between the BAM complex and substrate proteins, especially in early stage of the assembly process. In the case of peptide 9, which contains all of our proposed elements but did not have an inhibitory effect, there is the presence of an arginine residue at the polar residue next to hydrophobic residue in position 0 (0 Φ). As seen in Fig S5, S6, and S7, there are no positively charged amino acids in the polar residue positions in the -5 or last strands. This might be the reason why peptide 9, as well as peptide 24, the β-signal derived from the mitochondrial OMP Tom40 and contains a lysine at the polar position, did not display an inhibitory effect. Incorporating the reviewer's suggestions might elucidate conditions that should not be added to the elements, but this is not the focus of this paper and was not discussed to avoid complicating the paper.

1. The β-signal is known to form a β-strand, therefore it is unclear why the authors did not choose to chop OmpC up according to its strands, rather than by a fixed peptide size. What was the rationale for how the peptide lengths were chosen since many of them partially overlap known strands, and only partially (2 residues) overlap each other? It may not be too surprising that most of the inhibitory peptides consist of full strands (#4, 10, 21, 23).

A simple scan of known β-strands would have been an alternative approach, however this comes with the bias of limiting the experiments to predicted substrate (strand) sequences, and it presupposes that the secondary structure element would be formed by this tightly truncated peptide.

Instead, we allowed for the possibility that OMPs meet the BAM complex in an unfolded or partially folded state, and that the secondary structure (β-strand) might only form via β-argumentation after the substrate is placed in the context of the lateral gate. We therefore used peptides that mapped right across the entirety of OmpC, with a two amino acid overlap.

To clarify this important point regarding the unbiased nature of our screen, we have revised the text:

(Lines 147-151) "We used peptides that mapped the entirety of OmpC, with a two amino acid overlap. This we considered preferable to peptides that were restricted by structural features, such as β-strands, in consideration that β-strand formation may or may not have occurred in early-stage interactions at the BAM complex."

1. It would be good to have an idea of the propensity of the chosen peptides to form β-stands and participate in β-augmentation. We know from previous studies with darobactin and other peptides that they can inhibit OMP assembly by competing with substrates.

We appreciate the reviewer's suggestion. However, we have not conducted biophysical characterizations of the peptides to calculate the propensity of each peptide to form β-stands and participate in β-augmentation. The sort of detailed biophysical analysis done for Darobactin (by the Maier and Hiller groups, The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase Nature 593:125-129) was a Nature publication based on this single peptide. A further biophysical analysis of all of the peptides presented here goes well beyond the scope of our study.

1. The recognition motifs that the authors present span up to 9 residues which would suggest a relatively large binding surface, however, the structures of these regions are not large enough to accommodate these large peptides.

The β-signal motif (ζxGxx[Ω/Φ]x[Ω/Φ]) is an 8-residue consensus, some of the inhibitory peptides include additional residues before and after the defined motif of 8 residues, and the lateral gate of BamA has been shown interact with a 7-residue span (eg. Doyle et al, 2022). Cross-linking presented in our study showed BamD residues R49 and G65 cross-linked to the positions 0 and 6 of the internal signal in OmpC (Fig. 6D).

We appreciate this point of clarification and have modified the text to acknowledge that in the final registering of the peptide with its binding protein, some parts of the peptide might sit beyond the bounds of the BamD receptor’s binding pocket and the BamA lateral gate:

(Lines 458-471) "The β-signal motif (ζxGxx[Ω/Φ]x[Ω/Φ]) is an eight-residue consensus, and internal signal motif is composed of a nine-residue consensus. Recent structures have shown the lateral gate of BamA interacts with a 7-residue span of substrate OMPs. Interestingly, inhibitory compounds, such as darobactin, mimic only three resides of the C-terminal side of β-signal motif. Cross-linking presented here in our study showed that BamD residues R49 and G65 cross-linked to the positions 0 and 6 of the internal signal in OmpC (Fig. 6D). Both signals are larger than the assembly machineries signal binding pocket, implying that the signal might sit beyond the bounds of the signal binding pocket in BamD and the lateral gate in BamA. These finding are consistent with similar observations in other signal sequence recognition events, such as the mitochondrial targeting presequence signal that is longer than the receptor groove formed by the Tom20, the subunit of the translocator of outer membrane (TOM) complex (Yamamoto et al., 2011). The presequence has been shown to bind to Tom20 in several different conformations within the receptor groove (Nyirenda et al., 2013)."

Moreover, the distance between amino acids of BamD which cross-linked to the internal signal, R49 and Y62, is approximately 25 Å (pdbID used 7TT3). The distance of the maximum amino acid length of the internal signal of OmpC, from F280 to Y288, is approximately 22 Å (pdbID used 2J1N). This would allow for the signal to fit within the confines of the TRP motif of BamD.

Author response image 1.

1. The authors highlight that the sequence motifs are common among the inhibiting peptides, but do not test if this is a necessary motif to mediate the interactions. It would have been good to see if a library of non-OMP related peptides that match this motif could also inhibit or not.

With respect, this additional work would not address any biological question relevant to the function of BamD. To randomize sequences and then classify those that do or don’t fit the motif would help in refining the parameters of the β-signal motif, but that was not our intent.

We have identified the peptides from within the total sequence of an OMP, shown which peptides inhibit in an assembly assay, and then observed that the inhibitory peptides conform to a previously published (β-signal) motif.

1. In the studies that disrupt the motifs by mutagenesis, an effect was observed and attributed to disruption of the interaction of the 'internal signal'. However, the literature is filled with point mutations in OMPs that disrupt biogenesis, particular those within the membrane region. F280, Y286, V359, and Y365 are all residues that are in the membrane region that point into the membrane. Therefore, more work is needed to confirm that these mutations are in parts of a recognition motif rather than on the residues that are disrupting stability/assembly into the membrane.

As the reviewer pointed out, the side chains of the amino acids constituting the signal elements we determined were all facing the lipid side, of which Y286 and Y365 were important for folding as well as to be recognized. However, F280A and V359A had no effect on folding, but only on assembly through the BAM complex. The fact that position 0 functions as a signal has been demonstrated by peptidomimetics (Fig. 1) and point mutant analysis (Fig. 2). We appreciate this clarification and have modified the text to acknowledge that the all of the signal element faces the lipid side, which contributes to their stability in the membrane finally, and before that the BAM complex actively recognizes them and determines their orientation:

(Lines 519-526) After OMP assembly, all elements of the internal signal are positioned such that they face into the lipid-phase of the membrane. This observation may be a coincidence, or may be utilized by the BAM complex to register and orientate the lipid facing amino acids in the assembling OMP away from the formative lumen of the OMP. Amino acids at position 6, such as Y286 in OmpC, are not only component of the internal signal for binding by the BAM complex, but also act in structural capacity to register the aromatic girdle for optimal stability of the OMP in the membrane.

1. The title of Figure 3 indicates that disrupting the internal signal motif disrupts OMP assembly, however, the point mutations did not seem to have any effect. Only when both 280 and 286 were mutated was an effect observed. And even then, the trimer appeared to form just fine, albeit at reduced levels, indicating assembly is just fine, rather the rate of biogenesis is being affected.

We appreciate this point and have revised the title of Figure 3 to be:

(Lines 1070-1071) "Modifications in the putative internal signal slow the rate of OMP assembly in vivo."

1. In Figure 4, the authors attempt to quantify their blots. However, this seems to be a difficult task given the lack of quality of the blots and the spread of the intended signals, particularly of the 'int' bands. However, the more disturbing trend is the obvious reduction in signal from the post-urea treatment, even for the WT samples. The authors are using urea washes to indicate removal of only stalled substrates. However a reduction of signal is also observed for the WT. The authors should quantify this blot as well, but it is clear visually that both WT and the mutant have obvious reductions in the observable signals. Further, this data seems to conflict with Fig 3D where no noticeable difference in OmpC assembly was observed between WT and Y286A, why is this the case?

We have addressed this point by adding a statistical analysis on Fig. 4A. As the reviewer points out, BN-PAGE band quantification is a difficult task given the broad spread of the bands on these gels. Statistical analysis showed that the increase in intermediates (int) was statistically significant for Y286A at all times until 80 min, when the intermediate form signals decrease.

(Lines 1093-1096) "Statistical significance was indicated by the following: N.S. (not significant), p<0.05; , p<0.005; *. Exact p values of intermediate formed by Wt vs Y286A at each timepoint were as follows; 20 minutes: p = 0.03077, 40 minutes: p = 0.02402, 60 minutes: p = 0.00181, 80 minutes: p = 0.0545."

Further regarding the Int. band, we correct the statement as follows.

(Lines 253-254) "Consistent with this, the assembly intermediate which was prominently observed at the OmpC(Y286A) can be extracted from the membranes with urea;"

OMP assembly in vivo has additional periplasmic chaperones and factors present in order to support the assembly process. Therefore, it is likely that some proteins were assembled properly in vivo compared to their in vitro counterparts. Such a decrease has been observed not only in E. coli but also in mitochondrial OMP import (Yamano et al., 2010).

1. The pull-down assays with BamA and BamD should include a no protein control at the least to confirm there is no non-specific binding to the resin. Also, no detergent was mentioned as part of the pull downs that contained BamA or OmpC, nor was it detailed if OmpC was urea solubilized.

We have performed pull down experiments with a no-protein (Ni-NTA only) control as noted (Author response image 1). The results showed that the amount of OmpC carrying through on beads only was significantly lower than the amount of OmpC bound in the presence of BamD or BamA. The added OmpC was not treated with urea, but was synthesized by in vitro translation; the in vitro translated OmpC is the standard substrate in the EMM assembly assay (Supp Fig. S1) where it is recognized by the BAM complex. Thus, we used it for pull-down as well and, to make this clearer, we have revised as follows:

Author response image 2.

Pull down assay of radio-labelled OmpC with indicated protein or Ni-NTA alone (Ni-NTA) . T; total, FT; Flow throw, W; wash, E; Elute.

(Lines 252-265) "Three subunits of the BAM complex have been previously shown to interact with the substrates: BamA, BamB, and BamD (Hagan et al., 2013; Harrison, 1996; Ieva et al., 2011). In vitro pull-down assay showed that while BamA and BamD can independently bind to the in vitro translated OmpC polypeptide (Fig .S9A), BamB did not (Fig. S9B)."

11.

• The neutron reflectometry experiments are not convincing primarily due to the lack controls to confirm a consistent uniform bilayer is being formed and even if so, uniform orientations of the BamA molecules across the surface.

• Further, no controls were performed with BamD alone, or with OmpC alone, and it is hard to understand how the method can discriminate between an actual BamA/BamD complex versus BamA and BamD individually being located at the membrane surface without forming an actual complex.

• Previous studies have reported difficulty in preparing a complex with BamA and BamD from purified components.

• Additionally, little signal differences were observed for the addition of OmpC. However, an elongated unfolded polypeptide that is nearly 400 residues long would be expected to produce a large distinct signal given that only the C-terminal portion is supposedly anchored to BAM, while the rest would be extended out above the surface.

• The depiction in Figure 5D is quite misleading when viewing the full structures on the same scales with one another.

We have addressed these five points individually as follows.

i. The uniform orientation of BamA on the surface is guaranteed by the fixation through a His-tag engineered into extracellular loop 6 of BamA and has been validated in previous studies as cited in the text. Moreover, to explain this, we reconstructed another theoretical model for BamA not oriented well in the system as below. However, we found that the solid lines (after fitting) didn’t align well with the experimental data. We therefore assumed that BamA has oriented well in the membrane bilayer.

Author response image 3.

Experimental (symbols) and fitted (curves) NR profiles of BamA not oriented well in the POPC bilayer in D2O (black), GMW (blue) and H2O (red) buffer.

ii. There would be no means by which to do a control with OmpC alone or BamD alone as neither protein binds to the lipid layer chip. OmpC is diluted from urea and then the unbound OmpC is washed from the chip before NR measurements. BamD does not have an acyl group to anchor it to the lipid layer, without BamA to anchor to, it too is washed from the chip before NR measurements. We have reconstructed another theoretical model for both of BamA + BamD embedding in the membrane bilayer, and the fits were shown below. Apparently, the fits didn’t align well with the experimental data, which discriminate the BamA/BamD individually being located at the membrane surface without forming an actual complex.

Author response image 4.

Experimental (symbols) and fitted (curves) NR profiles of BamA+D embedding together in the POPC bilayer in D2O (black), GMW (blue) and H2O (red) buffer.

iii. The previous studies that reported difficulty in preparing a complex with BamA and BamD from purified components were assays done in aqueous solution including detergent solubilized BamA, or with BamA POTRA domains only. Our assay is superior in that it reports the binding of BamD to a purified BamA that has been reconstituted in a lipid bilayer.

iv. The relatively small signal differences observed for the addition of OmpC are expected, since OmpC is an elongated, unfolded polypeptide of nearly 400 residues long which, in the context of this assay, can occupy a huge variation in the positions at which it will sit with only the C-terminal portion anchored to BAM, and the rest moving randomly about and extended from the surface.

v. We appreciate the point raised and have now added a note in the Figure legend that these are depictions of the results and not a scale drawing of the structures.

1. In the crosslinking studies, the authors show 17 crosslinking sites (43% of all tested) on BamD crosslinked with OmpC. Given that the authors are presenting specific interactions between the two proteins, this is worrisome as the crosslinks were found across the entire surface of BamD. How do the authors explain this? Are all these specific or non-specific?

The crosslinking experiment using purified BamD was an effective assay for comprehensive analysis of the interaction sites between BamD and the substrate. However, as the reviewer pointed out, cross-linking was observed even at the sites that, in the context of the BAM complex, interact with BamC as a protein-protein interaction and would not be available for substrate protein-protein interactions. To complement this, analysis and to address this issue, we also performed the experiment in Fig. 6C.

In Fig. 6C, the interaction of BamD with the substrate is examined in vivo, and the results demonstrate that if BPA is introduced into the site, we designated as the substrate recognition site, it is cross-linked to the substrate. On the other hand, position 114 was found to crosslink with the substrate in vitro crosslinking, but not in vivo. It should be noted that position 114 has also been confirmed to form cross-link products with BamC, we believe that BamD-substrate interactions in the native state have been investigated. To explain the above, we have added the following description to the Results section.

(Lines 319-321) "Structurally, these amino acids locate both the lumen side of funnel-like structure (e.g. 49 or 62) and outside of funnel-like structure such as BamC binding site (e.g. 114) (fig. S12C). (Lines 350-357) Positions 49, 53, 65, and 196 of BamD face the interior of the funnel-like structure of the periplasmic domain of the BAM complex, while position 114 is located outside of the funnel-like structure (Bakelar et al., 2016; Gu et al., 2016; Iadanza et al., 2016). We note that while position 114 was cross-linked with OmpC in vitro using purified BamD, that this was not seen with in vivo cross-linking. Instead, in the context of the BAM complex, position 114 of BamD binds to the BamC subunit and would not be available for substrate binding in vivo (Bakelar et al., 2016; Gu et al., 2016; Iadanza et al., 2016)."

1. The study in Figure 6 focuses on defined regions within the OmpC sequence, but a more broad range is necessary to demonstrate specificity to these regions vs binding to other regions of the sequence as well. If the authors wish to demonstrate a specific interaction to this motif, they need to show no binding to other regions.

The region of affinity for the BAM complex was determined by peptidomimetic analysis, and the signal region was further identified by mutational analysis of OmpC. Subsequently, the subunit that recognizes the signal region was identified as BamD. In other words, in the process leading up to Fig. 6, we were able to analyze in detail that other regions were not the target of the study. We have revised the text to make clear that we focus on the signal region including the internal signal, and have not also analyzed other parts of the signal region:

(Lines 329-332) "As our peptidomimetic screen identified conserved features in the internal signal, and cross-linking highlighted the N-terminal and C-terminal TPR motifs of BamD as regions of interaction with OmpC, we focused on amino acids specifically within the β-signals of OmpC and regions of BamD which interact with β-signal."

1. The levels of the crosslinks are barely detectable via western blot analysis. If the interactions between the two surfaces are required, why are the levels for most of the blots so low?

These are western blots of cross-linked products – the efficiency of cross-linking is far less than 100% of the interacting protein species present in a binding assay and this explains why the levels for the blots are ‘so low’. We have added a sentence to the revised manuscript to make this clear for readers who are not molecular biologists:

(Lines 345-348) "These western blots reveal cross-linked products representing the interacting protein species. Photo cross-linking of unnatural amino acid is not a 100% efficient process, so the level of cross-linked products is only a small proportion of the molecules interacting in the assays."

15.

• Figure 7 indicates that two regions of BamD promote OMP orientation and assembly, however, none of the experiments appears to measure OMP orientation?

• Also, one common observation from panel F was that not only was the trimer reduced, but also the monomer. But even then, still a percentage of the trimer is formed, not a complete loss.

(i) We appreciate this point and have revised the title of Figure 7 to be:

(Lines 1137-1138) "Key residues in two structurally distinct regions of BamD promote β-strand formation and OMP assembly."

(ii) In our description of Fig. 7F (Lines 356-360) we do not distinguish between the amount of monomer and trimer forms, since both are reflective of the overall assembly rate i.e. assembly efficiency. Rather, we state that:

"The EMM assembly assay showed that the internal signal binding site was as important as the β-signal binding site to the overall assembly rates observed for OmpC (Fig. 7F), OmpF (fig. S15D), and LamB (fig. S15E). These results suggest that recognition of both the C-terminal β-signal and the internal signal by BamD is important for efficient protein assembly."

16.

• The experiment in Fig 7B would be more conclusive if it was repeated with both the Y62A and R197A mutants and a double mutant. These controls would also help resolve any effect from crowding that may also promote the crosslinks.

• Further, the mutation of R197 is an odd choice given that this residue has been studied previously and was found to mediate a salt bridge with BamA. How was this resolved by the authors in choosing this site since it was not one of the original crosslinking sites?

As stated in the text, the purpose of the experiment in Figure 7B is to measure the impact of pre-forming a β-strand in the substrate (OmpC) before providing it to the receptor (BamD). We thank the reviewer for the comment on the R197 position of BamD. The C-terminal domain of BamD has been suggested to mediate the BamA-BamD interface, specifically BamD R197 amino acid creates a salt-bridge with BamA E373 (Ricci et al., 2012). It had been postulated that the formation of this salt-bridge is not strictly structural, with R197 highlighted as a key amino acid in BamD activity and this salt-bridge acts as a “check-point” in BAM complex activity (Ricci et al., 2012, Storek et al., 2023). Our results agree with this, showing that the C-terminus of BamD acts in substrate recognition and alignment of the β-signal (Fig. 6, Fig S12). We show that amino acids in the vicinity of R197 (N196, G200, D204) cross-linked well to substrate and mutations to the β-signal prevent this interaction (Fig S12B, D). For mutational analysis of BamD, we looked then at the conservation of the C-terminus of BamD and determined R197 was the most highly conserved amino acid (Fig 6C). In order to account for this, we have adjusted the manuscript:

(Lines 376-377) "R197 has previously been isolated as a suppressor mutation of a BamA temperature sensitive strain (Ricci et al., 2012)."

(Lines 495-496) "This adds an additional role of the C-terminus of BamD beyond a complex stability role (Ricci et al., 2012; Storek et al., 2023)."

1. As demonstrated by the authors in Fig 8, the mutations in BamD lead to reduction in OMP levels for more than just OmpC and issues with the membrane are clearly observable with Y62A, although not with R197A in the presence of VCN. The authors should also test with rifampicin which is smaller and would monitor even more subtle issues with the membrane. Oddly, no growth was observed for the Vec control in the lower concentration of VCN, but was near WT levels for 3 times VCN, how is this explained?

While it would be interesting to correlate the extent of differences to the molecular size of different antibiotics such as rifampicin, such correlations are not the intended aim of our study. Vancomycin (VCN) is a standard measure of outer membrane integrity in our field, hence its use in our tests for membrane integrity.

We apologize to the reviewer as Figure 8 D-G may have been misleading. Figure 8D,E are using bamD shut-down cells expressing plasmid-borne BamD mutants. Whereas Figure 8F, G are the same strain as used in Figure 3. We have adjusted the figure as well as the figure legend: (Lines 1165-1169) D, E E coli bamD depletion cells expressing mutations at residues, Y62A and R197A, in the β-signal recognition regions of BamD were grown with of VCN. F, G, E coli cells expressing mutations to OmpC internal signal, as shown in Fig 3, grown in the presence of VCN. Mutations to two key residues of the internal signal were sensitive to the presence of VCN.

1. While Fig 8I indeed shows diminished levels for FY as stated, little difference was observed for the trimer for the other mutants compared to WT, although differences were observed for the dimer. Interestingly, the VY mutant has nearly WT levels of dimer. What do the authors postulate is going on here with the dimer to trimer transition? How do the levels of monomer compare, which is not shown?

The BN-PAGE gel system cannot resolve protein species that migrate below ~50kDa and the monomer species of the OMPs is below this size. We can’t comment on effects on the monomer because it is not visualized. The non-cropped gel image is shown here. Recently, Hussain et al., has shown that in vitro proteo-liposome system OmpC assembly progresses from a “short-lived dimeric” form before the final process of trimerization (Hussain et al., 2021). However, their findings suggest that LPS plays the final role in stimulation of dimer-to-trimer, a step well past the recognition step of the β-signals. Mutations to the internal signal of OmpC results in the formation of an intermediate, the substrate stalled on the BAM complex. This stalling, presumably, causes a hinderance to the BAM complex resulting in reduced timer and loss of dimer OmpF signal in the EMM of cells expressing OmpC double mutant strain, FY. cannot resolve protein species that migrate below ~50kDa and the monomer species of the OMPs is below this size. We can’t comment on effects on the monomer because it is not visualized. The non-cropped gel image is shown here. We have noted this in the revised text:

Author response image 5.

Non-cropped gel of Fig. 8I. the asterisk indicates a band observed in the sample loading wells at the top of the gel.

(Lines 417-418) "The dimeric form of endogenous OmpF was prominently observed in both the OmpC(WT) as well as the OmpC(VY) double mutant cells."

1. In the discussion, the authors indicate they have '...defined an internal signal for OMP assembly', however, their study is limited and only investigates a specific region of OmpC. More is needed to definitively say this for even OmpC, and even more so to indicate this is a general feature for all OMPs.

We acknowledge the reviewer's comment on this point and have expanded the statement to make sure that the conclusion is justified with the specific evidence that is shown in the paper and the supplementary data. We now state:

(Lines 444-447) "This internal signal corresponds to the -5 strand in OmpC and is recognized by BamD. Sequence analysis shows that similar sequence signatures are present in other OMPs (Figs. S5, S6 and S7). These sequences were investigated in two further OMPs: OmpF and LamB (Fig. 2C and D)."

Note, we did not state that this is a general feature for all OMPs. That would not be a reasonable proposition.

20.

• In the proposed model in Fig 9, it is hard to conceive how 5 strands will form along BamD given the limited surface area and tight space beneath BAM.

• More concerning is that the two proposal interaction sites on BamD, Y62 and R197, are on opposite sides of the BamD structure, not along the same interface, which makes this model even more unlikely.

• As evidence against this model, in Figure 9E, the two indicates sites of BamD are not even in close proximity of the modeled substrate strands.

We can address the reviewer’s three concerns here:

i. The first point is that the region (formed by BamD engaged with POTRA domains 1-2 and 5 of BamA) is not sufficient to accommodate five β-strands. Structural analysis reveals that the interaction between the N-terminal side of BamD and POTRA1-2 is substantially changed the conformation by substrate binding, and that this surface is greatly extended. This surface does have enough space to accommodate five beta-strands, as now documented in Fig. 9D, 9E using the latest structures (7TT5 and 7TT2) as illustrations of this. The text now reads:

(Lines 506-515) "Spatially, this indicates the BamD can serve to organize two distinct parts of the nascent OMP substrate at the periplasmic face of the BAM complex, either prior to or in concert with, engagement to the lateral gate of BamA. Assessing this structurally showed the N-terminal region of BamD (interacting with the POTRA1-2 region of BamA) and the C-terminal region of BamD (interacting with POTRA5 proximal to the lateral gate of BamA) (Bakelar et al., 2016; Gu et al., 2016; Tomasek et al., 2020) has the N-terminal region of BamD changing conformation depending on the folding states of the last four β-strands of the substrate OMP, EspP (Doyle et al., 2022). The overall effect of this being a change in the dimensions of this cavity change, a change which is dependent on the folded state of the substrate engaged in it (Fig 9 B-E)."

ii. The second point raised regards the orientation of the substrate recognition residues of BamD. Both Y62A and R197 were located on the lumen side of the funnel in the EspP-BAM transport intermediate structure (PDBID;7TTC); Y62A is relatively located on the edge of BamD, but given that POTRA1-2 undergoes a conformational change and opens this region, as described above, both are located in locations where they could bind to substrates. This was explained in the following text in the results section of revised manuscript.

(Lines 377-379) "Each residue was located on the lumen side of the funnel-like structure in the EspP-BAM assembly intermediate structure (PDBID; 7TTC) (Doyle et al., 2022)."

**Reviewer #2 (Public Review):"

Previously, using bioinformatics study, authors have identified potential sequence motifs that are common to a large subset of beta-barrel outer membrane proteins in gram negative bacteria. Interestingly, in that study, some of those motifs are located in the internal strands of barrels (not near the termini), in addition to the well-known "beta-signal" motif in the C-terminal region.

Here, the authors carried out rigorous biochemical, biophysical, and genetic studies to prove that the newly identified internal motifs are critical to the assembly of outer membrane proteins and the interaction with the BAM complex. The author's approaches are rigorous and comprehensive, whose results reasonably well support the conclusions. While overall enthusiastic, I have some scientific concerns with the rationale of the neutron refractory study, and the distinction between "the intrinsic impairment of the barrel" vs "the impairment of interaction with BAM" that the internal signal may play a role in. I hope that the authors will be able to address this.

Strengths:

1. It is impressive that the authors took multi-faceted approaches using the assays on reconstituted, cell-based, and population-level (growth) systems.

2. Assessing the role of the internal motifs in the assembly of model OMPs in the absence and presence of BAM machinery was a nice approach for a precise definition of the role.

Weaknesses:

1. The result section employing the neutron refractory (NR) needs to be clarified and strengthened in the main text (from line 226). In the current form, the NR result seems not so convincing.

What is the rationale of the approach using NR?

We have now modified the text to make clear that:

(Lines 276-280) "The rationale to these experiments is that NR provides: (i) information on the distance of specified subunits of a protein complex away from the atomically flat gold surface to which the complex is attached, and (ii) allows the addition of samples between measurements, so that multi-step changes can be made to, for example, detect changes in domain conformation in response to the addition of a substrate."

What is the molecular event (readout) that the method detects?

We have now modified the text to make clear that:

(Lines 270-274) "While the biochemical assay demonstrated that the OmpC(Y286A) mutant forms a stalled intermediate with the BAM complex, in a state in which membrane insertion was not completed, biochemical assays such as this cannot elucidate where on BamA-BamD this OmpC(Y286A) substrate is stalled."

What are "R"-y axis and "Q"-x axis and their physical meanings (Fig. 5b)?

The neutron reflectivity, R, refers to the ratio of the incoming and exiting neutron beams and it is measured as a function of Momentum transfer Q, which is defined as Q=4π sinθ/λ, where θ is the angle of incident and λ is the neutron wavelength. R(Q)is approximately given byR(Q)=16π2/ Q2 |ρ(Q)|2, where R(Q) is the one-dimensional Fourier transform of ρ(z), the scattering length density (SLD) distribution normal to the surface. SLD is the sum of the coherent neutron scattering lengths of all atoms in the sample layer divided by the volume of the layer. Therefore, the intensity of the reflected beams is highly dependent on the thickness, densities and interface roughness of the samples. This was explained in the following text in the method section of revised manuscript.

(Lines 669-678) "Neutron reflectivity, denoted as R, is the ratio of the incoming to the exiting neutron beams. It’s calculated based on the Momentum transfer Q, which is defined by the formula Q=4π sinθ/λ, where θ represents the angle of incidence and λ stands for the neutron wavelength. The approximate value of R(Q) can be expressed as R(Q)=16π2/ Q2 |ρ(Q)|2, where R(Q) is the one-dimensional Fourier transform of ρ(z), which is the scattering length density (SLD) distribution perpendicular to the surface. SLD is calculated by dividing the sum of the coherent neutron scattering lengths of all atoms in a sample layer by the volume of that layer. Consequently, factors such as thickness, volume fraction, and interface roughness of the samples significantly influence the intensity of the reflected beams."

How are the "layers" defined from the plot (Fig. 5b)?

The “layers” in the plot (Fig. 5b) represent different regions of the sample being studied. In this study, we used a seven-layer model to fit the experimental data (chromium - gold - NTA - HIS8 - β-barrel - P3-5 - P1-2. This was explained in the following text in the figure legend of revised manuscript. (Lines 1115-1116) The experimental data was fitted using a seven-layer model: chromium - gold - NTA - His8 - β-barrel - P3-5 - P1-2.

What are the meanings of "thickness" and "roughness" (Fig. 5c)?

We used neutron reflectometry to determine the relative positions of BAM subunits in a membrane environment. The binding of certain subunits induced conformational changes in other parts of the complex. When a substrate membrane protein is added, the periplasmic POTRA domain of BamA extends further away from the membrane surface. This could result in an increase in thickness as observed in neutron reflectometry measurements.

As for roughness, it is related to the interface properties of the sample. In neutron reflectometry, the intensity of the reflected beams is highly dependent on the thickness, densities, and interface roughness of the samples. An increase in roughness could suggest changes in these properties, possibly due to protein-membrane interactions or structural changes within the membrane.

(Lines 1116-1120) "Table summarizes of the thickness, roughness and volume fraction data of each layer from the NR analysis. The thickness refers to the depth of layered structures being studied as measured in Å. The roughness refers to the irregularities in the surface of the layered structures being studied as measured in Å."

What does "SLD" stand for?

We apologize for not explaining abbreviation when the SLD first came out. We explained it in revised manuscript. (Line 298)

1. In the result section, "The internal signal is necessary for insertion step of assembly into OM" This section presents an important result that the internal beta-signal is critical to the intrinsic propensity of barrel formation, distinct from the recognition by BAM complex. However, this point is not elaborated in this section. For example, what is the role of these critical residues in the barrel structure formation? That is, are they involved in any special tertiary contacts in the structure or in membrane anchoring of the nascent polypeptide chains?

We appreciate the reviewer's comment on this point. Both position 0 and position 6 appear to be important amino acids for recognition by the BAM complex, since mutations introduced at these positions in peptide 18 prevent competitive inhibition activity.

In terms of the tertiary structure of OmpC, position 6 is an amino acid that contributes to the aromatic girdle, and since Y286A and Y365A affected OMP folding as measured in folding experiments, it is perhaps their position in the aromatic girdle that contributes to the efficiency of β-barrel folding in addition to its function as a recognition signal. We have added a sentence in the revised manuscript:

(Lines 233-236) "Position 6 is an amino acid that contributes to the aromatic girdle. Since Y286A and Y365A affected OMP folding as measured in folding experiments, their positioning into the aromatic girdle may contributes to the efficiency of β-barrel folding, in addition to contributing to the internal signal."

The mutations made at position 0 had no effect on folding, so this residue may function solely in the signal. Given the register of each β-strand in the final barrel, the position 0 residues have side-chains that face out into the lipid environment. From examination of the OmpC crystal structure, the residue at position 0 makes no special tertiary contacts with other, neighbouring residues.  

Reviewer #1 (Recommendations For The Authors):

Minor critiques (in no particular order):

1. Peptide 18 was identified based on its strong inhibition for EspP assembly but another peptide, peptide 23, also shows inhibition and has no particular consensus.

We would correct this point. Peptide 23 has a strong consensus to the canonical β-signal. We had explained the sequence consensus of β-signal in the Results section of the text. In the third paragraph, we have added a sentence indicating the relationship between peptide 18 and peptide 23.

(Lines 152-168) "Six peptides (4, 10, 17, 18, 21, and 23) were found to inhibit EspP assembly (Fig. 1A). Of these, peptide 23 corresponds to the canonical β-signal of OMPs: it is the final β-strand of OmpC and it contains the consensus motif of the β-signal (ζxGxx[Ω/Φ]x[Ω/Φ]). The inhibition seen with peptide 23 indicated that our peptidomimetics screening system using EspP can detect signals recognized by the BAM complex. In addition to inhibiting EspP assembly, five of the most potent peptides (4, 17, 18, 21, and 23) inhibited additional model OMPs; the porins OmpC and OmpF, the peptidoglycan-binding OmpA, and the maltoporin LamB (fig. S3). Comparing the sequences of these inhibitory peptides suggested the presence of a sub-motif from within the β-signal, namely [Ω/Φ]x[Ω/Φ] (Fig. 1B). The sequence codes refer to conserved residues such that: ζ, is any polar residue; G is a glycine residue; Ω is any aromatic residue; Φ is any hydrophobic residue and x is any residue (Hagan et al., 2015; Kutik et al., 2008). The non-inhibitory peptide 9 contained some elements of the β-signal but did not show inhibition of EspP assembly (Fig. 1A).

Peptide 18 also showed a strong sequence similarity to the consensus motif of the β-signal (Fig. 1B) and, like peptide 23, had a strong inhibitory action on EspP assembly (Fig. 1A). Variant peptides based on the peptide 18 sequence were constructed and tested in the EMM assembly assay (Fig. 1C)."

1. It is unclear why the authors immediately focused on BamD rather than BamB, given that both were mentioned to mediate interaction with substrate. Was BamB also tested?

We thank the reviewer for this comment. Following the reviewer's suggestion, we have now performed a pull-down experiment on BamB and added it to Fig. S9. We also modified the text of the results as follows.

(Lines 262-265) "Three subunits of the BAM complex have been previously shown to interact with the substrates: BamA, BamB, and BamD (Hagan et al., 2013; Harrison, 1996; Ieva et al., 2011). In vitro pull-down assay showed that while BamA and BamD can independently bind to the in vitro translated OmpC polypeptide (Fig .S9A), BamB did not (Fig. S9B)."

1. For the in vitro folding assays of the OmpC substrates, labeled and unlabeled, no mention of adding SurA or any other chaperone which is known to be important for mediating OMP biogenesis in vitro.

We appreciate the reviewer’s concerns on this point, however chaperones such as SurA are non-essential factors in the OMP assembly reaction mediated by the BAM complex: the surA gene is not essential and the assembly of OMPs can be measured in the absence of exogenously added SurA. It remains possible that addition of SurA to some of these assays could be useful in detailing aspects of chaperone function in the context of the BAM complex, but that was not the intent of this study.

1. For the supplementary document, it would be much easier for the reader to have the legends groups with the figures.

Following the reviewer's suggestion, we have placed the legends of Supplemental Figures together with each Figure.

1. Some of the figures and their captions are not grouped properly and are separated which makes it hard to interpret the figures efficiently.

We thank the reviewer for this comment, we have revised the manuscript and figures to properly group the figures and captions together on a single page.

1. The authors begin their 'Discussion' with a question (line 454), however, they don't appear to answer or even attempt to address it; suggest removing rhetorical questions.

As per the reviewers’ suggestion, we removed this question.

1. Line 464, 'unbiased' should be removed. This would imply that if not stated, experiments are 'negatively' biased.

We removed this word and revised the sentence as follows:

(Lines 431-433) "In our experimental approach to assess for inhibitory peptides, specific segments of the major porin substrate OmpC were shown to interact with the BAM complex as peptidomimetic inhibitors."

1. Lines 466-467; '...go well beyond expected outcomes.' What does this statement mean?

Our peptidomimetics led to unexpected results in elucidating the additional essential signal elements. The manuscript was revised as follows:

(Lines 433-435) "Results for this experimental approach went beyond expected outcomes by identifying the essential elements of the signal Φxxxxxx[Ω/Φ]x[Ω/Φ] in β-strands other than the C-terminal strand."

1. Line 478; '...rich information that must be oversimplified...'?

We appreciate the reviewer’s pointed out. For more clarity, the manuscript was revised as follows:

(Lines 450-453) "The abundance of information which arises from modeling approaches and from the multitude of candidate OMPs, is generally oversimplified when written as a primary structure description typical of the β-signal for bacterial OMPs (i.e. ζxGxx[Ω/Φ]x[Ω/Φ]) (Kutik et al., 2008)."

1. There are typos in the supplementary figures.

We have revised and corrected the Supplemental Figure legends.  

Reviewer #2 (Recommendations For The Authors):

1. In Supplementary Information, I recommend adding the figure legends directly to the corresponding figures. Currently, it is very inconvenient to go back and forth between legends and figures.

Following the reviewer's suggestion, we have placed the legends of Supplemental Figures together with each Figure.

1. Line 94 (p.3): "later"

Lateral?

Yes. We have corrected this.

1. Line 113 (p.3): The result section, "Peptidomimetics derived from E. coli OmpC inhibit OMP assembly" Rationale of the peptide inhibition assay is not clear. How can the peptide sequence that effectively inhibit the assembly interpreted as the b-assembly signal? By competitive binding to BAM or by something else? What is the authors' hypothesis in doing this assay?

In revision, we have added following sentence to explain the aim and design of the peptidomimetics:

(Lines 140-145) "The addition of peptides with BAM complex affinity, such as the OMP β-signal, are capable of exerting an inhibitory effect by competing for binding of substrate OMPs to the BAM complex (Hagan et al., 2015). Thus, the addition of peptides derived from the entirety of OMPs to the EMM assembly assay, which can evaluate assembly efficiency with high accuracy, expects to identify novel regions that have affinity for the BAM complex."

1. Line 113- (p.3) and Fig. S1: The result section, "Peptidomimetics derived from E. coli OmpC inhibit OMP assembly"

Some explanation seems to be needed why b-barrel domain of EspP appears even without ProK?

We appreciate the reviewer’s pointed out. We added following sentence to explain:

(Lines 128-137) "EspP, a model OMP substrate, belongs to autotransporter family of proteins. Autotransporters have two domains; (1) a β-barrel domain, assembled into the outer membrane via the BAM complex, and (2) a passenger domain, which traverses the outer membrane via the lumen of the β-barrel domain itself and is subsequently cleaved by the correctly assembled β-barrel domain (Celik et al., 2012). When EspP is correctly assembled into outer membrane, a visible decrease in the molecular mass of the protein is observed due to the self-proteolysis. Once the barrel domain is assembled into the membrane it becomes protease-resistant, with residual unassembled and passenger domains degraded (Leyton et al., 2014; Roman-Hernandez et al., 2014)."

1. Line 186 (p.6): "Y285"

Y285A?

We have corrected the error, it was Y285A.

1. Lines 245- (p. 7)/ Lines 330- (p. 10)

It needs to be clarified that the results described in these paragraphs were obtained from the assays with EMM.

We appreciate the reviewer’s concerns on these points. For the first half, the following text was added at the beginning of the applicable paragraph to indicate that all of Fig. 4 is the result of the EMM assembly assay.

(Line 241) "We further analyzed the role of internal β-signal by the EMM assembly assay. At the second half, we used purified BamD but not EMM. We described clearly with following sentence."

(Lines 316-318) "We purified 40 different BPA variants of BamD, and then irradiated UV after incubating with 35S-labelled OmpC."

Die IEA korrigiert ihre Prognose zum Ölbedarf 2024 nach oben. Die Nachfrage werde um 180.000 Barrel pro Tag steigern. Das Angebot wird die Nachfrage übertreffen. Die OPEC geht von einer noch größeren Steigerung aus https://www.reuters.com/business/energy/iea-raises-2024-oil-demand-growth-forecast-again-2024-01-18/

Author Response

The following is the authors’ response to the original reviews.

We are very grateful to both reviewers for taking the time to review our manuscript and data in great detail. We thank you for the fair assessment of our work, the helpful feedback, and for recognizing the value of our work. We have done our best to address your concerns below:

eLife assessment This work reports a valuable finding on glucocorticoid signaling in male and female germ cells in mice, pointing out sexual dimorphism in transcriptomic responsiveness. While the evidence supporting the claims is generally solid, additional assessments would be required to fully confirm an inert GR signaling despite the presence of GR in the female germline and GR-mediated alternative splicing in response to dexamethasone treatment in the male germline. The work may interest basic researchers and physician-scientists working on reproduction and

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Cincotta et al set out to investigate the presence of glucocorticoid receptors in the male and female embryonic germline. They further investigate the impact of tissue-specific genetically induced receptor absence and/or systemic receptor activation on fertility and RNA regulation. They are motivated by several lines of research that report inter and transgenerational effects of stress and or glucocorticoid receptor activation and suggest that their findings provide an explanatory mechanism to mechanistically back parental stress hormone exposure-induced phenotypes in the offspring.

Strengths:

A chronological immunofluorescent assessment of GR in fetal and early life oocyte and sperm development.

RNA seq data that reveal novel cell type specific isoforms validated by q-RT PCR E15.5 in the oocyte.

2 alternative approaches to knock out GR to study transcriptional outcomes. Oocytes: systemic GR KO (E17.5) with low input 3-tag seq and germline-specific GR KO (E15.5) on fetal oocyte expression via 10X single cell seq and 3-cap sequencing on sorted KO versus WT oocytes both indicating little impact on polyadenylated RNAs

2 alternative approaches to assess the effect of GR activation in vivo (systemic) and ex vivo (ovary culture): here the RNA seq did show again some changes in germ cells and many in the soma.

They exclude oocyte-specific GR signaling inhibition via beta isoforms.

Perinatal male germline shows differential splicing regulation in response to systemic Dex administration, results were backed up with q-PCR analysis of splicing factors. Weaknesses:

COMMENT #1: The presence of a protein cannot be entirely excluded based on IF data

We agree that very low levels of GR could escape the detection by IF and confocal imaging. We feel that our IF data do match transcript data in our validation studies of the GR KO using (1) qRT-PCR on fetal ovary in Fig 2E and (2) scRNA-seq in germ cells and ovarian soma in Fig S2B.

COMMENT #2: (staining of spermatids is referred to but not shown).

You are correct that this statement was based on a morphological identification of spermatids using DAPI morphology. We have performed a co-stain for GR with the spermatocyte marker SYCP3, and the spermatid/spermatozoa marker PNA (Peanut Agglutinin; from Arachis hypogaea) in adult testis tissue. We have updated Figure 4D to reflect this change, as well as the corresponding text in the Results section.

COMMENT #3: The authors do not consider post-transcriptional level a) modifications also triggered by GR activation b) non-coding RNAs (not assessed by seq).

We thank the reviewer for raising this very important point about potential post-transcriptional (non-genomic) effects of GR in the fetal oocyte. We agree that while our RNA-seq results show only a minimal transcriptional response, we cannot rule out a non-canonical signaling function of GR, such as the regulation of cellular kinases (as reviewed elsewhere1), or the regulation of non coding RNAs at the post-transcriptional level, and we have amended the discussion to include a sentence on this point. However, while we fully acknowledge the possibility of GR regulating non-genomic level cellular signaling, we chose not to explore this option further based on the lack of any overall functional effect on meiotic progression when GR signaling was perturbed- either by KO (Figure 2D) or dex-mediated activation (Figure S3C).

COMMENT #4: Sequencing techniques used are not total RNA but either are focused on all polyA transcripts (10x) or only assess the 3' prime end and hence are not ideal to study splicing

We thank the reviewer for raising this concern, however this statement is not correct and we have clarified this point in the Results section to explain how the sequencing libraries of the male germ cell RNA-seq were prepared. We agree that certain sequencing techniques (such as 3’ Tag-Seq) that generate sequencing libraries from a limited portion of an entire transcript molecule are not appropriate for analysis of differential splicing. This was not the case, however, for the RNA-seq libraries prepared on our male germ cells treated with dexamethasone. These libraries were constructed using full length transcripts that were reverse transcribed using random hexamer priming, thus accounting for sequencing coverage across the full transcript length. As a result, this type of library prep technique should be sufficient for capturing differential splicing events along the length of the transcript. We do, however, point out that these libraries were constructed on polyA-enriched transcripts. Thus while we obtained full length transcript coverage for these polyA transcripts, any differential splicing taking place in non poly-adenylated RNA moieties were not captured. While we are excited about the possibility of exploring GR-mediated splicing regulation of other RNA species in the future, we chose to focus the scope of our current study on polyA mRNA molecules specifically.

COMMENT #5: The number of replicates in the low input seq is very low and hence this might be underpowered

While the number of replicates (n=3-4 per condition) is sufficient for performing statistical analysis of a standard RNA-seq experiment, we do acknowledge and agree with the reviewer that low numbers of FACS-sorted germ cells from individual embryos combined with the low input 3’ Tag-Seq technique could have led to higher sample variability than desired. Given that we validated our bulk RNA-seq analysis of GR knockout ovaries using an orthogonal single-cell RNA-seq approach, we feel that our conclusions regarding a lack of transcriptional changes upon GR deletion remain valid.

COMMENT #6: Since Dex treatment showed some (modest) changes in oocyte RNA - effects of GR depletion might only become apparent upon Dex treatment as an interaction.

We may be missing the nuance of this point, but our interpretation of an effect that is seen only when the KO is treated with Dex would be that the mechanism would not be autonomous in germ cells but indirect or off-target.

COMMENT #7: Effects in oocytes following systemic Dex might be indirect due to GR activation in the soma.

As both the oocytes and ovarian soma express GR during the window of dex administration, we agree that it is possible that the few modest changes seen in the oocyte transcriptome are the result of indirect effects following robust GR signaling in the somatic compartment. However, given that these modest oocyte transcript changes in response to dex treatment did not significantly alter the ability of oocytes to progress through meiosis, we chose not to explore this mechanism further.

COMMENT #8: Even though ex vivo culture of ovaries shows GR translocation to the nucleus it is not sure whether the in vivo systemic administration does the same.

AND

The conclusion that fetal oocytes are resistant to GR manipulation is very strong, given that "only" poly A sequencing and few replicates of 3-prime sequencing have been analyzed and information is lacking on whether GR is activated in germ cells in the systemically dex-injected animals.

If we understand correctly, the first part refers to a technical limitation and the second part takes issue with our interpretation of the data. For the former, we appreciate this astute insight on the conundrum of detecting a response to systemic dex in fetal oocytes, which is generally monitored by nuclear translocation of GR. As shown in Figure 1A and 1B, GR localization is overwhelmingly nuclear in fetal oocytes of WT animals at E13.5 without addition of any dex. We could not, therefore, use GR translocation as a proxy for activation in response to dex treatment. We instead used ex vivo organ culture to monitor localization changes, as we were able to maintain fetal ovaries ex vivo in hormone-depleted and ligand negative conditions. As shown in Fig. 3, these defined culture conditions elicited a shift of GR to the cytoplasm of fetal oocytes. This led us to conclude that GR is capable of translocating between nucleus and cytoplasm in fetal oocytes, and we were able to counteract this loss in nuclear localization by providing dex ligand in the media.

We feel that our conclusion that oocytes are resistant to manipulation of glucocorticoid signaling despite their possession of the receptor and capacity for nuclear translocation is substantiated by multiple results: meiotic phenotyping, bulk RNA-seq and scRNA-seq analysis of both GR KO and dex dosed mice. Our basis for testing the timing and fidelity of meiotic prophase I was the coincident onset of GR expression in female germ cells at E13, and the disappearance of GR in neonatal oocytes as they enter meiotic arrest. The lack of transcriptional changes observed in oocytes in response to dex has made it even more challenging to demonstrate a bona fide “activation” of GR. Observation of a dose-dependent induction of the canonical GR response gene Fkbp5 in the somatic cells of the fetal ovary (Figure S3A and 3A) affirmed that dex traverses the placenta. We agree with the reviewer that it remains possible that dex or GR KO could lead to changes in epigenetic marks or small RNAs in oocytes, and have mentioned these possibilities in the discussion, but we note that even epigenetic perturbations during oocyte development such as the loss of Tet1 or Dnmt1 result in measurable changes in the transcriptome and the timing of meiotic prophase 2–4.

COMMENT #9: This work is a good reference point for researchers interested in glucocorticoid hormone signaling fertility and RNA splicing. It might spark further studies on germline-specific GR functions and the impact of GR activation on alternative splicing. While the study provides a characterization of GR and some aspects of GR perturbation, and the negative findings in this study do help to rule out a range of specific roles of GR in the germline, there is still a range of other potential unexplored options. The introduction of the study eludes to implications for intergenerational effects via epigenetic modifications in the germline, however, it does not mention that the indirect effects of reproductive tissue GR signaling on the germline have indeed already been described in the context of intergenerational effects of stress.

The reviewer raises an excellent point that we have not made sufficient distinction in our manuscript between prior studies of gestational stress and preconception stress and the light that our work may shed on those findings. We have revised the introduction to clarify this difference, and added reference to an outstanding study that identifies glucocorticoid-induced changes to microRNA cargo of extracellular vesicles shed by epididymal epithelial cells that when transferred to mature sperm can induce changes in the HPA axis and brain of offspring 5. Interestingly, this GR-mediated effect in the epididymal epithelial cells concurs with our observation in the adult testis that GR can be detected only cKit+ spermatogonia but not in subsequent stages of spermatids.

COMMENT #10: Also, the study does not assess epigenetic modifications.

We agree with the reviewer that exploring the role of GR in regulating epigenetic modifications within the germline is an area of extreme interest given the potential links between stress and transgenerational epigenetic inheritance. As this is a broader topic that requires a more thorough and comprehensive set of experiments, we have intentionally chosen to keep this work separate from the current study, and hope to expand upon this topic in the future.

COMMENT #11: The conclusion that the persistence of a phenotype for up to three generations suggests that stress can induce lasting epigenetic changes in the germline is misleading. For the reader who is unfamiliar with the field, it is important to define much more precisely what is referred to as "a phenotype". Furthermore, this statement evokes the impression that the very same epigenetic changes in the germline have been observed across multiple generations.

We see how this may be misleading, and we have amended the text of the introduction and discussion accordingly to avoid the use of the term “phenotype”.

COMMENT #12: The evidence of the presence of GR in the germline is also somewhat limited - since other studies using sequencing have detected GR in the mature oocyte and sperm.

As described above in response to Comment #2, we have included immunostaining of adult testis in a revised Figure 4D and shown that we detect GR in PLZF+ and cKIT+ spermatogonia. We also show low/minimal expression in some (SYCP3+) early meiotic spermatocytes, but not in (Lectin+) spermatids. We are not aware of any studies that have shown expression of GR protein in the mature oocyte.

COMMENT #13: The discussion ends again on the implications of sex-specific differences of GR signaling in the context of stress-induced epigenetic inheritance. It states that the observed differences might relate to the fact that there is more evidence for paternal lineage findings, without considering that maternal lineage studies in epigenetic inheritance are generally less prevalent due to some practical factors - such as more laborious study design making use of cross-fostering or embryo transfer.

We thank the reviewer for this valid point, and we have amended the discussion section.

Reviewer #2 (Public Review):

Summary:

There is increasing evidence in the literature that rodent models of stress can produce phenotypes that persist through multiple generations. Nevertheless, the mechanism(s) by which stress exposure produces phenotypes are unknown in the directly affected individual as well as in subsequent offspring that did not directly experience stress. Moreover, it has also been shown that glucocorticoid stress hormones can recapitulate the effects of programmed stress. In this manuscript, the authors test the compelling hypothesis that glucocorticoid receptor (GR)-signaling is responsible for the transmission of phenotypes across generations. As a first step, the investigators test for a role of GR in the male and female germline. Using knockouts and GR agonists, they show that although germ cells in male and female mice have GR that appears to localize to the nucleus when stimulated, oocytes are resistant to changes in GR levels. In contrast, the male germline exhibits changes in splicing but no overt changes in fertility.

Strengths:

Although many of the results in this manuscript are negative, this is a careful and timely study that informs additional work to address mechanisms of transmission of stress phenotypes across generations and suggests a sexually dimorphic response to glucocorticoids in the germline. The work presented here is well-done and rigorous and the discussion of the data is thoughtful. Overall, this is an important contribution to the literature.

Reviewer #1 (Recommendations For The Authors):

RECOMMENDATION #1: To assess whether in females the systemic Dex administration directly activates GR in oocytes it would be great to assess GR activation following Dex administration, and ideally to see the effects abolished when Dex is administered to germline-specific KO animals.

In regard to the recommendation to assess GR activation in response to systemic dex administration, we refer the reviewer back to our response in Comment #8 highlighting the difficulties defining and measuring GR activation in the germline.

This therefore has made it difficult to assess whether any of the modest effects seen in response to dex are abolished in our germline-specific KO animals. While repeating our RNA-seq experiment in dex-dosed germline KO animals would address whether the ~60 genes induced in oocytes are the result of oocyte-intrinsic GR activity, we have decided not to explore this mechanism further due to the overall lack of a functional effect on meiotic progression in response to dex (Figure S3C).

RECOMMENDATION #2: To further strengthen the link between GR and alternative splicing it would be great to see the dex administration experiment repeated in germline specific GR KO's.

While we understand the reviewer’s suggestion to explore whether deletion of GR in the spermatogonia is sufficient to abrogate the dex-mediated decreases in splice factor expression, we chose not to explore the details of this mechanism given that deletion of GR in the male germline does not impair fertility (Figure 6).

RECOMMENDATION #3: I am wondering how much a given reduction in one of the splicing factors indeed affects splicing events. Can the authors relate this to literature, or maybe an in vitro experiment can be done to see whether the level of differential splicing events detected is in a range that can be expected in the case of the magnitude of splicing factor reduction?

It has been shown in many instances in the literature that a full genetic deletion of a single splice factor leads to impairments in spermatogenesis, and ultimately infertility 6–16. We suspect that dex treatment leads to fewer differential splicing events than a full splice factor deletion, given that dex treatment causes a broader decrease in splice factor expression without entirely abolishing any single splice factor. We have amended the discussion section to include this point. While we share the reviewer’s curiosity to compare the effects of dex vs genetic deletion of splicing machinery on the overall magnitude of differential splicing events, we unfortunately do not have access to mice with a floxed splice factor at this time. While we have considered knocking out one or more splice factors in an ex vivo cultured testis to compare alongside dex treatment, our efforts to date have proven unsuccessful due to high cell death upon culture of the postnatal testis for more than 24 hours.

RECOMMENDATION #4: It is unclear from the methods whether in germline-specific KO's also the controls received tamoxifen.

We thank the reviewer for catching this missing piece of information. All control embryos that were assessed received an equivalent dose of tamoxifen to the germline-specific KO embryos. The only difference between cKOs and controls was the presence of the Cre transgene. We have updated the Materials and Methods 3’ Tag-Seq sample preparation section to include the sentence: “Both GRcKO/cKO and control GRflox/flox embryos were collected from tamoxifen-injected dams, and thus were equally exposed to tamoxifen in utero”.

Reviewer #2 (Recommendations For The Authors):

I just have only a few comments/questions.

RECOMMENDATION #5: It is somewhat surprising that GR is expressed in female germ cells, yet there doesn't seem to be a requirement. Is there any indication of what it does? Is the long-term stability of the germline compromised?

We thank the reviewer for these questions, and we agree that it was quite surprising to find a lack of GR function in the female germline despite its robust expression. The question of whether loss of GR affects the long-term stability of the female germline is interesting, given that similar work in GR KO zebrafish has shown impairments to female reproductive capacity, yet only upon aging 17–19.

While we have shared interest in this question, technical limitations thus far have prevented us from properly assessing the effect of GR loss in aged females. Homozygous deletion of GR results in embryonic lethality at approximately E17.5. Conditional deletion of GR using Oct4-CreERT2 with a single dose of tamoxifen (2.5 mg / 20g mouse) at E9.5 results in complete deletion of GR by E10.5, although dams consistently suffer from dystocia and are no longer able to deliver viable pups. While using the more active tamoxifen metabolite (4OHT) at 0.1 mg / 20g has allowed for successful delivery, the resulting deletion rate is very poor (see qPCR results in panel below, left). While using half the dose of standard tamoxifen (1.25 mg / 20g mouse) at E9.5 has on rare occasions led to a successful delivery, the resulting recombination efficiency is insufficient (Author response image 1 right panel).

Author response image 1.

While a Blimp1-Cre conditional KO model was used to assess male fertility on GR deletion, we believe this model may not be ideal for studying fertility in the context of aging. While Blimp1-Cre is highly specific to the germ cells within the gonad, there are many cell types outside of the gonad that express Blimp1, including the skin and certain cells of the immune system. It is unclear, particularly over the course of aging, whether any effects on fertility seen would be due to an oocyte-intrinsic effect, or the result of GR loss elsewhere in the body. While we hope to explore the role of GR in the aging oocyte further using alternative Cre models in the future, this is currently outside the scope of this work.

RECOMMENDATION #6: Figure 5b: what is the left part of that panel? Is it the same volcano plot for germ cells as shown in part a but with splicing factors?

We apologize if this panel was unclear. Yes, the left panel of Figure 5B is in fact the same volcano plot in 5A, labeled with splicing factors instead of top genes. We have edited Figure 5B and corresponding figure legend to clarify this.

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