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Reply to the reviewers

[Reviewer's comments]

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

Summary In this article Roure et al address the role of BMP during formation of the ascidian palps, using Ciona intestinalis. Overexpression of BMP (specifically ADMP) from early stages of development results in complete suppression of palp formation, and early loss of the palp forming region (also called anterior neural border ANB). Using p-Smad1/5/8 antibody staining they show a marker of the ANB (FoxC) is expressed in a region negative for BMP signals. Inhibition of BMP signals is not sufficient to produce ectopic ANB. However, treatment with FGF protein from very early stages (8-cell stage) plus inhibition of BMP signaling (from 8-cell stage) increased FoxC expression. Looking at later stages of development the authors show that in a U-shaped expression domain of Foxg, Smad1/5/8 is active in the ventral-most part, which is expected to form the ventral-most palp. BMP2 treatment from gastrula stages results in loss of the ventral most palp expression of Isl and repression of ventral Foxg expression. Inhibition of BMP signaling from gastrula or neurula stages results in failure of a U-shaped pattern of Isl expression to resolve into the three palp expression domains, and by late tailbud stages, Sp6/7/8/9 (proposed as a repressor of Foxg in the inter-palp territory) expression is reduced and the numbers of specific cell-types making up the palps is increased. These cells are present in a single large palp of dorsal identity. Thus, inhibition of BMP from early gastrula stages results in a single palp made of more cells than the three palps of control larvae, presumably due to recruitment of cells usually present between the palps. The authors then show a similar phenotype in another ascidian species Phallusia mammillata. Using their previous RNA-Seq data of embryos treated with BMP4, they looked for potential novel palp markers and identify a further eight novel markers of the palps. Looking further into this data and at a list of 68 genes expressed in palps (but not exclusively) they find that in whole embryo RNA-Seq data 70% were regulated by BMP signaling, mostly repressed, but some activated by BMP. 30 of these genes were regulated by Notch. Apart from the confusion I explained in my comments below, the data seems to be carefully presented and interpreted. Overall, this manuscript presents a more detailed analysis of the role of BMP signaling during ascidian palp formation, but it remains to be precisely understood.

[Response]

We thank the reviewer for the evaluation of our work.

Major comments

1) I am a little confused about the timing of the protein treatments. In Figure 2, the authors show nicely that at the neurula stages, P-Smad1/5/8 staining abuts the FoxC ANB territory. Then at late neurula P-Smad1/5/8 is detected in the ventral-most part of the Foxg U-shaped part of the palp forming region, presumably the ventral most palp. However, the protein treatments with BMP (and FGF) are carried out from the 8-cell stage, which seems a bit drastic and embryos look difficult to orientate (e.g. Fig. 3D).

[Response]

We first would like to clarify the issue raised from Figure 3. Actually, Figure 3D was the only case where the embryo was shown from the side (the description as a lateral view was inadvertently omitted in the legend). We have now modified Figure 3 by properly showing only dorsal (neural plate) views and lateral views in insets when necessary. In addition, we have added schemes of embryos depicting the main tissues we have examined (palps, CNS and epidermis) and their localization depending on the treatments.

Regarding the timing of treatments, we performed them at the 8-cell stage to make them manageable to perform. At the latest, bFGF treatment should be performed at the 16-cell stage (before neural induction at the 32-cell stage), while BMP2 treatment should be performed at the 64-cell stage (before the onset of Foxc/partial effect at early gastrula (St. 10)). In principle, sequential treatment (first bFGF, then BMP2) could thus be performed. Since earlier treatments, produce the same effects, we reasoned that combined treatments from the 8-cell stage should be equivalent and would avoid fastidious repeated manipulation of the embryos that could negatively impact their development. We are convinced that the way we performed the treatment has no impact on our results (except for the treatment by bFGF alone on Foxc as already discussed in the text) and conclusions.

While BMP-treatment from early stages inhibits all palp gene expression and any sign of palp formation (Figure 1), treatment with BMP from the early gastrula stage, when Smad1/5/8 is detected only in mesendoderm cells and before it is detected in any ectoderm, is sufficient only to block ventral palp formation and cause a partial down-regulation of FoxC expression in the ANB. Thus, there seems to be a discrepancy between the roles proposed for BMP during ANB and palp formation as judged by P-Smad1/5/8 staining and the temporal evidence from BMP- and BMP-inhibitor treatment. Do the authors have some explanation for why they need to treat at least one hour before the BMP-mediated patterning mechanism (as indicated from the P-Smad1/5/8 staining) is taking place? For example, could the authors check how long it takes DMH1 to inhibit P-Smad1/5/8 positive staining? Or BMP to strongly induce P-Smad1/5/8? This seems to be a simple experiment and might go some way to explaining why they need to treat embryos much earlier than I would have thought necessary.

[Response]

We understand the reviewer's concerns, but we do not think that there are major discrepancies in the timing of events. The main rationale is to consider the onset of expression for the main genes of interest. We have examined their dynamics of expression in details, but we do not show them since our conclusions are in agreement with a previous report (Figure 1 from Liu and Satou, 2019). We have summarized the data in the modified Figure 2. Foxc can be detected from early gastrula stages (St. 10) when the palp precursors consist of a single row of 4 cells. This is the exact developmental time when the treatment with BMP2 has partial effects (Figure 4). Once the cells divide to make 2 rows of 4 cells robustly expressing Foxc (St. 12), BMP2 treatment has no effect on Foxc. Similarly, DMH1 treatment has no effect from late neurula stage (St. 16) (Figure 4) that corresponds to the onset of Sp6/7/8/9 expression. We thus consider that modulating BMP pathway has no effect once key regulatory genes have acquired a robust expression in their normal domains. We have enhanced these points in the main text (lines 205-208, lines 228-229).

We think the above discussion should address the points raised by the reviewer. In the contrary, we are willing to perform the suggested experiments.

2) It does not make sense to me that BMP treatment from gastrula stage blocks only ventral palp formation (Figure 4) and ventral Foxg expression (Fig. 5G). In particular, it is the ventral palp region which is positive for P-Smad1/5/8 (Fig.2I,J) so I would not expect the ventral palp to be the most sensitive to BMP-treatment.

[Response]

We were, like the reviewer, surprised by the phenotype. The time window to obtain this phenotype is quite narrow, and most likely deals with the full acquisition of the palp fate ('consolidation' of Foxc expression, onset of Foxg). This is actually a phenotype that we have not characterized in details. And such a characterization may help clarify the role of BMP: does BMP regulate papilla/inter-papilla fates only for the ventral palp or for all three palps? Does BMP 'only' regulate the dorso-ventral identities of the palps?

To better understand the role of BMP in palp formation, we propose to describe this specific phenotype: loss of ventral palp induced by BMP2 treatment at St. 10. We propose to test the following hypotheses. What is the fate of the ventral palp? Conversion into epidermis (more ventral fate)? Conversion into inter-papillar fate? What is the identity of the 2 remaining presumptive palps? Do they still have a dorsal identity? Are they converted into ventral palps? This is part of the proposed experiments for a revision.

Minor comments line 185 I see what the authors are trying to say but I don't agree that BMP limits the domain of FoxC expression as inhibition of BMP has no effect on FoxC. Rather BMP has to be kept out of the ANB in order to allow ANB formation.

[Response]

We have modified the sentence (lines 195-196).

The relationship between Foxg and Sp6/7/8/9 expression is not really clear and it would be better to do this with double ISH if the authors want to show mutually exclusive expression domains, or at least provide a summary figure.

[Response]

We have modified Figure 5 by adding schematic representations of our understanding of the expression patterns in relation to the different precursors of the palp lineage.

In case the reviewer does not find this clarification sufficient, we propose to perform the double fluorescent in situ hybridizations as part of the revision plan.

Line 218, I do not see the data showing that Isl is expressed at a U-shape at st. 23, it seems to be expressed in three dots, unless embryos are treated with DMH1.

[Response]

We apologize for the misunderstanding since the sentence was not clear. We referred to the U-shaped Isl expression under BMP inhibition. Indeed, Isl starts to be expressed in 3 separate domains in the palp forming region, and not following a U-shape as its upstream regulator Foxg (Liu and Satou, 2019). We amended the sentence (lines 234-235).

Figure 6B, G. It could be nice to show a close up of the palps to see elongated cells.

[Response]

The close up pictures have now been added in the modified Figure 6.

Figure 6K. It is better to use a statistical test to support the authors conclusions.

[Response]

As suggested, we have performed a statistical evaluation (Mann-Whitney U test) of the cell counts. The p-values are presented in Figure 6Q. The slight increase of Celf3/4/5/6 is not statistically significant, but it does not impact our conclusion that the number of papilla cells increases following BMP inhibition.

It could be nice to provide a timeline for Smad1/5/8 signaling and the role for BMP signals that are proposed in this manuscript as a summary diagram.

[Response]

Following the suggestion, we have added summary diagrams in Figure 2 for BMP signaling in relation to lineages and gene expression.

lines 66-74 is lacking references.

[Response]

This is now corrected (lines 70-80).

Reviewer #1 (Significance (Required)):

Significance While it is still not clear how BMP signals are established (which ligands for example) and their precise role in palp formation, this manuscript adds more information to our current understanding of the role of BMP signaling during palp formation. In particular it shows that BMP signals need to be kept out of the ANB for its formation and that it is required to resolve the later forming palp territory into three discrete palp regions. However, there is some way to go before this is fully understood. This article will certainly be of interest to ascidian developmental biologists trying to understand the formation and patterning of the larval PNS. It may also be of some interest to evolutionary biologists trying to understand the relationship between the telencephalon territory of vertebrates and the palp forming territory of ascidians as some links have been proposed between these two developmental territories (e.g. line 78).

[Reviewer's comments]

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

Summary. The manuscript presents a detailed examination of how dynamic changes in BMP signaling during the development of the ascidian larval palps. Early in development BMP inhibition is responsible for the formation of a large field within the neuroectoderm that includes, among other fates, the presumptive palps. As development progresses, the territories of BMP activity/inhibition appear to be spatially refined within the palp-forming territory to specify palp versus interpalp fate. The experiments are presented with sufficient replication and statistical rigor.

[Response]

We thank the reviewer for the evaluation of our work.

Major Comments.

1. The researchers should look at otx expression in pFOG>Admp overexpressing embryos. It is difficult to assess from Figure 1, but it appears possible the the entire anterior sensory vesicle (not just the palps) are absent in the pFOG>Admp embryos (can the authors say briefly whether other ectodermal structures such as the atrial primordia or the oral siphon are still present?). Thus, is it possible that the entire a-lineage is disrupted? This would be an important distinction to make: are the defects attributed to experimental BMP activation specific to the palps, or are they more widespread in the anterior neuroectoderm? If the entire a-lineage is mis-fated, might this change the interpretation of the role of BMP inhibition? For example, might the formation of the palps depend on the proper development of the neighboring anterior neural plate? To address this concern, the authors should use a different driver to restrict Admp overexpression only to the palp forming region.

[Response]

In Figure 1, we show that Celf3/4/5/6, a general neural marker was still expressed in pFog>Admp embryos. We explain, in the Figure 1 legend, that this most likely corresponds to the CNS. It does not demonstrate that the anterior sensory vesicle (a-line induced CNS lineage) is still present. Unfortunately, Otx cannot be used as a suitable marker since it is also expressed in the posterior sensory vesicle (A-line lineage) (Hudson et al., 2003). Other a-line markers do exist. However, determining their expression at tailbud stages may not be conclusive since it is most likely that the patterning of the sensory vesicle (hence the expression of these markers) is modified after BMP activation. We have presented in former Figure 3 and Figure S1, strong evidence that the a-line neural lineage is intact at the neural plate stage. To better communicate these data, we have combined then in a modified Figure 3 that includes all markers examined and interpretative embryonic schemes. We show that, following BMP2 treatment, Otx and Celf3/4/5/6 were downregulated in the palp lineage but otherwise normal. Consequently, the a-line CNS lineage is most likely not affected by BMP pathway activation. This does not mean that its later derivatives form normally, but this is an issue that we have not addressed. A previous report indicates that BMP activation leads to Six1/2 repression and, possibly, the absence of oral siphon primordium (based on the images, no description in this paper) (Figure 1 from Abitua et al., 2015).

We think that we have addressed the concern of the reviewer, but would like to comment on the suggested experiment. It is very difficult to find a driver that would allow BMP activation only in the palp lineage (by overexpressing a constitutive active BMP receptor for example). a-line neural linage and palp lineage are intimately linked and separate at gastrula stages (St. 10). The regulatory sequences of Foxc, the first palp specific gene that we know, would thus be interesting. But it is most likely too late according to our whole embryo protein treatments (Figure 4). In agreement with this assumption, overexpressing Bmp2/4 (another BMP ligand) using the regulatory sequences of Dmrt (a master regulator of the palp+a-line CNS lineage expressed just before Foxc) does not apparently abolish palp formation (Extended Data Figure 5 from Abitua et al., 2015).

1. The authors hypothesize that papilla versus inter-papilla fate is controlled by differential BMP signaling. Is it possible to show differential P-Smad staining in papilla versus inter-papilla territories, as in Figure 2 for earlier gastrula-stage embryos? This data would make the authors hypothesis much more compelling. It appears that the authors have the necessary reagents.

[Response]

The actual lineage and fate segregation of papilla and inter-papilla lineage has not been determined as far as we know. Our current understanding comes from indirect evidence from gene expression and gene function, in particular from the study of Foxg and Sp6/7/8/9 by Liu and Satou (2009). Papillae originate from the 3 Foxg/Isl positive spots that are visible at very early tailbud stages. At earlier stages, Isl is not expressed and Foxg is expressed with a U-shape (Figure 5). Within this U, it is most likely that the segregation of papilla and inter-papilla fates takes place when Sp6/7/8/9 starts being expressed at late neurula stages. It is thought that Sp6/7/8/9+/Foxg+ cells will become inter-papilla cells while Sp6/7/8/9-/Foxg+ will become papilla. Our data indicate that BMP signaling is active in the future ventral papilla. We have mapped these data on schematics in the modified Figure 2.

Minor Comments.

1. There is no mention of panels Figure 1 U and V in the text. In the figure legend they are misidentified as panels S and T.

[Response]

This has been corrected.

Very small issue with English usage that occurs throughout the manuscript. The authors should check the use of "palps" versus "palp", particularly when expressions such as the following are used: "palps formation", "palps network", "palps lineage", "palps differentiation", "palps molecular markers", "palps neuronal markers", "palps phenotypes", etc . For example, the sentence, "Here, we show that BMP signaling regulates two phases of palps formation in Ciona intestinalis", should read instead "Here, we show that BMP signaling regulates two phases of palp formation in Ciona intestinalis".

[Response]

Thank you, we have corrected these mistakes.

It would be worth mentioning possible relationships between the tunicate palps and the adhesive glands for larval fish and amphibians. Are there common mechanisms? All of these are anterior ectoderm derivatives.

[Response]

Thank you for the suggestion. We have added a section on that topic in the discussion (line 358).

Please consider providing references in the Introduction for the sentences which end on the following lines of text: 36 ( . . . is the sister group of vertebrates), 46 ( . . . and sensory properties), 48 ( . . . the secretion of adhesive materials), 57 ( . . . on the nervous system in chordates), 68 ( . . . also known as Ap2-like), 74 ( . . . anterior neural territories)

[Response]

References have now been added.

To provide extra emphasis and to help the figures to stand alone with their respective legends, can you mention in the legend for Fig. 2 that D and E are controls? Also, can a brief legend be provided for S2 to give overall indication of staging, scale, orientation, etc.?

[Response]

Actually, the original Fig 2D and 2E correspond to treated embryos as explained in the legend. For clarity, these embryos have been separated from control embryos in the modified Figure 2.

Figure S2 has modified and a legend has been added.

Reviewer #2 (Significance (Required)):

Significance.

This study presents an advance in our understanding of the fine-structure regulation of BMP signaling in sculpting neuroectoderm derivatives. While this study is potentially of broad interest, the authors fail to fully discuss the comparative aspects of this study in the context of conserved chordate developmental mechanisms. This could be remedied without too much difficulty in the Discussion section.

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

Summary: This paper explores the role of BMP signaling for palp formation in ascidians using gain and loss of function approaches. The paper shows that while BMP at early (gastrula) stages prevents formation of the Foxc-positive palp ectoderm in Ciona, at later stages it appears to be essential for separation of the palps (possibly by promoting differentiation of interpapillary cells). The paper further shows that BMP plays similar roles in a different ascidian, Phallusia mammillata. Using previously published RNA-Seq results for the latter species after BMP up-regulation, the authors were able to identify additional BMP-responsive genes expressed in the palp region of ascidians.

[Response]

We thank the reviewer for the evaluation of our work.

Major comments: However, while the effect of BMP overexpression at early stages has been confirmed by two independent strategies (electroporation of the BMP agonist ADMP and BMP2 treatment), the effects of late BMP activation as well as the effects of BMP inhibition at both early and late stages have been studied exclusively by pharmacological treatments with a single BMP signaling agonist (BMP2) and antagonist (DMH1). To substantiate these findings and rule out unspecific side effects, it would have been desirable to verify them with alternative strategies.

[Response]

The reviewer may have missed some of our data. We have shown that BMP inhibition through overexpression of the secreted antagonist Noggin via electroporation using the early ectodermal driver pFog gives the same phenotypes as DMH1 treatment. The effects on Foxc * were presented in Figure S1, and are now presented in the modified Figure 3 (line 170). We also showed that the morphological Cyrano phenotype was observed with Noggin overexpression (modified Figure 6H). We now present a novel Figure S1 with expression of Isl and Celf3/4/5/6* following Noggin overexpression, and stress the use of this independent way of inhibiting BMP (lines 260-264). Given that early or late BMP inhibition lead to the same phenotype, we do not consider that overexpressing Noggin at gastrula stages is necessary.

Regarding BMP activation from gastrula stages, we have only used BMP2 treatment. It may be possible to overexpress Admp using promoters active in the palp lineage such as the ones of Dmrt, Foxc or Foxg. However, it may be difficult to phenocopy the phenotype obtained using BMP2 protein (loss of ventral palp), for two reasons. First, the precise timing to reach high BMP activation is not tightly controlled using such a method. Hence, all drivers should be tested. Second, the different promoters are active progressively later in development and in more and more restricted regions. Consequently, we consider that this requires a huge effort to validate a method (BMP protein treatment) that we already validated for the early effects and that has been used in several publications.

Therefore, while this study provides some new insights into the role of BMP in the specification of the palp forming region and subsequent palp development in ascidians, the evidence provided is relatively weak. Moreover, the scope of the study is quite limited. While identifying some BMP-responsive genes expressed in the palp region and describing the effects of BMP dysregulation on palp morphology, the study does not provide further insights into the underlying mechanisms how BMP patterns this region or affects subsequent palp formation.

[Response]

We are surprised by the appreciation of the reviewer describing our work as 'some new insights'. To our knowledge, this is the first report addressing the role of BMP signaling in palp formation at the molecular level. The only previous report by Darras and Nishida (2001) describes solely the morphology of the palps following overexpression of Bmp2/4 and Chordin overexpression by mRNA injection. We have brought significant novel findings 1) two important steps in palp formation with a precise description of the cellular and molecular actors, and a proposed function for BMP at each step, 2) evidence for conservation of this process in different ascidian species and 3) significant enrichment in the molecular description of this process. Moreover, the reviewer does not ask for specific items, we thus feel in the impossibility to offer satisfaction.

Minor comments:

• 63: ...as the anterior...

[Response]

Corrected.

• 68, 71, 74: references missing

[Response]

References have now been added.

• 73: better: anterior neural territories and placodes

[Response]

Corrected.

• 76: palp territories also share molecular signature with anterior (eg. olfactory) placodes, not only telencephalon

[Response]

Corrected.

• 106: awkward sentence

[Response]

Corrected.

• 114: at what stage was ADMP electroporated?

[Response]

Electroporation of plasmid DNA is performed in the fertilized egg. Transcription of the transgene is controlled by the driver. In this case, with pFog, it occurs from the 16-cell stage. This precision has been added in line 121.

• 134: to facilitate comparison between stages it would be useful to label cells in Fig. 2(eg. which are a-line and b-line cells? Where is the border between them?)

[Response]

As suggested by the reviewer, we have modified Figure 2 with embryo outlines and schemes to better appreciate where BMP signaling is active.

• 152: since Foxc and Foxg overlap with pSMAD1/5/8 at neurula but not gastrula stages, do you know whether this is due to a dorsal expansion of BMP activity or a ventral expansion of Foxc/Foxg expression? Again, labeling of the nuclei would help

[Response]

The change corresponds to a dorsal expansion of P-Smad1/5/8. Our conclusion comes from combining nuclear staining (not shown for simplicity) and available fate maps. The results are presented in schematic diagrams of embryos in frontal views in the modified Figure 2.

• 174: the description is not clear here; what proportion of embryos did show reduction versus expansion of expression?. Why is the reduction shown in Fig.3 D asymmetrical?

[Response]

The proportions are now indicated in line 184.

We apologize for the impression led by Fig 3D. Actually, it was the only case where the embryo was shown from the side (the description as a lateral view was inadvertently omitted in the legend). It did not show an asymmetric repression but an ectopic expression. We have now modified Figure 3 by properly showing only dorsal (neural plate) views and lateral views in insets when necessary. In addition, we have added schemes of embryos depicting the main tissues we have examined (palps, CNS and epidermis) and their localization depending on the treatments. We hope that the results are now clearly presented.

• 198: ... of endogenous...

[Response]

Corrected (line 213).

• 208: I suggest to highlight the regions of changes in Fig. with asterisks/arrows etc.

[Response]

We have added schematic embryos to highlight expression changes in the modified Figure 5.

• 218: contrary to what is stated here, there is no depiction of u-shaped Isl1 expression in control embryos of Fig. 4

[Response]

As also pointed by reviewer 1, we apologize for the misunderstanding since the sentence was not clear. We referred to the U-shaped Isl expression under BMP inhibition. Indeed, Isl starts to be expressed in 3 separate domains in the palp forming region, and not following a U-shape as its upstream regulator Foxg (Liu and Satou, 2019). We amended the sentence (lines 234-235).

• 220: the cell shapes referred to here cannot be seen in Fig. 4 (too small)

[Response]

We have modified Figure 6 to include close up of the palps.

• 271: the description here is confusing: first you talk about 53 genes and the mention palp expression of 12/26. Where does number 26 come from? And why was in situ done then for 27 additional genes? Also, while the comparison with previously published RNA-Seq data was valuable in uncovering additional BMP-sensitive palp markers, it does not provide any substantial new insights into how BMP patterns this territory.

[Response]

We have amended the sentence to make it clearer (lines 291-295).

• line 624: where

[Response]

Thank you. Corrected line 731.

• Fig. 2: to facilitate comparison between stages it would be useful to label cells (eg. which are a-line and b-line cells? Where is the border between them?)

[Response]

Already responded above.

-Fig. 3: Why is the expression in D asymmetrical? In the main text you write that expression is expanded in some embryos but reduced in others - Please show examples also of the expanded phenotype and give numbers

[Response]

Already responded above.

• Fig. 6: small panels in I, L, N need to be explained (single channels), white signal needs to be explained (overlap ?)

[Response]

We used white for better display of separate single channels. Given the confusion and the good quality of the 2 color fluorescent in situ images, we removed these panels in the modified Figure 6.

White in K and L correspond to overlap (explained in the legend).

• Fig. S2: legend is missing

[Response]

This has been amended.

Reviewer #3 (Significance (Required)):

Since the study does not provide substantial new insights into the mechanisms how BMP patterns the palp forming region or affects subsequent palp formation in ascidians, it will be of interest mostly for a specialized audience in the field of developmental biology.

[Response]

We do not agree with the reviewer as discussed above. The description of the role of BMP signaling in the specification of the ANB and its subsequent patterning in ascidians has interesting evolutionary implications and should be of interest for a broader audience.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

Summary:

This paper explores the role of BMP signaling for palp formation in ascidians using gain and loss of function approaches. The paper shows that while BMP at early (gastrula) stages prevents formation of the Foxc-positive palp ectoderm in Ciona, at later stages it appears to be essential for separation of the palps (possibly by promoting differentiation of interpapillary cells). The paper further shows that BMP plays similar roles in a different ascidian, Phallusia mammillata. Using previously published RNA-Seq results for the latter species after BMP up-regulation, the authors were able to identify additional BMP-responsive genes expressed in the palp region of ascidians.

Major comments:

However, while the effect of BMP overexpression at early stages has been confirmed by two independent strategies (electroporation of the BMP agonist ADMP and BMP2 treatment), the effects of late BMP activation as well as the effects of BMP inhibition at both early and late stages have been studied exclusively by pharmacological treatments with a single BMP signaling agonist (BMP2) and antagonist (DMH1). To substantiate these findings and rule out unspecific side effects, it would have been desirable to verify them with alternative strategies.

Therefore, while this study provides some new insights into the role of BMP in the specification of the palp forming region and subsequent palp development in ascidians, the evidence provided is relatively weak. Moreover, the scope of the study is quite limited. While identifying some BMP-responsive genes expressed in the palp region and describing the effects of BMP dysregulation on palp morphology, the study does not provide further insights into the underlying mechanisms how BMP patterns this region or affects subsequent palp formation.

Minor comments:

• 63: ...as the anterior...
• 68, 71, 74: references missing
• 73: better: anterior neural territories and placodes
• 76: palp territories also share molecular signature with anterior (eg. olfactory) placodes, not only telencephalon
• 106: awkward sentence
• 114: at what stage was ADMP electroporated?
• 134: to facilitate comparison between stages it would be useful to label cells in Fig. 2(eg. which are a-line and b-line cells? Where is the border between them?)
• 152: since Foxc and Foxg overlap with pSMAD1/5/8 at neurula but not gastrula stages, do you know whether this is due to a dorsal expansion of BMP activity or a ventral expansion of Foxc/Foxg expression? Again, labeling of the nuclei would help
• 174: the description is not clear here; what proportion of embryos did show reduction versus expansion of expression?. Why is the reduction shown in Fig.3 D asymmetrical?
• 198: ... of endogenous...
• 208: I suggest to highlight the regions of changes in Fig. with asterisks/arrows etc.
• 218: contrary to what is stated here, there is no depiction of u-shaped Isl1 expression in control embryos of Fig. 4
• 220: the cell shapes referred to here cannot be seen in Fig. 4 (too small)
• 271: the description here is confusing: first you talk about 53 genes and the mention palp expression of 12/26. Where does number 26 come from? And why was in situ done then for 27 additional genes? Also, while the comparison with previously published RNA-Seq data was valuable in uncovering additional BMP-sensitive palp markers, it does not provide any substantial new insights into how BMP patterns this territory.
• line 624: where
• Fig. 2: to facilitate comparison between stages it would be useful to label cells (eg. which are a-line and b-line cells? Where is the border between them?) -Fig. 3: Why is the expression in D asymmetrical? In the main text you write that expression is expanded in some embryos but reduced in others - Please show examples also of the expanded phenotype and give numbers
• Fig. 6: small panels in I, L, N need to be explained (single channels), white signal needs to be explained (overlap ?)
• Fig. S2: legend is missing

Significance

Since the study does not provide substantial new insights into the mechanisms how BMP patterns the palp forming region or affects subsequent palp formation in ascidians, it will be of interest mostly for a specialized audience in the field of developmental biology.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary.

The manuscript presents a detailed examination of how dynamic changes in BMP signaling during the development of the ascidian larval palps. Early in development BMP inhibition is responsible for the formation of a large field within the neuroectoderm that includes, among other fates, the presumptive palps. As development progresses, the territories of BMP activity/inhibition appear to be spatially refined within the palp-forming territory to specify palp versus interpalp fate. The experiments are presented with sufficient replication and statistical rigor.

Major Comments.

1. The researchers should look at otx expression in pFOG>Admp overexpressing embryos. It is difficult to assess from Figure 1, but it appears possible the the entire anterior sensory vesicle (not just the palps) are absent in the pFOG>Admp embryos (can the authors say briefly whether other ectodermal structures such as the atrial primordia or the oral siphon are still present?). Thus, is it possible that the entire a-lineage is disrupted? This would be an important distinction to make: are the defects attributed to experimental BMP activation specific to the palps, or are they more widespread in the anterior neuroectoderm? If the entire a-lineage is mis-fated, might this change the interpretation of the role of BMP inhibition? For example, might the formation of the palps depend on the proper development of the neighboring anterior neural plate? To address this concern, the authors should use a different driver to restrict Admp overexpression only to the palp forming region.
2. The authors hypothesize that papilla versus inter-papilla fate is controlled by differential BMP signaling. Is it possible to show differential P-Smad staining in papilla versus inter-papilla territories, as in Figure 2 for earlier gastrula-stage embryos? This data would make the authors hypothesis much more compelling. It appears that the authors have the necessary reagents.

Minor Comments.

1. There is no mention of panels Figure 1 U and V in the text. In the figure legend they are misidentified as panels S and T.
2. Very small issue with English usage that occurs throughout the manuscript. The authors should check the use of "palps" versus "palp", particularly when expressions such as the following are used: "palps formation", "palps network", "palps lineage", "palps differentiation", "palps molecular markers", "palps neuronal markers", "palps phenotypes", etc . For example, the sentence, "Here, we show that BMP signaling regulates two phases of palps formation in Ciona intestinalis", should read instead "Here, we show that BMP signaling regulates two phases of palp formation in Ciona intestinalis".
3. It would be worth mentioning possible relationships between the tunicate palps and the adhesive glands for larval fish and amphibians. Are there common mechanisms? All of these are anterior ectoderm derivatives.
4. Please consider providing references in the Introduction for the sentences which end on the following lines of text: 36 ( . . . is the sister group of vertebrates), 46 ( . . . and sensory properties), 48 ( . . . the secretion of adhesive materials), 57 ( . . . on the nervous system in chordates), 68 ( . . . also known as Ap2-like), 74 ( . . . anterior neural territories)
5. To provide extra emphasis and to help the figures to stand alone with their respective legends, can you mention in the legend for Fig. 2 that D and E are controls? Also, can a brief legend be provided for S2 to give overall indication of staging, scale, orientation, etc.?

Significance

This study presents an advance in our understanding of the fine-structure regulation of BMP signaling in sculpting neuroectoderm derivatives. While this study is potentially of broad interest, the authors fail to fully discuss the comparative aspects of this study in the context of conserved chordate developmental mechanisms. This could be remedied without too much difficulty in the Discussion section.

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Referee #1

Evidence, reproducibility and clarity

Summary

In this article Roure et al address the role of BMP during formation of the ascidian palps, using Ciona intestinalis. Overexpression of BMP (specifically ADMP) from early stages of development results in complete suppression of palp formation, and early loss of the palp forming region (also called anterior neural border ANB). Using p-Smad1/5/8 antibody staining they show a marker of the ANB (FoxC) is expressed in a region negative for BMP signals. Inhibition of BMP signals is not sufficient to produce ectopic ANB. However, treatment with FGF protein from very early stages (8-cell stage) plus inhibition of BMP signaling (from 8-cell stage) increased FoxC expression. Looking at later stages of development the authors show that in a U-shaped expression domain of Foxg, Smad1/5/8 is active in the ventral-most part, which is expected to form the ventral-most palp. BMP2 treatment from gastrula stages results in loss of the ventral most palp expression of Isl and repression of ventral Foxg expression. Inhibition of BMP signaling from gastrula or neurula stages results in failure of a U-shaped pattern of Isl expression to resolve into the three palp expression domains, and by late tailbud stages, Sp6/7/8/9 (proposed as a repressor of Foxg in the inter-palp territory) expression is reduced and the numbers of specific cell-types making up the palps is increased. These cells are present in a single large palp of dorsal identity. Thus, inhibition of BMP from early gastrula stages results in a single palp made of more cells than the three palps of control larvae, presumably due to recruitment of cells usually present between the palps.

The authors then show a similar phenotype in another ascidian species Phallusia mammillata. Using their previous RNA-Seq data of embryos treated with BMP4, they looked for potential novel palp markers and identify a further eight novel markers of the palps. Looking further into this data and at a list of 68 genes expressed in palps (but not exclusively) they find that in whole embryo RNA-Seq data 70% were regulated by BMP signaling, mostly repressed, but some activated by BMP. 30 of these genes were regulated by Notch.

Apart from the confusion I explained in my comments below, the data seems to be carefully presented and interpreted. Overall, this manuscript presents a more detailed analysis of the role of BMP signaling during ascidian palp formation, but it remains to be precisely understood.

Major comments

1. I am a little confused about the timing of the protein treatments. In Figure 2, the authors show nicely that at the neurula stages, P-Smad1/5/8 staining abuts the FoxC ANB territory. Then at late neurula P-Smad1/5/8 is detected in the ventral-most part of the Foxg U-shaped part of the palp forming region, presumably the ventral most palp. However, the protein treatments with BMP (and FGF) are carried out from the 8-cell stage, which seems a bit drastic and embryos look difficult to orientate (e.g. Fig. 3D). While BMP-treatment from early stages inhibits all palp gene expression and any sign of palp formation (Figure 1), treatment with BMP from the early gastrula stage, when Smad1/5/8 is detected only in mesendoderm cells and before it is detected in any ectoderm, is sufficient only to block ventral palp formation and cause a partial down-regulation of FoxC expression in the ANB. Thus, there seems to be a discrepancy between the roles proposed for BMP during ANB and palp formation as judged by P-Smad1/5/8 staining and the temporal evidence from BMP- and BMP-inhibitor treatment. Do the authors have some explanation for why they need to treat at least one hour before the BMP-mediated patterning mechanism (as indicated from the P-Smad1/5/8 staining) is taking place? For example, could the authors check how long it takes DMH1 to inhibit P-Smad1/5/8 positive staining? Or BMP to strongly induce P-Smad1/5/8? This seems to be a simple experiment and might go some way to explaining why they need to treat embryos much earlier than I would have thought necessary.
2. It does not make sense to me that BMP treatment from gastrula stage blocks only ventral palp formation (Figure 4) and ventral Foxg expression (Fig. 5G). In particular, it is the ventral palp region which is positive for P-Smad1/5/8 (Fig.2I,J) so I would not expect the ventral palp to be the most sensitive to BMP-treatment.

Minor comments

line 185 I see what the authors are trying to say but I don't agree that BMP limits the domain of FoxC expression as inhibition of BMP has no effect on FoxC. Rather BMP has to be kept out of the ANB in order to allow ANB formation.

The relationship between Foxg and Sp6/7/8/9 expression is not really clear and it would be better to do this with double ISH if the authors want to show mutually exclusive expression domains, or at least provide a summary figure.

Line 218, I do not see the data showing that Isl is expressed at a U-shape at st. 23, it seems to be expressed in three dots, unless embryos are treated with DMH1.

Figure 6B, G. It could be nice to show a close up of the palps to see elongated cells.

Figure 6K. It is better to use a statistical test to support the authors conclusions.

It could be nice to provide a timeline for Smad1/5/8 signaling and the role for BMP signals that are proposed in this manuscript as a summary diagram.

lines 66-74 is lacking references.

Significance

While it is still not clear how BMP signals are established (which ligands for example) and their precise role in palp formation, this manuscript adds more information to our current understanding of the role of BMP signaling during palp formation. In particular it shows that BMP signals need to be kept out of the ANB for its formation and that it is required to resolve the later forming palp territory into three discrete palp regions. However, there is some way to go before this is fully understood. This article will certainly be of interest to ascidian developmental biologists trying to understand the formation and patterning of the larval PNS. It may also be of some interest to evolutionary biologists trying to understand the relationship between the telencephalon territory of vertebrates and the palp forming territory of ascidians as some links have been proposed between these two developmental territories (e.g. line 78).

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Reply to the reviewers

Reviewer 1

This paper identifies a role for the hereditary spastic paraplegia protein spatacsin in lysosome morphology, positioning and dynamics, and undertakes detailed mechanistic studies to try to identify the mechanism for this effect. In doing so the paper elucidates further mechanistic information about the properties of two other hereditary spastic paraplegia proteins, spastizin and AP5Z1. The work is done in mammalian cells and uses a combination of over-expression, depletion and biochemical studies. The main findings are:

1. The authors present evidence that spatacsin is an ER-localised protein.
2. Murine embryonic fibroblasts lacking spatacsin have a reduced number of tubular lysosomes and the remaining lysosomes are less motile. In general, a relationship between tubular lysosome morphology and lysosome motility, often in association with the endoplasmic reticulum (ER), is demonstrated. These tubular lysosomes are catalytically active and acidic.
3. In terms of mechanism of this effect, by combining a yeast-two hybrid and siRNA phenotypic screen, the authors identify a number of spatacsin-interacting proteins that also regulate lysosomal tubulation. The most important of these for the purposes of this paper is UBR4, an E3 ubiquitin ligase.
4. The authors show that spatacsin and UBR4 promote degradation of AP5Z1, and that this property required the ability of spatacsin to interact with UBR4. Somewhat surprisingly, as AP5Z1 is a coat protein, this degradation appeared to occur within the lumen of the lysosome - the authors speculate how this could be in the discussion.
5. The authors then demonstrate that AP5Z1 and spastizin, both hereditary spastic paraplegia proteins, compete for binding with spatacsin.
6. The relationship between spatacsin, spastizin, AP5Z1 and motor proteins in then examined. There is a known interaction between spastizin and KIF13A and expression of a dominant negative KIF13A protein reduced lysosomal tubulation. The authors then demonstrate an interaction between AP5Z1 and the p150Glued dynein/dynactin complex member, then showed that expression of a dominant negative p150Glued protein reduced lysosomal tubulation.
7. Finally, that authors demonstrate the relevance of these findings to neurons, the target cells of hereditary spastic paraplegia, by showing that lysosomal tubulation and axonal transport are reduced in mouse neurons lacking spastacsin, and that depletion of UBR4 or AP5Z1 affected these as expected from the experiments above.

Major comments:

Overall I believe that the key conclusions of this paper are generally convincing and that the work is of high quality. However, I do have some reservations:

1. The localisation of spatacsin on the ER. It is always difficult to be convinced about colocalization of a diffuse punctate marker and the ER. From the STED experiments in figure 1, while it definitely seems that there is some spatacsin on the ER, there also appears to be some spatacsin puncta that are not. I'd like to know if these puncta represent lysosome-associated spatacsin. This is important for interpretation of the subsequent experiments (see point 3 below). I also think quantification of these co-localisation will increase confidence in the results. In addition, a caveat of the immunofluorescence studies is that they use over-expressed spatacsin. I appreciate that there are no good antibodies to endogenous spatacsin, but I don't think this limitation is sufficiently acknowledged. As the claim of ER-localisation is critical for the proposed mechanistic model, and in the absence of experiments with endogenously tagged spatacsin, this makes the biochemical fractionation studies of figure 1C very important. To make these more convincing I would prefer to see additional control markers to verify the separation of lysosomal and ER compartments - e.g. lamp1, lamp2, an ER tubular marker such as a REEP5 or a reticulon.

Authors response : We agree with the reviewer that the localization of spatacsin is critical, and we appreciate the knowledge of the reviewer concerning the lack of good antibodies to endogenous spatacsin. We better acknowledged this limitation in our revised manucript (p. 5 and p. 15). We performed extra experiments to convincingly show that spatacsin is indeed localized at the ER. First, we performed 3-color STED experiments to visualize in the same cell spatacsin, the ER and lysosomes. The preliminary data seem to indicate that some spatacsin is associated with lysosomes at ER-lysosomes contact site. We plan to add quantifications of colocalization between spatacsin and ER staining at STED resolution to better support the fact that spatacsin is a protein of the ER.

Moreover, as requested, we have performed a western blot with Lamp2 and REEP5 antibodies on the ER- and lysosome-enriched fractions (New Figure 1B). This western blot shows that a significant proportion of Lamp2 is present in the ER-enriched fraction, which may be explained by the strong association of ER with late endosomes and lysosomes. Yet the lysosome-enriched fractions that contained no ER markers do not present spatacsin staining, suggesting that spatacsin is either in the ER or in lysosomes associated with the ER that are not positive for cathepsin D. We reformulated the text of Figure 1 according to the new included data (p. 5-6).

The authors generally do a good job of quantifying their results. However, this is lacking for the biochemical experiments (immunoblotting and IP) in figures 4 and 5, and I would prefer to see these quantified (the quantification should include data from repeat experiments so that we can judge the reproducibility of the results).

Authors response : We agree that our presentation did not indicate that the western blots were repeated several times. We have added quantifications for the western blots present in Figures 4 and 5.

On page 10, referring to the proximity ligation results, the authors comment: "This suggests that the spatacsin-spastizin interaction occurs at contact sites between the ER and lysosomes to allow spastizin recruitment to lysosomes". I'm not sure this statement is fully supported, as mentioned at point 1 above it is possible that some steady state spatacsin is at lysosomes. To fully support this, we'd need to see the PLA signal also convincingly co-localise with an ER marker.

Authors response : We will perform extra PLA experiment to indeed show that the spots where spatacsin and spastizin colocalize with an ER marker. This data will be added in Figure 5.

In figure 6C and D the effect of spastizin on lysosomal tubulation and dynamics is investigated. Wartmannin treatment is used to do this, as it is known to remove spastizin from lysosomes. However, this is a very indirect manipulation that could have many other consequences and it would be better to demonstrate this directly by showing the effect of depletion of spastizin on lysosomal morphology/dynamics. I also think the role of AP5Z1 in tubulation/dynamics would be better supported with additional experiments to deplete the protein - at present only over-expression is examined.

Authors response: *We added new data to answer this comment. Downregulation of spastizin using siRNA led to lower number of tubular lysosomes and decreased the proportion of dynamic lysosomes, showing that spastizin is required to regulate lysosome motility (Figure 6B-6C Supplementary Figure 7B). We have also added new data regarding downregulation of AP5Z1 (Figure 6A-6C-Supplementary 7A). Both overexpression and downregulation of AP5Z1 using siRNA decreased the number of tubular lysosomes and decreased the proportion of dynamic lysosomes (Figure 6A-6C-Supplementary Figure 6C-D). *

This observation suggests that the levels of AP5Z1 must be tightly regulated to control lysosome motility. We added discussion about this point as well (p.12-13).

While the experiments showing that over-expression of dominant negative forms of KIF13A and p150Glued affect lysosomal tubulation/dynamics provide good circumstantial evidence that spatacsin influences these lysosomal properties via its interactions with spastizin and AP5Z1 (which bind to these motor proteins), the authors have not shown that the interaction of the motor proteins with spastizin and AP5Z1 is required for this ability to regulate lysosome tubulation/dynamics. This means that the model presented in figure 7 is not fully supported by the data. If the authors have been able to map the binding regions for these interactions then perhaps this could be investigated with rescue experiments, although I appreciate that this is potentially a major piece of work and perhaps outside the scope of this paper. An alternative would be that the authors acknowledged this part of the model as somewhat speculative.

Authors response : We agree with the reviewer that our data do not show that KIF13A and p150Glued interact directly with spastizin and AP5Z1 to regulate lysosome dynamics. It has previously been shown that the adaptor complex AP2 interacts with p150glued via the ear domain of AP2 b subunit (Kononenko et al, 2017). It is therefore likely that the interaction of adaptor complex 5 with p150-Glued also occurs via AP5B1 subunit, and thus interaction of AP5Z1 with p150 glued would be indirect. *We discussed this point carefully (p.16). *

*Regarding the interaction of Spastizin with KIF13A, it was identified by yeast-two hybrid screen and validated by GST-pulldown (Sagona et al, 2010). This showed that KIF13A interacts with the C-terminal domain of Spastizin, and we discussed this point. To confirm that KIF13A interaction with spastizin is required to promote its role in tubular lysosome formation and dynamics, we can perform an experiment where we downregulate endogenous mouse spastizin using siRNA and express either full length human spastizin to rescue the effect of the siRNA, or overexpress a human spastizin lacking its C-terminal domain required for the interaction with KIF13A (where we would expect no rescue). This would strengthen our conclusion on the role of KIF13A in link with spastizin to regulate the formation and dynamics of tubular lysosomes. We could add these data in Figure 6 (or Supplementary Figure 7). *

• Are the experiments adequately replicated and statistical analysis adequate?

In general I am not convinced that the statistical tests are applied rigorously in this paper. Most experiments are done three times, but the "n" used for statistical testing is typically chosen as, e.g. the number of cells, number of lysosomes, rather than number of biological repeat experiments. This means that inter-experimental variability is not rigorously taken into account. A more rigorous practice would be to use the mean measures for each of three biological repeats and apply the statistical tests to the three means, so n=3 if three repeats were done. Superplots would be a nice way to graphically display these data.

Authors response : We agree with the comments of the reviewer regarding data presentation. We have therefore changed the presentation of all graphs of the manuscript using superplots that allow us to show all the points that were analyzed as well as the mean value for each biological replicate, and performed statistical analyses by comparing the biological replicates as proposed in Lord et al, JCB 2020 (10.1083/jcb.202001064).

Minor comments:

1. In supplementary figure 3D I cannot honestly say that I see the smaller band.

Authors response : We agree that this western blot is not clear. We will provide a new western blot.

When first called out, I expected supplementary tables 1 and 2 to show the list of interactors with wild-type spatacsin and spatacsind32-34 respectively, but this is not what they show.

Author response : We have added two supplementary data tables (Now Supplementary Tables 1 and 2) to give the list of interactors of wild-type C-terminal domain of spatacsin and spatacsinD32-34, respectively.

Supplementary Tables 3 and 4 now refer to the analysis of the downregulation experiments by respectively the neural network method and the tubular lysosome detection method.

The experiments in Figure 4A are a little problematic in the way that they are called out. The first call refers to just a small subset of the data in the figure, and the figure is then called out at various points later in the paper. This is quite confusing. Is there any way this could be simplified?

Authors response :We agree with the reviewer that Figure 4A was called at various points of the manuscript. This was to avoid duplicating data into two separate figures. However, we have modified the presentation of Figure 4 and Figure 5. We have included new Figure 4C to show that downregulation of UBR4 prevents the degradation of AP5Z1 upon overexpression of Spatacsin-GFP, but also in basal conditions in wild-type fibroblasts. The co-IP that was originally presented in Figure 4A has now been moved into Supplementary Figure 6A.

The section on page 10: "Spatacsin also interacts with spastizin, and is required to recruit spastizin to lysosomes (Hirst et al., 2021). ........ We hypothesized that spatacsin interaction with spastizin was required for spastizin localization to lysosomes." Is odd, as the authors seem to be hypothesising an observation that they have just said has already been demonstrated.

Authors response : We agree that these sentences were odd. We have rephrased the paragraph (p. 11).

Can the authors explain why there is so little interaction between wild-type KIF13A and spastizin?

Authors response : The interaction domain of spastizin with KIF13A is close to the motor domain according to the two-hybrid data published by Sagona et al (2010). The dominant negative construct of KIF13A that is devoid of the motor domain (KIF13A-ST) may thus facilitate access of spastizin to binding domain. We have commented on this point in the text (p.13).

In figure 6G p150Glued signal is also present in the control IP lane, which casts doubt on the specificity of the interaction. Could the authors generate a cleaner result?

Authors response : We have repeated the experiment 3 times, always with some p150Glued signal present in the control IP. Of note, as stated in the method section, we have increased the concentration of NaCl in the washing of this co-IP to decrease non-specific binding of p150glued to control beads, but we could not get cleaner results so far. We will try to get cleaner western blot to illustrate Figure 6G.

I would be interested to see how AP5Z1 expression differs between neurons with and without spatacsin- we would expect similar results to those shown in the MEFS.

*Authors response : We have not checked the levels of AP5Z1 in neurons with and without spatacsin yet. However, the complete knockout of spatacsin strongly modifies the levels of its partners. We previously showed that spastizin levels are decreased by >90% in Spg11 knockout brain (Branchu et al, 2017). Furthermore, the levels of AP5Z1 have been shown to be decreased by ~50% in fibroblasts of SPG11 and SPG15 patients (Hirst et al, 2015). *

*Our work shows that spatacsin promotes the degradation of AP5Z1 by lysosomes. It is possible that other degradation mechanism(s) may exist and could explain the lower levels of AP5Z1 in knockout cells. We discussed this point (p.15). *

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

In this study Pierga et al. report that SPG11 (spatacsin) is an ER-resident protein involved in the regulation of ER-lysosome contact sites (in particular tubular lysosomes) and subsequent faster motility of tubular lysosomes, as well in the degradation of AP5Z1 (SPG48), which forms a heterotrimeric complex with SPG15 (spastizin) and SPG11. This complex has been localized by several groups on the cytoplasmic side of LAMP-1-positive lysosomes. In addition, mutations in SPG11, SPG15, and SPG48 patients share various clinical features and were supported by biochemical/cell biological data from Spg11 and Spg15 KO mouse models and cultured cells both from patients and mice, respectively, demonstrating e.g. accumulation of autolysosome storage material, defects in the autophagic lysosome reformation process, and the loss of cortical motoneurons and Purkinje cells.

Major concerns:

i) Fig. 1, 2, 3: major disadvantage of this study is the analysis of overexpressed proteins (SPG11-V5, GFP-Sec61, and Lamp1-mCherry) which might contribute to the observed strong expression of SPG11-V5 in the ER/ER-enriched fraction. The results should be compared with the endogenous expressed proteins.

Authors response :* As stated by reviewer 1, there are no good antibodies to endogenous spatacsin, and therefore we have to rely on expression of tagged spatacsin to study its localization by immunohistochemistry. For the colocalization with the ER, we stained the latter by GFP-Sec61 that is a widely used marker for this compartment. To confirm our results, we plan to try to perform new STED imaging with REEP5 antibody to stain the ER, and Lamp1 antibody to label lysosomes, avoiding overexpression of proteins to label the subcellular compartments. Furthermore, as it is not possible to localize endogenous spatacsin by immunostaining, we addressed its localization by biochemical fractionation and western blots comparing wild-type and Spg11 knockout samples. *

For Figure 2, the data presented were indeed obtained using transfection of Lamp1-mCherry. However, we confirmed our observation of Figure 2A using alternative staining of lysosomes (Lysotracker or loading of lysosomes with Texas-Red Dextran). We therefore think that our data presented in figure 2 are valid, and that the effect we observed on tubular lysosomes was not affected by expression of Lamp1-mCherry.

In Figure 3, the lysosome were labelled with Texas-Red Dextran, and thus all the data presented in figure 3 do not rely on overexpression.

In Fig. 1C the lack of the mature Cathepsin D form which is proteolytically generated only in lysosomes from the higher molecular mass precursor is misleading and should be related to presence of lysosomal membrane proteins.

Authors response: As requested, we have performed a western blot to show the lysosomal membrane protein Lamp2 on the ER- and lysosome-enriched fractions (Figure 1B). This western blot shows that a significant proportion of Lamp2 is actually present in the ER-enriched fraction, which may be explained by the strong association of ER with late endosomes and lysosomes previously described (Friedman et al, 2013). Yet the lysosome-enriched fractions that contained no ER markers do not present spatacsin staining, suggesting that spatacsin is either in the ER or in lysosomes associated with the ER. We reformulated the text of Figure 1 according to the new included data (p 5-6). The 3-colours STED experiment that we plan to perform to answer reviewer 1 comments will help discriminate between these possibilities.

Fig. 1D: the TEM image shows only a single lysosome and proposed ER contact zones in wt-MEFs without comparison with Spg11 KO MEFs (only in the quantification). Without double immunogold labeling of SPG11 (and their lack on SPG11 KO cell lysosomes) and known ER contact-site proteins this image and the conclusion are insufficient.

Authors response : We have added an image of a lysosome taken from a knockout fibroblast (Figure 1E). As stated above there are no good antibodies to spatacsin for immunostaining, so it will not be possible to perform double immunogold labelling. This prevents us from claiming that spatacsin is a protein enriched at contact site. We therefore modulated our result section and discussion accordingly (p.5-6 and p.16).

ii) The rationale for the selection of the deleted Spg11 region D32-34 is not clear. What are the symptoms of this Spg11 knock-in mouse? A more detailed description of the phenotype is required Is the phenotype including the accumulation of LC3-positive material similar to the phenotype of the SPG11 KO mouse which has been published by Varga et al.(2015) and Branchu et al. (2017) ? If not, is the new mechanisms reported here not so important?

Author response : We have added new data (Supplementary Figure 3E-F) showing motor and cognitive impairment in mice expressing truncating spatacsin, although the motor dysfunction is slightly less marked than in Spg11 knockout animals. We also checked for accumulation of autophagy markers. We did not use LC3, but p62 that labels substrates to be degraded by autophagy. We observed accumulation of p62 in Spg11 knockout and in Spg11D32-34/D32-34 mouse neurons (Supplementary Figure 3G). These data support the functional importance of the domain encoded by exons 32 to 34 of Spg11. We commented on this in the text (p.9).

iii) p8/Fig. 3F/Suppl.Fig.3F- the most important part of the manuscript: what are the parameters of lysosomal staining in images that were used to identify genes important for lysosome tubulation by the neural network?

Authors response : For screening in Figure 3, lysosomes were stained by loading fibroblasts with Texas-Red Dextran overnight, followed by a wash of at least 4 hours. The neural network was first trained to discriminate between control and Spg11-/- fibroblasts, using any parameters of the lysosomal staining, not necessarily lysosome tubulation. This is a completely unsupervised and unbiased method, but one of its drawbacks is that we do not know which parameters were used by the network to discriminate between control and Spg11-/- fibroblasts. Therefore, we validated the classification performed by the neural network on a data set independent from the training set before using it for the screening. We rephrased the paragraph to make it clearer (p.9).

I cannot understand how the authors predict the probability of the cell to be considered as an Spg11 KO fibroblast (why not as an Spg11 D32-34 knock-in fibroblast?) as the basis for the selection of interaction candidates.

Author response : The neural network was trained on sets of images obtained from wild-type and Spg11 KO fibroblasts, which were expected to represent extreme lysosomal phenotypes linked to spatacsin function. We could therefore predict the probability of cells to be considered as Spg11 KO, not as Spg11 *D32-34 fibroblasts. We clarified this in the text (p9). *

A simple statement that the neural network approach identified those genes is too weak and requires more convincing experimental data. It has to be shown at least for the 8 positive genes in both approaches how the siRNA treatments of these genes phenocopied the lysosomal changes and of course the effect of the downregulation on the protein level of their products both in wild-type control and Spg11 D32-34 knock-in MEF. The Suppl. Fig.3F is completely unclear. How were the Y2H interaction partner validated? Did the authors use the identified 8 interaction candidates as full length bait to demonstrate the interaction with the Spg11 exons 32-34 ?

Author response : The purpose of the siRNA screen was to quickly identify putative candidates important for the regulation of lysosome dynamics. We identified 8 candidates possibly implicated in lysosomes dynamics based on the two analysis methods. We have added in Supplementary Figure 4 C-D the effect of both siRNA on lysosomal function by the two methods of analysis compared to the effect of siSPG11. However, here we aimed to identify candidates and we do not claim that every one of these eight proteins were indeed implicated in the regulation of lysosome dynamics. We corrected the text, accordingly, stating that the products of the 8 identified genes are good candidates to regulate lysosomal function (p.10). We validated the role of one of the identified candidates, UBR4, and we showed that the UBR4 siRNA indeed downregulates the protein level (Figure 4C). We only validated the interaction of spatacsin Cter with UBR4 by co-immunoprecipitation (Figure 4B).

*For the 7 remaining candidates, full characterization would indeed be required to validate their role and elucidate their mechanisms of action, but this is out of the scope of this manuscript. *

p8/Fig.3F: the genes identified in both approaches have to be listed in the Fig. 3F-Table.

Authors response : We have added in new Figure 3F the list of the 8 candidate genes that could contribute to regulate lysosome function.

The GO process- ubiquitin-dependent protein catabolic process is neither positive for the neural network nor for the directed analysis but positive for both analyses? Please explain. Similarly, the GO process proteolysis involved in cellular protein catabolic process -is not positive for the neural network analysis but again positive for both analyses.

Authors response : We agree with the reviewer that Table 3F in its older version could be a bit confusing. GO analysis is based on “enrichment” of biological processes within a list of proteins. As we did not have the same number of proteins in the 3 analyses provided in original Table 3F, we got variability in the identified biological processes. To simplify, we have therefore chosen to present only the GO analysis for the 8 candidates that were most likely implicated in lysosomal dynamics according to our two analyses of the siRNA screen which is the most relevant for our study (new Figure 3G).

For Fig. 3G the mutant ubiquitin-K0 staining in wild-type MEF cells has to be shown as well as for the Spg11 ki/KO MEFs (+ quantification of the respective data)

Authors response : As stated by Reviewer 4, the expression of lysine-null ubiquitin may impact many different cellular pathways. We therefore removed this part of the data in order to simplify the manuscript (p.10)

iv) The interpretation of the Y2H-interactome analysis by the authors is hard to follow. They searched with the exon 32-34 cDNA for binding partner, selected 3 degradative GO processes and showed by overexpression of a mutant Ub-K0 plasmid in wild-type MEFs a decreased number of tubular lysosomes, as well as their dynamics (without showing the control data in Spg11 KO or ki-MEFs). Thus, poly-ub of proteins should be in some way responsible for a lysosomal phenotype of Spg11ki MEFs.

Now they went to AP5Z1, the second binding partner of SPG11, which is reduced in its abundance upon overexpression of Spg11-GFP. I would expect to do the respective control experiment to show that in the absence of SPG11 or in the knock-in cells the amount of AP5Z1 has to increase. However, in the studies by the Huebner group by deletion of Spg11 or the other binding partner Spg15, no increase of AP5Z1 protein levels has been observed. The authors have to comment on this discrepancy.

*Authors response : We agree that this is an important point to discuss, and we failed to do it in our first version. *

*The complete knockout of spatacsin strongly modifies the levels of its partners. We previously showed that spastizin levels are decreased by >90% in Spg11 knockout (Branchu 2017). Furthermore, the levels of AP5Z1 have been shown to be decreased by ~50% in fibroblasts of SPG11 and SPG15 patients (Hirst et al, 2015). *

Our work shows that spatacsin promotes the degradation of AP5Z1 by lysosomes. It is possible that other degradation mechanism may exist, and could explain the lower levels of AP5Z1 in knockout cells. Furthermore, it was proposed that AP5Z1 stability may depend on the presence of spatacsin and spastizin (Hirst et al., 2013)*. Therefore spatacsin may contribute to tightly regulate AP5Z1 levels by contributing both to its stability, and to its degradation. We have carefully discussed this point (p.16). Furthermore, the experiments requested by reviewer 2 in point (vi) that we are planning to perform will help clarify the mechanisms of AP5Z1 degradation both in presence and absence of spatacsin. *

Then the authors found that the selected interaction partner of the exon 32-34 sequence, UBR4, does not bind to the Spg11-GFP construct lacking the domain encoded by exons 32-34 but to the C-terminal domain of Spg11-GFP. Unfortunately, all these IP-experiments were shown as cut and paste figures, preventing the direct comparison between the input and the IP protein amounts (since the information is missing what percentage of the input and the IP has been loaded per lane, the evaluation and significance of these Co-IPs are unclear).

Authors response : We have added in the Figure legend the fact that the input represents 5% of lysate added to the immunoprecipitation assays

v) p9: AP5 (Z1) is a cytoplasmic protein and can be localized on the cytoplasmic surface of lysosomes. How should the GFP-mcherry-AP5Z1 protein enter the lumen of lysosomes justifying the quenching of the GFP signal? A positive control has to be included in the experiment shown in Fig. 4E demonstrating the effect of MG132 under identical conditions of a protein substrate for proteasomal degradation.

Authors response :* We agree this is an important control. We plan to add a control showing accumulation of ubiquitin in lysates upon MG132 treatment to show it was indeed effective. *

vi) Fig. 5A: In contrast to GFP-mcherry-AP5Z1, spastizin-GFP is localized at the cytoplasmic surface of lysosomes (co-staining with LAMP1-mcherry) in wild-type MEFs. In regard to the incomplete data commented under "minor points Fig.4/Suppl.Fig.4", I suggest to perform a simple control experiment with overexpressed GFP-spastizin and mCherry-AP5Z1 in wild-type MEFs (at the best also in Spg11 KO MEF) with and without bafA treatment, which will clearly demonstrate whether single components of the trimeric Spg11, spastizin-AP5Z1 complex are degraded independently of each other in lysosomes.

*Authors response : As stated above, we will perform this control experiment, and will add the data in Figure 5 in future revision. This will help clarify the mechanism of degradation of AP5Z1 and spastizin both in presence and absence of spatacsin. Discussion of this point will also help to clarify the point iv raised by reviewer #2. *

vii) why did the authors neither mention nor discuss the described role of SPG11 in autophago-lysosome reformation (ALR)?

*Authors response : We did not discuss ALR in our first version as we did not investigate autophagic conditions. However, due to the well-described role of spatacsin in ALR, we agree that we should discuss ALR in our manuscript, and we added a paragraph (p.15). *

Minor points

• Figure 1 A, B, D, and G: ER-lysosome contact sites. The quantification of the co-localization of spatacsin-V5 with the ER marker protein GFP-Sec61b has to be given.

Authors response :* We plan to add quantification data performed on STED images showing localization of Spatacsin-GFP together with ER and lysosomal markers. This data will be added in Figure 1. *

Moreover, the authors analyzed overexpressed tagged-proteins only. The results should be compared with the endogenous proteins.

Authors response :* As stated above, there are no good antibodies to endogenous spatacsin for immunostaining. We will add new STED images with antibodies against endogenous Reep5 and Lamp1 to label the ER and lysosomes together with overexpressed spatacsin. Regarding endogenous spatacsin, we could only investigate its localization by subcellular fractionation and western blots comparing wild-type and Spg11 knockout samples. We added biochemical data suggesting that spatacsin is enriched either in the ER or in lysosome membrane associated with the ER. These data have been added in Figure 1 and in text (p.5) and we added a paragraph in discussion regarding spatacsin subcellular localization (p.15). *

p8/Figure 3: what does the 'analysis of trained neural networks' mean?

Authors response : We did not analyzed the trained neural network, but we used this trained neural network to perform image analysis. We clarified the text (p.10).

Figure 4: what happens with the other AP5 subunits?

Authors response : This is a very interesting question. We will test whether overexpression of spatacsin-GFP induces a degradation of some other AP5 subunit, provided we get specific antibody. We will add the data in Figure 4A.

Fig.4F/Suppl.Fig4: live images of GFP-mcherry-AP5Z1 + lysotracker staining have to be shown both for wild-type MEFs with and without bafilomycin A treatment(as in Fig.4F), and in Spg11 KO and Ki MEFs +/- bafA.

Authors response : We will add these data in Figure 4 (WT Mefs +/- Baf A) and in Supplementary Figure 5 (Spg11KO and SPG11D32-34 Mefs +/- Baf).

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

This manuscript highlights an interesting localization of spatacsin in the endoplasmic reticulum (ER)-lysosomes contact sites. In addition, it implicates spatacsin in regulating tubular dynamic lysosomes. Mechanistically, the authors propose that spatacsin interacts with UBR4 to promote the autophagic degradation of its binding partner AP5Z1 at the lysosomes. In turn, this would also regulate the amount of spastizin at the lysosomes, which is known to interact with anterograde motors. The authors further show that AP5Z1 interacts with p150Glued. Thus, the balance between AP5Z1 and spastizin at the lysosomes would determine lysosomal trafficking directionality.

Major Comments

1. Several crucial results of the manuscript are based on quantifications performed on immunofluorescence stainings. Data points in graphs show individual cells or individual lysosomes and the authors apply statistical tests on replicates that cannot be considered biologically independent, since they come from the same experiment or even the same cell. It is recommended to show superplots where both the individual data and the average of each independent experiment is indicated as recommended by Lord et al. (J Cell Biol 2020 219 (6): e202001064.). Statistics should be performed only on independent biological replicates.

Authors response : We agree with the comments of the reviewer regarding data presentation. We have therefore changed the presentation of all graphs of the manuscript using superplots that allow us to show all the points that were analyzed as well as the mean value for each biological replicate, and performed statistical analyses by comparing the biological replicates as proposed in Lord et al, JCB 2020 (10.1083/jcb.202001064).

The authors have used yeast two-hybrid to search for spatacsin interactors. Although in the manuscript they refer to supplementary tables that should show these interactors, the available Tables are confusing and refer to the following downregulation experiments.

Author response : We have added two supplementary data tables (Now Supplementary Tables 1 and 2) to give the list of interactors of wild-type C-terminal domain of spatacsin and spatacsinD32-34, respectively.

Supplementary Tables 3 and 4 now refer to the analysis of the downregulation experiments by respectively the neural network method and the tubular lysosome detection method.

An experiment to demonstrate that endogenous UBR4 and spatacsin interact by co-immunoprecipitation would be crucial.

Authors response : We agree with the reviewer that it would be important to test whether endogenous spatacsin and UBR4 are interacting by co-immunoprecipitation. So far we have not managed to immunoprecipitate either endogenous spatacsin or endogenous UBR4 with the antibodies we tested, which prevents us to test the interactions of endogenous proteins by co-immunoprecipitation. We are not sure we can provide this result.

Several important experiments to unravel the mechanistic role of spatacsin (Figure 4 and 5) are performed upon overexpression. This is a major limitation of the study and the authors should address it as much as possible. Western blots and immunoprecipitations are shown that appear to have been performed only once and have no quantification. As an example, in Fig 4A the difference in levels of AP5Z1 upon spatacsin overexpression or UBR4 downregulation are very minor. I would be very careful in drawing big conclusions, without additional repetitions and additional experiments in an endogenous setting.

*Authors response : We agree that a lot of our experiments used overexpression. We have now added to the manuscript new data obtained in MEFs where we downregulated spastizin or AP5Z1 (Figure 6). They confirm the role of spastizin in the regulation of lysosome dynamics. Furthermore, our new data show that levels of AP5Z1 must be tightly regulated as both overexpression and downregulation of AP5Z1 affects lysosome dynamics (p.12). We also discussed these data carefully (p.16 ). *

Furthermore, we agree that our presentation did not indicate that the western blots were repeated several times. We have now added quantifications for the western blots presented in Figures 4 and 5. Furthermore, we have also added the data showing that downregulation of UBR4 led to higher levels of AP5Z1 in control fibroblasts (Figure 4C).

The authors suggest a model by which UBR4 recruited by spatacsin is involved in autophagic degradation of AP5Z1. The data shown do not support this conclusion. First, in Figure 4A downregulation of UBR4 does not increase levels of AP5Z1 above the control in lane 1, but only when spatacsin is overexpressed. The effect of downregulation of UBR4 in wilt-type cells on AP5Z1 should be investigated. Secondly, there is no experiment directly proving that the stability of AP5Z1 depends on UBR4.

Authors response : We have added new western blots (and quantification) in Figure 4C showing that downregulation of UBR4 increased levels of AP5Z1 in control conditions. The fact that downregulation of UBR4 increased levels of AP5Z1 in control conditions suggests that UBR4 contributes to regulating the levels of AP5Z1. However, we do not show whether UBR4 directly promotes the degradation of UBR4, which has been added in the discussion (p15). To test whether UBR4 affects the stability of AP5Z1, we will monitor whether downregulation of UBR4 by siRNA increases the half-life of AP5Z1. These data will be added on Figure 4.

The authors suggest that the interaction of spatacsin with spastizin or AP5Z1 are in competition. This is an interesting hypothesis, however to conclusively demonstrate this, pull-down experiments in KO cells and not upon extreme overexpression should be performed.

Authors response : We agree that testing the interaction of spatacsin with its partners in SPG15 KO or AP5Z1 KO fibroblasts would be a very good control of our hypothesis. However, we previously showed that the levels of AP5Z1 are lower in SPG15 KO than in control fibroblasts (Hirst et al, 2015), which introduces a bias in the analysis. We therefore plan to concentrate on AP5Z1 fibroblasts and investigate whether interaction of spatacsin with spastizin is modified in these cells. An alternative would be to monitor the effect of siRNA downregulating AP5Z1 on the interaction between spatacsin and spastizin. We will add these data in Figure 5.

Minor comments

1. In figure 1G and 1H the overlapping area between lysosomes and ER is quantified. Considering that the ER occupies a large portion of the field a 90{degree sign} flipped control for both WT and KO would be important to sort out random colocalization. In this direction, it would be also essential to show that the total amount of lysosomes is not different in WT and KO, especially because in figure 1A the lysosomes in WT and KO seem to be different not just in shape but also in number and size. A different number or size of lysosomes affects this analysis.

Authors response :* We added quantifications in Supplementary Figure 1F showing that 90° flipped controls are indeed not capturing the same proportion of contacts between the ER and lysosomes. We also added quantifications in Supplementary Figure 1D-E showing that the average size of lysosomes and the number of lysosomes per unit area are similar in control and Spg11 KO fibroblasts and mentioned it in the text (p.6). If the lysosomal staining appears different in Spg11 KO fibroblasts it is because lysosomes are clustered around the nucleus, an observation that we reported previously (Boutry et al, 2019). *

In the second chapter of the Results, the authors state: "we observed by live imaging a higher number of lysosomes with tubular shape in Spg11+/+ compared to Spg11-/- cells", however the number of elongated lysosomes is quantified per area. Why the number of elongated lysosomes is not quantified over the total amount of lysosomes?

Authors response : The point raised by the reviewer is a fair point. The purpose of our analysis was to compare the number of lysosomes with tubular shape in control and Spg11 KO cells. As the number of lysosomes per unit area is invariant between control and Spg11 KO cells as shown in new data included in Supplementary Figure 1D, normalization to total number of lysosomes or to cell surface reflects the same difference in phenotype.

The In the fourth chapter of the Results, the authors state:" In wild-type MEFs, mCherry was colocalized with lysosomes. In contrast, GFP that is sensitive to pH was poorly colocalized with lysosomes, suggesting that AP5Z1 was mainly inside the acidic subcellular compartment (Figure 4F)." If the aim of the authors is to shown that AP5Z1 is mainly into the lysosome, the amount AP5Z1-mcherry inside and outside the lysosome need to be compared, with a proper statistical analysis. There is also a lot of GFP signal in the cytosol. Why is that?

*Authors response : We agree with the reviewer, we will add quantification of the proportion of AP5Z1-mCherry inside lysosomes on Supplementary Figure 5. *

Regarding the GFP-AP5Z1 signal in the cytosol, AP5Z1 has no transmembrane domain and may thus exist as a cytosolic protein. Since GFP is quenched in the acidic environment of lysosomes, the GFP fluorescence of the mCherry-GFP-AP5Z1 protein is outside lysosomes, and it appears partly cytosolic. Of note, there is also some cytosolic mCherry signal that is less visible due to the high level of mCherry fluorescence in lysosomes. We will clarify this point with the quantification of the proportion of mCherry signal compared to GFP inside the lysosomes and add it in Figure 4.

construct used in the paper is a C-terminal tagged version of spatacsin. The authors should consider to test an N-terminal tagged construct at least for the localization experiments.

Authors response : We added an immunostaining image of Spatacsin with an N-terminal tag (Supplementary Figure 1B) and mentioned it in the text (p.6). As spatacsin with a C-terminal tag, it presents a diffuse distribution that poorly co-localizes with lysosomes.

Figure 5C: a negative control and the quantification are missing.

Authors response : A non-transfected cell is present on Figure 5C, visible thanks to the Lamp1 immunostaining, and that we considered as a negative control. In this non-transfected cell, we detected no PLA signal. We added an asterisk to point the non-transfected cell on Figure 5C. Quantification will also be added in the revised version after we have performed the PLA experiment required by Reviewer 1.

Reviewer #3 (Significance (Required)):

Since spatacsin, AP5Z1 and spastizin are all implicated in hereditary spastic paraplegia, the data are of potential interest not only for basic cell biology, but also to understand the pathogenesis of the disease. In addition, the manuscript proposes a novel model regulating trafficking of dynamic lysosomes.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Pierga et al. reveal subtle differences in lysosome morphology, ER-contact, and trafficking in the absence of Spatascin. These data are replicated with a truncated Spatascin, presumably a loss of function. Two-hybrid screening of the deleted sequence from this truncation for interactors and then asked whether these hits could phenocopy the lysosome morphology changes. This led to an assertion for a role for ubiquitination in these effects. Rather than these hits the group then investigates previously known Spatascin interactors and reports similar complex but subtle abnormalities via overexpression or knockdown of these. While data show overlapping phenotypes by modulation Spatascin, AP5z1, and Spastizin, the manuscript is confusing, leaps from experiment to experiment, and does not provide novel rigorous mechanisitic insight. It conflates all the discrete lysosomes aspects into a collective to link them. The title is over-stated and not appropriate for the experiments.

The localization of endogenous Spatascin is lacking - over-expression is prone to artifact and the punctate data on the V5 suggests much more work is needed to understand where in the cell it is. It would seem much more work is needed here.

Authors response : As stated by reviewer 1, there are no good antibody to endogenous spatacsin, and therefore we have to rely on expression of tagged spatacsin to study its localization by immunofluorescence. When performing the images, we avoided the cells with the highest ovexpression of tagged spatacsin. Yet, we agree that this is still overexpression. That’s why we included subcellular fractionation data where we can detect endogenous spatacsin (Figure 1A-1B). These data confirmed that spatacsin is enriched in the ER or in lysosome fraction tightly associated with the ER.

Furthermore, the EM data (1E) would suggest the far majority of lysosomes are in contact with ER - these seems uncharacteristic.

Authors response : The EM data in figure 1E indeed shows that the majority of lysosomes are in contact with the ER, as previously shown by other groups (Friedman et al, 2013, Höglinger et al, 2019).

The phenotypes analyzed are very subtle, and while statistically significant the biological impact is unclear - in many cases individual lysosomes (or lysosome-ER contacts) are considered as an 'n'. While these results are probed across multiple independent experiments the batch effects and how uniform per cell the events are is unclear.

Authors response : We agree with the comments of the reviewer regarding data presentation. ‘n’ represented individual cells, but did not actually take into account the variability across experiments. We have therefore changed the presentation of all graphs of the manuscript using superplots that allow us to show all the points that were analyzed as well as the mean value for each biological replicate, and performed statistical analyses by comparing the biological replicates as proposed in Lord et al, JCB 2020 (10.1083/jcb.202001064).

In fig 2H critical data are missing - the effect of Spatascin KO on the transition between these morphologies should be considered as in G. Otherwise the relevance is unclear.

Authors response : We have added this quantification on Figure 2I. It shows that transition of morphology of lysosomes from round to tubular in Spg11 KO cells is still associated with a change of speed, although the average speed attained is halved compared to conditions where spatacsin is present. This shows that loss of spatacsin does not abolish morphological transition of lysosomes but limit their speed in the tubular shape. We commented on this new data in the text (p.8).

The impact of over-expressing a lysine-null Ub ( Fig 3) is far too crude and non-specific to have meaning here. It is assumed that the only proteins affected are those of interest. This is consistent with much of the paper where "true-true-and unrelated" is more likely than the presumption of causality.

Authors response : It is true that the expression of lysine-null ubiquitin is really crude and may impact many different cellular pathways. Furthermore, the results obtained with the lysine-null ubiquitin do not contribute to the rest of the paper. We therefore removed the original Fig3G, H, I and Fig 4B and updated the text accordingly (p.10).

The blots in Fig4 are a relatively poor quality and not quantified over repetition.

*Authors response :Spatacsin and spastizin are large proteins, and there is not much choice for antibodies able to detect these proteins. Yet we have validated their specificity by western blot using knockout cells (spatacsin) (Supplementary Figure 4 A-B) or siRNA (spastizin) (Supplementary Figure 7B). We agree that our presentation did not indicate that the western blots were repeated several times. We have added quantifications for the western blots present in Figures 4 and 5. We also changed some illustrative western blots to improve quality. *

Controls are missing and Fig5 suffers from a reliance on over-expression - there is a massive over-expression of AP5Z1 which may be affected the stoichiometry of these overall interactions, but with an n=1 its hard to know and its not clear what these data add. Again, while statistically significant (5E and F) due to the nature of data analysis (every lysosome=n of 1) it is not clear how biologically significant UBR4 siRNA or AP5Z1 over-expression is - as the accumulation of AP5Z1 in these two conditions is orders of magnitude apart - again likely unrelated.

Authors response : We added quantification for this western blot (Supplementary Figure 6A).

*As stated above we have changed the representation of the graphs. Each point represents one cell, and we included the mean value for each biological replicate. *

Preventing degradation of AP5Z1 by UBR4 siRNA or overexpression of AP5Z1 do not indeed have the same effect on total AP5Z1 but do have a similar effect on the interaction of spatacsin with its partners evaluated by co-immunoprecipitation, as illustrated by the quantifications that we have added. We clarified this in the text (p.12). As requested by reviewer 3, we will also investigate the effect of AP5Z1 knockout or downregulation on the interaction between spatacsin and spastizin assessed by co-immunoprecipitation. These data will be added in Figure 5 and will strengthen our conclusions.

Fig 6 begins to conflate the fact that different lysosome morphologies appear to have different trafficking properties even in WT cells and that many of these targets affect morphology - therefore to conclude a direct effect on trafficking seems inappropriate.

Authors response : In original Figure 6, we showed that Kif13A-ST and p150CC1 changed the proportion of tubular lysosomes (previous Figure 6 and H), and the data showing that these constructs changed the trafficking of lysosomes were presented in Supplementary Figure 5 B-C. We have now moved the data showing the effect of Kif13A-ST and p150CC1 in the main Figure (Figure 6F and 6I) to facilitate the interpretation of the data. Therefore, expression of Kif13A-ST and p150CC1 do not only affect the morphology of lysosomes, but also impaired their trafficking. We thus do not extrapolate lysosome dynamics from their morphology, we actually quantify lysosome dynamics.

Fig 7 extends this into polar cells (neurons) but still it is not clear whether form (morphology) dictates function (likelihood of trafficking or directionality.

Authors response : We did not only analyzed neurons because they are polarized cells, but because neurons are the main cells affected by neurodegeneration observed in absence of spatacsin (Branchu et al, 2017). We added new data on Figure 7 showing that tubular lysosomes in axons are actually more dynamic than round lysosomes, as observed in fibroblasts. We added these data in Figure 7 and text (p.13).

Investigation of lysosome trafficking in axons also allowed us to investigate the directionality of movement, which is difficult in MEFs. We clarified this point in the text (p.13).

In sum, there is a lot of data that collectively points to a partial localization of Spatascin at Er-lysosome contacts and an influence on morphology and trafficking of lysosomes in the cell, but at the end of the day very new mechanism is brought to light.

Authors response : The mechanisms regulating trafficking of lysosomes are far from being fully resolved. Our manuscript shows that spatacsin contributes to this regulation by modulating the degradation of AP5Z1. This in turn regulate the lysosomal association of AP5Z1 and spastizin that interact with motor proteins to control lysosomal dynamics.

Reviewer #4 (Significance (Required)):

This manuscript is directed to the basic cell biology community - involving ER, lysosome, and microtubule dependent trafficking. There are some new analytical tools employed and many co-factors and binding partners of Spatascin considered but frankly too many to adequately and rigorously control for. Because of this the manuscript is very unfocused, hard to follow and makes too many assumptions about shared dynamics ? necessarily arising from shared morphology - lysosomes are highly dynamic and can be affected by virtually any change in intracellular trafficking or protein/membrane transport. This is not appropriately considered.

Authors response : We have clarified our manuscript to show that dynamics is not necessarily arising from a tubular morphology. It turns out that lysosomes with a tubular morphology indeed are more dynamic that lysosomes with a round morphology. Importantly, in all our experiments dealing with lysosomal dynamics, we have actually included a quantification of lysosome dynamics using time lapse imaging as detailed in methods (p.21).

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Referee #4

Evidence, reproducibility and clarity

Pierga et al. reveal subtle differences in lysosome morphology, ER-contact, and trafficking in the absence of Spatascin. These data are replicated with a truncated Spatascin, presumably a loss of function. Two-hybrid screening of the deleted sequence from this truncation for interactors and then asked whether these hits could phenocopy the lysosome morphology changes. This led to an assertion for a role for ubiquitination in these effects. Rather than these hits the group then investigates previously known Spatascin interactors and reports similar complex but subtle abnormalities via overexpression or knockdown of these. While data show overlapping phenotypes by modulation Spatascin, AP5z1, and Spastizin, the manuscript is confusing, leaps from experiment to experiment, and does not provide novel rigorous mechanisitic insight. It conflates all the discrete lysosomes aspects into a collective to link them. The title is over-stated and not appropriate for the experiments.

The localization of endogenous Spatascin is lacking - over-expression is prone to artifact and the punctate data on the V5 suggests much more work is needed to understand where in the cell it is. It would seem much more work is needed here. Furthermore, the EM data (1E) would suggest the far majority of lysosomes are in contact with ER - these seems uncharacteristic.

The phenotypes analyzed are very subtle, and while statistically significant the biological impact is unclear - in many cases individual lysosomes (or lysosome-ER contacts) are considered as an 'n'. While these results are probed across multiple independent experiments the batch effects and how uniform per cell the events are is unclear.

In fig 2H critical data are missing - the effect of Spatascin KO on the transition between these morphologies should be considered as in G. Otherwise the relevance is unclear.

The impact of over-expressing a lysine-null Ub ( Fig 3) is far too crude and non-specific to have meaning here. It is assumed that the only proteins affected are those of interest. This is consistent with much of the paper where "true-true-and unrelated" is more likely than the presumption of causality.

The blots in Fig4 are a relatively poor quality and not quantified over repetition.

Controls are missing and Fig5 suffers from a reliance on over-expression - there is a massive over-expression of AP5Z1 which may be affected the stoichiometry of these overall interactions, but with an n=1 its hard to know and its not clear what these data add. Again, while statistically significant (5E and F) due to the nature of data analysis (every lysosome=n of 1) it is not clear how biologically significant UBR4 siRNA or AP5Z1 over-expression is - as the accumulation of AP5Z1 in these two conditions is orders of magnitude apart - again likely unrelated.

Fig 6 begins to conflate the fact that different lysosome morphologies appear to have different trafficking properties even in WT cells and that man of these targets affect morphology - therefore to conclude a direct effect on trafficking seems inappropriate. Fig 7 extends this into polar cells (neurons) but still it is not clear whether form (morphology) dictates function (likelihood of trafficking or directionality.

In sum, there is a lot of data that collectively points to a partial localization of Spatascin at Er-lysosome contacts and an influence on morphology and trafficking of lysosomes in the cell, but at the end of the day very new mechanism is brought to light.

Significance

This manuscript is directed to the basic cell biology community - involving ER, lysosome, and microtubule dependent trafficking. There are some new analytical tools employed and many co-factors and binding partners of Spatascin considered but frankly too many to adequately and rigorously control for. Because of this the manuscript is very unfocused, hard to follow and makes too many assumptions about shared morphology necessarily arising from shared morphology - lysosomes are highly dynamic and can be affected by virtually any change in intracellular trafficking or protein/membrane transport. This is not appropriately considered.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

This manuscript highlights an interesting localization of spatacsin in the endoplasmic reticulum (ER)-lysosomes contact sites. In addition, it implicates spatacsin in regulating tubular dynamic lysosomes. Mechanistically, the authors propose that spatacsin interacts with UBR4 to promote the autophagic degradation of its binding partner AP5Z1 at the lysosomes. In turn, this would also regulate the amount of spastizin at the lysosomes, which is known to interact with anterograde motors. The authors further show that AP5Z1 interacts with p150Glued. Thus, the balance between AP5Z1 and spastizin at the lysosomes would determine lysosomal trafficking directionality.

Major Comments

1. Several crucial results of the manuscript are based on quantifications performed on immunofluorescence stainings. Data points in graphs show individual cells or individual lysosomes and the authors apply statistical tests on replicates that cannot be considered biologically independent, since they come from the same experiment or even the same cell. It is recommended to show superplots where both the individual data and the average of each independent experiment is indicated as recommended by Lord et al. (J Cell Biol 2020 219 (6): e202001064.). Statistics should be performed only on independent biological replicates.
2. The authors have used yeast two-hybrid to search for spatacsin interactors. Although in the manuscript they refer to supplementary tables that should show these interactors, the available Tables are confusing and refer to the following downregulation experiments. An experiment to demonstrate that endogenous UBR4 and spatacsin interact by co-immunoprecipitation would be crucial.
3. Several important experiments to unravel the mechanistic role of spatacsin (Figure 4 and 5) are performed upon overexpression. This is a major limitation of the study and the authors should address it as much as possible. Western blots and immunoprecipitations are shown that appear to have been performed only once and have no quantification. As an example, in Fig 4A the difference in levels of AP5Z1 upon spatacsin overexpression or UBR4 downregulation are very minor. I would be very careful in drawing big conclusions, without additional repetitions and additional experiments in an endogenous setting.
4. The authors suggest a model by which UBR4 recruited by spatacsin is involved in autophagic degradation of AP5Z1. The data shown do not support this conclusion. First, in Figure 4A downregulation of UBR4 does not increase levels of AP5Z1 above the control in lane 1, but only when spatacsin is overexpressed. The effect of downregulation of UBR4 in wilt-type cells on AP5Z1 should be investigated. Secondly, there is no experiment directly proving that the stability of AP5Z1 depends on UBR4.
5. The authors suggest that the interaction of spatacsin with spastizin or AP5Z1 are in competition. This is an interesting hypothesis, however to conclusively demonstrate this, pull-down experiments in KO cells and not upon extreme overexpression should be performed.

Minor comments

1. In figure 1G and 1H the overlapping area between lysosomes and ER is quantified. Considering that the ER occupies a large portion of the field a 90{degree sign} flipped control for both WT and KO would be important to sort out random colocalization. In this direction, it would be also essential to show that the total amount of lysosomes is not different in WT and KO, especially because in figure 1A the lysosomes in WT and KO seem to be different not just in shape but also in number and size. A different number or size of lysosomes affects this analysis.
2. In the second chapter of the Results, the authors state: "we observed by live imaging a higher number of lysosomes with tubular shape in Spg11+/+ compared to Spg11-/- cells", however the number of elongated lysosomes is quantified per area. Why the number of elongated lysosomes is not quantified over the total amount of lysosomes?
3. The In the fourth chapter of the Results, the authors state:" In wild-type MEFs, mCherry was colocalized with lysosomes. In contrast, GFP that is sensitive to pH was poorly colocalized with lysosomes, suggesting that AP5Z1 was mainly inside the acidic subcellular compartment (Figure 4F)." If the aim of the authors is to shown that AP5Z1 is mainly into the lysosome, the amount AP5Z1-mcherry inside and outside the lysosome need to be compared, with a proper statistical analysis. There is also a lot of GFP signal in the cytosol. Why is that?
4. construct used in the paper is a C-terminal tagged version of spatacsin. The authors should consider to test an N-terminal tagged construct at least for the localization experiments.
5. Figure 5C: a negative control and the quantification are missing.

Significance

Since spatacsin, AP5Z1 and spastizin are all implicated in hereditary spastic paraplegia, the data are of potential interest not only for basic cell biology, but also to understand the pathogenesis of the disease. In addition, the manuscript proposes a novel model regulating trafficking of dynamic lysosomes.

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 #2

Evidence, reproducibility and clarity

In this study Pierga et al. report that SPG11 (spatacsin) is an ER-resident protein involved in the regulation of ER-lysosome contact sites (in particular tubular lysosomes) and subsequent faster motility of tubular lysosomes, as well in the degradation of AP5Z1 (SPG48), which forms a heterotrimeric complex with SPG15 (spastizin) and SPG11. This complex has been localized by several groups on the cytoplasmic side of LAMP-1-positive lysosomes. In addition, mutations in SPG11, SPG15, and SPG48 patients share various clinical features and were supported by biochemical/cell biological data from Spg11 and Spg15 KO mouse models and cultured cells both from patients and mice, respectively, demonstrating e.g. accumulation of autolysosome storage material, defects in the autophagic lysosome reformation process, and the loss of cortical motoneurons and Purkinje cells.

Major concerns:

1. Fig. 1, 2, 3: major disadvantage of this study is the analysis of overexpressed proteins (SPG11-V5, GFP-Sec61,and Lamp1-mCherry) which might contribute to the observed strong expression of SPG11-V5 in the ER/ER-enriched fraction. The results should be compared with the endogenous expressed proteins. In Fig. 1C the lack of the mature Cathepsin D form which is proteolytically generated only in lysosomes from the higher molecular mass precursor is misleading and should be related to presence of lysosomal membrane proteins. Fig. 1D: the TEM image shows only a single lysosome and proposed ER contact zones in wt-MEFs without comparison with Spg11 KO MEFs (only in the quantification). Without double immunogold labeling of SPG11 (and their lack on SPG11 KO cell lysosomes) and known ER contact-site proteins this image and the conclusion are insufficient.
2. The rationale for the selection of the deleted Spg11 region 32-34 is not clear. What are the symptoms of this Spg11 knock-in mouse? A more detailed description of the phenotype is required! Is the phenotype including the accumulation of LC3-positive material similar to the phenotype of the SPG11 KO mouse which has been published by Varga et al.(2015) and Branchu et al. (2017) ? If not, is the new mechanisms reported here not so important?
3. p8/Fig. 3F/Suppl.Fig.3F- the most important part of the manuscript: what are the parameters of lysosomal staining in images that were used to identify genes important for lysosome tubulation by the neural network? I cannot understand how the authors predict the probability of the cell to be considered as an Spg11 KO fibroblast (why not as an Spg11 32-34 knock-in fibroblast?) as the basis for the selection of interaction candidates. A simple statement that the neural network approach identified those genes is too weak and requires more convincing experimental data. It has to be shown at least for the 8 positive genes in both approaches how the siRNA treatments of these genes phenocopied the lysosomal changes and of course the effect of the downregulation on the protein level of their products both in wild-type control and Spg11 32-34 knock-in MEF. The Suppl. Fig.3F is completely unclear. How were the Y2H interaction partner validated? Did the authors use the identified 8 interaction candidates as full length bait to demonstrate the interaction with the Spg11 exons 32-34 ? p8/Fig.3F: the genes identified in both approaches have to be listed in the Fig. 3F-Table. The GO process- ubiquitin-dependent protein catabolic process is neither positive for the neural network nor for the directed analysis but positive for both analyses? Please explain. Similarly, the GO process proteolysis involved in cellular protein catabolic process -is not positive for the neural network analysis but again positive for both analyses. For Fig. 3G the mutant ubiquitin-K0 staining in wild-type MEF cells has to be shown as well as for the Spg11 ki/KO MEFs (+ quantification of the respective data)
4. The interpretation of the Y2H-interactome analysis by the authors is hard to follow. They searched with the exon 32-34 cDNA for binding partner, selected 3 degradative GO processes and showed by overexpression of a mutant Ub-K0 plasmid in wild-type MEFs a decreased number of tubular lysosomes, as well as their dynamics (without showing the control data in Spg11 KO or ki-MEFs). Thus, poly-ub of proteins should be in some way responsible for a lysosomal phenotype of Spg11ki MEFs. Now they went to AP5Z1, the second binding partner of SPG11, which is reduced in its abundance upon overexpression of Spg11-GFP. I would expect to do the respective control experiment to show that in the absence of SPG11 or in the knock-in cells the amount of AP5Z1 has to increase. However, in the studies by the Huebner group by deletion of Spg11 or the other binding partner Spg15, no increase of AP5Z1 protein levels has been observed. The authors have to comment on this discrepancy. Then the authors found that the selected interaction partner of the exon 32-34 sequence, UBR4, does not bind to the Spg11-GFP construct lacking the domain encoded by exons 32-34 but to the C-terminal domain of Spg11-GFP. Unfortunately, all these IP-experiments were shown as cut and paste figures, preventing the direct comparison between the input and the IP protein amounts (since the information is missing what percentage of the input and the IP has been loaded per lane, the evaluation and significance of these Co-IPs are unclear).
5. p9: AP5 (Z1) is a cytoplasmic protein and can be localized on the cytoplasmic surface of lysosomes. How should the GFP-mcherry-AP5Z1 protein enter the lumen of lysosomes justifying the quenching of the GFP signal? A positive control has to be included in the experiment shown in Fig. 4E demonstrating the effect of MG132 under identical conditions of a protein substrate for proteasomal degradation.
6. Fig. 5A: In contrast to GFP-mcherry-AP5Z1, spastizin-GFP is localized at the cytoplasmic surface of lysosomes (co-staining with LAMP1-mcherry) in wild-type MEFs. In regard to the incomplete data commented under "minor points Fig.4/Suppl.Fig.4", I suggest to perform a simple control experiment with overexpressed GFP-spastizin and mCherry-AP5Z1 in wild-type MEFs (at the best also in Spg11 KO MEF) with and without bafA treatment, which will clearly demonstrate whether single components of the trimeric Spg11, spastizin-AP5Z1 complex are degraded independently of each other in lysosomes.
7. why did the authors neither mention nor discuss the described role of SPG11 in autophago-lysosome reformation (ALR)?

Minor points

• Figure 1 A, B, D, and G: ER-lysosome contact sites. The quantification of the co-localization of spatacsin-V5 with the ER marker protein GFP-Sec61b has to be given. Moreover, the authors analyzed overexpressed tagged-proteins only. The results should be compared with the endogenous proteins.
• p8/Figure 3: what does the 'analysis of trained neural networks' mean?
• Figure 4: what happens with the other AP5 subunits?
• Fig.4F/Suppl.Fig4: live images of GFP-mcherry-AP5Z1 + lysotracker staining have to be shown both for wild-type MEFs with and without bafilomycin A treatment(as in Fig.4F), and in Spg11 KO and Ki MEFs +/- bafA.

Significance

see above.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This paper identifies a role for the hereditary spastic paraplegia protein spatacsin in lysosome morphology, positioning and dynamics, and undertakes detailed mechanistic studies to try to identify the mechanism for this effect. In doing so the paper elucidates further mechanistic information about the properties of two other hereditary spastic paraplegia proteins, spastizin and AP5Z1. The work is done in mammalian cells and uses a combination of over-expression, depletion and biochemical studies. The main findings are:

1. The authors present evidence that spatacsin is an ER-localised protein.
2. Murine embryonic fibroblasts lacking spatacsin have a reduced number of tubular lysosomes and the remaining lysosomes are less motile. In general, a relationship between tubular lysosome morphology and lysosome motility, often in association with the endoplasmic reticulum (ER), is demonstrated. These tubular lysosomes are catalytically active and acidic.
3. In terms of mechanism of this effect, by combining a yeast-two hybrid and siRNA phenotypic screen, the authors identify a number of spatacsin-interacting proteins that also regulate lysosomal tubulation. The most important of these for the purposes of this paper is UBR4, an E3 ubiquitin ligase.
4. The authors show that spatacsin and UBR4 promote degradation of AP5Z1, and that this property required the ability of spatacsin to interact with UBR4. Somewhat surprisingly, as AP5Z1 is a coat protein, this degradation appeared to occur within the lumen of the lysosome - the authors speculate how this could be in the discussion.
5. The authors then demonstrate that AP5Z1 and spastizin, both hereditary spastic paraplegia proteins, compete for binding with spatacsin.
6. The relationship between spatacsin, spastizin, AP5Z1 and motor proteins in then examined. There is a known interaction between spastizin and KIF13A and expression of a dominant negative KIF13A protein reduced lysosomal tubulation. The authors then demonstrate an interaction between AP5Z1 and the p150Glued dynein/dynactin complex member, then showed that expression of a dominant negative p150Glued protein reduced lysosomal tubulation.
7. Finally, that authors demonstrate the relevance of these findings to neurons, the target cells of hereditary spastic paraplegia, by showing that lysosomal tubulation and axonal transport are reduced in mouse neurons lacking spastacsin, and that depletion of UBR4 or AP5Z1 affected these as expected from the experiments above.

Major comments:

Overall I believe that the key conclusions of this paper are generally convincing and that the work is of high quality. However, I do have some reservations:

1. The localisation of spatacsin on the ER. It is always difficult to be convinced about colocalization of a diffuse punctate marker and the ER. From the STED experiments in figure 1, while it definitely seems that there is some spatacsin on the ER, there also appears to be some spatacsin puncta that are not. I'd like to know if these puncta represent lysosome-associated spatacsin. This is important for interpretation of the subsequent experiments (see point 3 below). I also think quantification of these co-localisation will increase confidence in the results. In addition, a caveat of the immunofluorescence studies is that they use over-expressed spatacsin. I appreciate that there are no good antibodies to endogenous spatacsin, but I don't think this limitation is sufficiently acknowledged. As the claim of ER-localisation is critical for the proposed mechanistic model, and in the absence of experiments with endogenously tagged spatacsin, this makes the biochemical fractionation studies of figure 1C very important. To make these more convincing I would prefer to see additional control markers to verify the separation of lysosomal and ER compartments - e.g. lamp1, lamp2, an ER tubular marker such as a REEP5 or a reticulon.
2. The authors generally do a good job of quantifying their results. However, this is lacking for the biochemical experiments (immunoblotting and IP) in figures 4 and 5, and I would prefer to see these quantified (the quantification should include data from repeat experiments so that we can judge the reproducibility of the results).
3. On page 10, referring to the proximity ligation results, the authors comment: "This suggests that the spatacsin-spastizin interaction occurs at contact sites between the ER and lysosomes to allow spastizin recruitment to lysosomes". I'm not sure this statement is fully supported, as mentioned at point 1 above it is possible that some steady state spatacsin is at lysosomes. To fully support this, we'd need to see the PLA signal also convincingly co-localise with an ER marker.
4. In figure 6C and D the effect of spastizin on lysosomal tubulation and dynamics is investigated. Wartmannin treatment is used to do this, as it is known to remove spastizin from lysosomes. However, this is a very indirect manipulation that could have many other consequences and it would be better to demonstrate this directly by showing the effect of depletion of spastizin on lysosomal morphology/dynamics. I also think the role of AP5Z1 in tubulation/dynamics would be better supported with additional experiments to deplete the protein - at present only over-expression is examined.

5. While the experiments showing that over-expression of dominant negative forms of KIF13A and p150Glued affect lysosomal tubulation/dynamics provide good circumstantial evidence that spatacsin influences these lysosomal properties via its interactions with spastizin and AP5Z1 (which bind to these motor proteins), the authors have not shown that the interaction of the motor proteins with spastizin and AP5Z1 is required for this ability to regulate lysosome tubulation/dynamics. This means that the model presented in figure 7 is not fully supported by the data. If the authors have been able to map the binding regions for these interactions then perhaps this could be investigated with rescue experiments, although I appreciate that this is potentially a major piece of work and perhaps outside the scope of this paper. An alternative would be that the authors acknowledged this part of the model as somewhat speculative.

6. Are the data and the methods presented in such a way that they can be reproduced?

Yes - Are the experiments adequately replicated and statistical analysis adequate?

In general I am not convinced that the statistical tests are applied rigorously in this paper. Most experiments are done three times, but the "n" used for statistical testing is typically chosen as, e.g. the number of cells, number of lysosomes, rather than number of biological repeat experiments. This means that inter-experimental variability is not rigorously taken into account. A more rigorous practice would be to use the mean measures for each of three biological repeats and apply the statistical tests to the three means, so n=3 if three repeats were done. Superplots would be a nice way to graphically display these data.

Minor comments:

1. In supplementary figure 3D I cannot honestly say that I see the smaller band.
2. When first called out, I expected supplementary tables 1 and 2 to show the list of interactors with wild-type spatacsin and spatacsind32-34 respectively, but this is not what they show.
3. The experiments in Figure 4A are a little problematic in the way that they are called out. The first call refers to just a small subset of the data in the figure, and the figure is then called out at various points later in the paper. This is quite confusing. Is there any way this could be simplified?
4. The section on page 10: "Spatacsin also interacts with spastizin, and is required to recruit spastizin to lysosomes (Hirst et al., 2021). ........ We hypothesized that spatacsin interaction with spastizin was required for spastizin localization to lysosomes." Is odd, as the authors seem to be hypothesising an observation that they have just said has already been demonstrated.
5. Can the authors explain why there is so little interaction between wild-type KIF13A and spastizin?
6. In figure 6G p150Glued signal is also present in the control IP lane, which casts doubt on the specificity of the interaction. Could the authors generate a cleaner result?

7. I would be interested to see how AP5Z1 expression differs between neurons with and without spatacsin- we would expect similar results to those shown in the MEFS.

8. Are prior studies referenced appropriately?

Yes. - Are the text and figures clear and accurate?

Yes - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Overall I thought the presentation was good. However, this is a complex paper and anything that the authors can do to simplify the textual descriptions of the experiments would be helpful. There are quite a few long multiphrase/multiclause sentences that could perhaps be broken up or simplified, e.g. I had to read the following three or four times to understand it: "Downregulation of UBR4 that prevented degradation of AP5Z1mediated by spatacsin (Figure 4A) led to higher interaction of spatacsin with AP5Z1 and decreased the interaction of spatacsin with spastizin (Figure 4A)."

Referees cross-commenting

Thanks for the opportunity to comment on the other reviews. It does seem that there is a consistent theme that reviewers are concerned about the over-reliance on over-expression experiments and the need for additional experiments using endogenous antibodies or protein depletion methodologies to strengthen the data. In addition, I and at least one other reviewer feel that it is not adequate to use number of cells as the "n" for statistical testing, and that true biological repeats are needed.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

I think this paper represents a significant conceptual advance in our understanding of the mechanisms by which lysosomal dynamics are controlled in non-polarised cells and neurons. In addition, it elucidates mechanisms that may underlie multiple forms of hereditary spastic paraplegia, a hereditary form of motor neuron disease.<br /> - Place the work in the context of the existing literature (provide references, where appropriate).

This is a significant conceptual advance on the current literature on spatacsin and on the molecular mechanisms controlling lysosomal morphology/dynamics. The paper elucidates important mechanistic details of the relationship between three key proteins involved in hereditary spastic paraplegia, while also shedding light on the basic biology of lysosomal morphology and dynamics. - State what audience might be interested in and influenced by the reported findings.

Basic cell biologists interested in the ER, in lysosomes, in ER-organelle contacts. Scientists interested in the causation of hereditary spastic paraplegias. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Membrane traffic, lysosome function, ER-endosome contacts, hereditary spastic paraplegia.

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Reply to the reviewers

Manuscript number: RC-2022-01683

Corresponding author(s): Prof Andrew Macdonald, Dr Ethan Morgan

1. General Statements [optional]

We are very appreciative of the helpful and insightful comments provided by the reviewers, which will greatly aid in improving this manuscript. We were delighted by the many positive comments we received, highlighting the “high quality” data and praising our “detailed and carefully constructed” experiments, which together reveal an “interesting and novel mechanism” of HPV-driven cervical carcinogenesis.

To summarise our key findings, we identify the host ATP-sensitive potassium ion (KATP) channel as a critical driver of proliferation and HPV oncoprotein expression in HPV+ cervical cancer cells. Use of pharmacological inhibitors and activators of KATP channels revealed that HPV oncoprotein expression correlates with channel activity, findings that were validated via siRNA/shRNA knockdown and overexpression strategies. Indeed, HPV was found to enhance expression of the ABCC8 gene (encoding the SUR1 KATP channel subunit), likely in a manner dependent on the E7 oncoprotein. We also reveal that channel knockdown impeded HPV+ cervical cancer cell proliferation, both in cell culture monolayer and, importantly, in tumour xenograft experiments. This loss of proliferation may be associated with induction of a G1 cell cycle arrest. Finally, we demonstrate that KATP channels are capable of activating ERK1/2 signalling and, in turn, the AP-1 transcription factor, leading to recruitment of AP-1 to the HPV18 promoter. Significantly, this study is the first to our knowledge to explicitly demonstrate modulation of ion channel expression and activity by HPV, and that this can contribute to host cell transformation. We believe the potential for use of the clinically available KATP channel inhibitors as novel therapies for HPV-associated malignancies should therefore be evaluated in future studies.

Outlined below, in a point-by-point manner, are the changes we have already incorporated to improve the precision and clarity of the manuscript, as well as the additional data we intend to add in order to strengthen our findings.

2. Description of the planned revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)): Addressing the following major points would help to strengthen the impact of the work:

1. The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?

We agree that an assessment of cell viability following the treatments/transfections performed will strengthen the manuscript. We will therefore perform the suggested CellTiterGlo assay using both HeLa and SiHa cells after glibenclamide treatment, SUR1 knockdown, and E6/E7 knockdown.

1. There is a major concern regarding whether SUR1 protein is produced at a biologically relevant level in SiHa and HeLa cells, in which most of the experiments in the paper were conducted. Protein levels are assessed in Fig 2 by immunostaining in raft cultures and in a cervical cancer tissue microarray. However, protein levels are otherwise not examined, especially in SiHa and HeLa cells. Is SUR1 protein produced in these cells? Are its levels reduced by the knockdown approaches? The fold change RNA data presented in figure 2A does not convincingly address this question, since even an 8-fold increase of ABCC8 mRNA over a low background level might not have biological significance. It would be very helpful to measure SUR1 protein in several of the experiments in HeLa and SiHa cells.

We accept the concern of the reviewer regarding the absence of an assessment of SUR1 protein levels in HeLa and SiHa cells. There is a critical lack of high-quality antibody reagents available for the detection of SUR1, a common phenomenon within the ion channel field. We have therefore been unable to reliably detect SUR1 via immunoblot using the antibodies we have tested thus far. Nevertheless, we would argue that our other experiments in these cell lines demonstrate that not only are KATP channels expressed at biologically relevant levels but, more importantly, are active in these cell lines. Patch clamping electrophysiology, the gold-standard technique for assessing ion channel functionality, was performed in HeLa cells and the changes in current observed following inhibitor/activator application suggests that the channels are active, and by extension, that SUR1 protein must be present. Furthermore, DiBAC4(3) assays were performed following inhibitor treatments, channel stimulation, SUR1 knockdown, and HPV E7 knockdown. Although this involves an assessment of plasma membrane polarisation, and therefore is not a direct measurement of KATP channel activity, the changes observed are consistent with our expectations (e.g. increased DiBAC4(3) fluorescence with glibenclamide treatment, indicative of membrane depolarisation, is consistent with a loss of K+ ion efflux via KATP channels). However, we recognise that providing protein expression data in HeLa and SiHa cells would make our conclusions more convincing. We will therefore continue our search for antibody reagents that will allow us to reliably detect SUR1 protein in HPV+ cell lines by western blot. We will also pursue other detection methods in these cell lines, including immunofluorescence and immunohistochemistry. We hope that we are successful in our optimisation, allowing us to validate that SUR1 protein levels are reduced following our knockdown approaches.

1. The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.

To address off-target effects, we will repeat some of the experiments performed in this manuscript using individual siRNAs. HeLa and SiHa cells will be transfected with each of the four siRNAs individually and the impact on HPV E6 and E7 expression examined by RT-qPCR and western blot. Further, we will also repeat colony formation assays and DiBAC4(3) assays to ensure that each siRNA has a similar effect on proliferation and membrane polarisation respectively.

1. Finally, since many of the experiments rely on knockdown approaches that show similar readouts, a rescue experiment (restore sh or si-resistant SUR1 and assess the impact on the phenotype) would confirm that the effects being observed are due to changes in SUR1 levels and not to off-target effects.

We agree that rescue experiments would greatly strengthen the manuscript. Our laboratory has significant experience in carrying out this type of experiment and as such we would be very happy to perform them. We will reintroduce siRNA-resistant SUR1 following knockdown of endogenous SUR1 levels and confirm that E6/E7 expression and proliferation are restored.

CROSS-CONSULTATION COMMENTS I note several areas of common feedback among the reviews. Several reviewers commented on the large number of experiments and that the work is of interest to researchers working on HPV and cancer therapeutics. Several reviewers shared concerns about cell viability upon HPV oncoprotein knockdown and about toxicity in various experiments. Several reviewers also raised concerns about the validation of SUR1 protein levels in several experiments. These concerns seem to me to be critical to address to strengthen the manuscript. I note that Reviewer #3's suggestion of making E7 knockout cells (presumably in HPV+ cancer cell lines) is unlikely to be possible because the cells require E7 for survival.

We agree that the two main areas of concern shared among the reviewers (cell viability assays and validation of SUR1 protein levels) are issues that should be addressed in a revised version of this manuscript. We will endeavour to perform the experiments described above, which we hope will significantly enhance the impact of our work.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Major Comments: Authors did not explain how HPV E7 would upregulate ABCC8 transcription or elevate SUR1 protein (Figure 4). Depletion of E7 is known to produce lethal effect in cervical cancer cell lines. No experiment was done to assess cytotoxicity. Hence it is not clear from the available evidence if the SUR1 is reduced by direct E7 mediated event or indirectly by general cytotoxicity induced by E7 knock down.

We thank the reviewer for their suggestions. We agree that it would be useful to provide some mechanistic insight into how E7 upregulates ABCC8 transcription. We therefore plan to analyse the promoter region of ABCC8 to identify potential transcriptional regulators. ChIP-qPCR and luciferase reporter constructs containing the ABCC8 promoter region will be used to unravel the importance of any candidate TFs identified. An assessment of the impact of E7 on the expression and/or activity of these TFs may also be performed. Finally, we will combine E7 overexpression in a HPV- cell line with knockdown/inhibition of a candidate TF to confirm our findings.

Experiments to assess cell viability/cytotoxicity will be performed as outlined in our response to Reviewer #1.

Authors did not analyze expression level and role of p53, pRB proteins, the direct targets of E6 and E7 proteins, on cell cycle regulation following SUR1 siRNA or Glibenclamide-treatment in cervical cancer cell lines.

We agree that the protein levels of p53 and pRb, the primary targets of E6 and E7 respectively, should be analysed in HeLa and SiHa cells following SUR1 knockdown and glibenclamide treatment and will endeavour to perform this.

Minor Comments:

Additional immunofluorescence or histological analysis is necessary to assess the potential cytotoxic effects of E7 siRNA, SUR1 siRNA or KATP inhibitors (Glibenclamide) in cervical cancer cell lines.

We agree that an assessment of cell viability will strengthen the manuscript and we plan to address this as outlined in our response to Reviewer #1.

3. Description of the revisions that have already been incorporated in the transferred manuscript

In addition to the points raised by the reviewers, we identified a minor error that occurred during assembly of the manuscript. Incorrect images of colony formation assays were provided in the original version of Figure 5B. This has now been amended in the transferred manuscript.

__Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __Overall, the data are of high quality and the individual results are consistent with each other and are convincing. However, the authors have understandably focused on two HPV-positive cancer cell lines (affected by modulating KATP levels) and one HPV-negative cancer cell line (which is not affected in the same way). The ability to extrapolate to conclusions about cervical or HPV-positive cancers in general is therefore limited and many of the authors' statements should be tempered to reflect the experiments they have conducted.

We thank the reviewer for their positive comments regarding our data. We agree that by mainly focussing our studies on cervical cancer cell lines, we limit the potential for extrapolation to other HPV-associated malignancies. We were careful to qualify many of our statements in the original submission to reflect this: e.g. lines 314-315: “Collectively, these data demonstrate that KATP channels are important drivers of proliferation in HPV+ cervical cancer cells.” Post review, we have altered references to HPV+ cancers in general to more accurately reflect the data presented, as outlined below:

• Lines 112-113: We hope that the targeting of KATP channels may prove to be beneficial in the treatment of HPV-associated __cervical __neoplasia.____

• Lines 430-431: As such, we believe that the clinically available inhibitors of KATP channels could constitute a potential novel therapy for HPV-associated malignancies HPV+ cervical cancer.

  Addressing the following major points would help to strengthen the impact of the work:

• The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?

All experiments involving siRNA-mediated knockdown were performed at 72 hours post-transfection. We apologise for omitting this information, and it has now been added to the ‘Materials and Methods’ section.

Minor comments: The text and figures are clear and statistics are appropriate. The authors should include at what time point post siRNA transfection the experiments were conducted.

As above, this information has been added to the ‘Materials and Methods’ section.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)):____ __E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

Regarding the number of biological replicates, as described under the ‘Statistical analysis’ subheading of the ‘Materials and Methods’ section, “all experiments were performed a minimum of three times, unless stated otherwise”. Where data is presented as bar graphs, this information is also included in the figure legend and each biological replicate is represented by a single data point where possible. In the case of western blots, a statement of the number of biological repeats has been added under the ‘Western blot analysis’ subheading of the ‘Materials and Methods’ section:

• Lines 614-615: “A minimum of three biological repeats were performed in all cases and representative blot images are displayed.”

  Minor Comments: They did not provide physiological functions of K+ATP channel. I consider this information should be important part of the introduction.

We agree that this would provide important contextual information and apologise for omitting this in the original submission. Details of some well-characterised functions of KATP channels, outside of their potential role in regulating cell proliferation, have been added to the introduction (lines 99-102).

The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

In an attempt to resolve the issue highlighted, we have removed a reference in the text to the layers of the epithelium in which SUR1 expression is upregulated, as detailed below:

• Lines 195-197: This demonstrated a marked increase in SUR1 protein expression in __the suprabasal layers of __HPV18+ rafts in comparison to NHK raft cultures, consistent across both donors (Fig 2C).

  There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

The HPV18-containing keratinocytes used in this study are the same model system we have used in previous investigations (Wasson et al. (2017) Oncotarget 8(61): 103581–103600; Morgan et al (2018) PLoS Pathog 14(4): e1006975). In these studies, which focus on the HPV life cycle rather than HPV+ cancer, Southern blot analysis of HPV episomes and western blots for E6/E7 protein levels were performed, providing validation of the presence of HPV in these cells. We have added a reference to our previous work in the text (line 191).

As this manuscript primarily focusses on HPV+ cancer rather than HR-HPV infection, we believe an assessment of the role of KATP channels on HPV episome maintenance and/or genome replication to be beyond the scope of this study.

4. Description of analyses that authors prefer not to carry out

__Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __Addressing the following major points would help to strengthen the impact of the work:

The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.

Our plan to address potential off-target effects of the siRNAs used is outlined above. Regarding the shRNA data, we use two different shRNAs in the majority of experiments presented. These were found to have highly similar impacts on E6/E7 expression (Figure 3E & 3G) and proliferation (Supp Figure 4). We therefore do not believe that further experiments to eliminate off-target effects of the shRNA are necessary.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)):____ __Major Comments: Overall, the authors performed many experiments to reveal an interesting and novel mechanism. (1) SUR1 expression and activity is necessary for HPV16 and-18 E6 and E7 expression. (2) HPV-16/18 E7 upregulates expression of ABCC8/SUR1 transcription. (3) SUR1 containing K+ATP channel then phosphorylates ERK. (4) Activated ERK then phosphorylates JUN/AP1. (5) Next, activated JUN/AP1 promotes E7 or E6E7 expression from HPV URR. However, in this cyclic feedforward regulation of these genes there is no control mechanism. Then how is homeostasis maintained in HPV infected lesions?

We thank the reviewer for praising the “interesting and novel mechanism” we have uncovered. The primary focus of our study is HPV+ cervical cancers, rather than HPV-infected lesions. The data we present in Figure 2 indicates that upregulation of KATP channel expression and/or activity is likely also occurring during HR-HPV infection, given we see e.g. increased SUR1 staining in HPV18-containing organotypic rafts. However, given our primary focus, we believe a more thorough assessment of KATP channels in HR-HPV infection, including any potential homeostatic regulation mechanisms, is outside the scope of our current study and inclusion of additional life cycle data would dilute the main conclusions of this manuscript. It is nonetheless a potentially exciting area for future work, and could form part of a separate, focussed manuscript.

E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

We believe that bands for E6 and E7 protein in DMSO-treated samples in Figure 1E and 1J are broadly consistent. However, we appreciate that E6 and E7 protein expression appears to be lower in DMSO samples in Figure 1H. All experiments involving diazoxide stimulation were performed under conditions of serum starvation, as indicated in the legends for Figures 1 and 8. Oncoprotein expression is known to be regulated by a series of host transcription factors, many of which become active in response to growth factor stimulation, such as AP-1 and SP1 (see: Tan et al. (1992) Nucleic Acids Res. 20(2):251-6; Hoppe-Seyler et al. (1992) Nucleic Acids Res. 20(24):6701-6; Butz K et al. (1993) J Virol. 67(11):6476-86). Serum starvation was therefore used to disentangle the upregulation of HPV gene expression by KATP channels from the myriad of other host signalling pathways demonstrated to drive E6/E7 expression. A side effect of this was a reduction in basal E6 and E7 protein levels in DMSO-treated cells. In addition, shorter exposure times were deliberately selected to prevent overexposure of protein bands corresponding to 50uM diazoxide treated cells. We apologise for any confusion caused.

Data from inhibitor treatments has been corroborated by knockdown experiments throughout the manuscript. The loss of E6/E7 expression with glibenclamide treatment was confirmed by SUR1 siRNA and shRNA knockdowns (Figure 3D-G) and Kir6.2 knockdown (Supp Figure 3C-D). The glibenclamide-induced loss of proliferation observed in HeLa and SiHa cells was also validated using the same approaches (Figure 5D-F, Supp Figure 3E-G, Supp Figure 4). Indeed, the cell cycle dysregulation experiments in Figure 7 were all performed with both inhibitor treatments and SUR1 siRNA knockdown.

The increase of G1 population, determined by flow cytometry, of HeLa cells treated with Glib or SUR1 siRNA is relative to controls appears to be small and not supported by similar study on other HPV+ or HPV_ vervical cancer cell lines. Importantly the mechanism of this increased G1 in HeLa cell line is not clear. The immunoblot data about the role of cyclins are not sufficient.

The flow cytometry experiments with glibenclamide treatment and SUR1 knockdown in HeLa cells were also performed with HPV16+ SiHa cells (Figure 7A-B) and the effects are highly consistent between the two cell lines. We believe that elucidating a more detailed mechanism of the observed G1 arrest is beyond the scope of this manuscript. Analysis of the mRNA and protein expression of cyclins was intended to provide corroboration of our findings via flow cytometry (i.e. a specific reduction in G1-phase cyclins corresponds to an accumulation of cells in G1 phase), rather than provide mechanistic insight.

What is the physiological effect of cyclin D1 in the context of HR-HPV infection (Figure 7)? In the event of HPV E7 mediated pRB degradation in cervical cancer cell lines, the inactivation of pRB by cyclin D1 does not appear to be physiologically relevant, may not account for difference in growth. It is known in literature that Cyclins A2 and B1 are often elevated by E7 activity. If SUR1 siRNA reduces E7-transcription and protein levels as shown in earlier results, why cyclinB1 and A2 protein level did not change?

As discussed, this manuscript primarily focusses on the role of KATP channels in HPV+ cervical cancer cells. An investigation into the effects on cyclin D1 expression in the context of HR-HPV infection, whilst an important question, is beyond the scope of this study and should form part of a standalone manuscript.

Regarding the expression of cyclins A2 and B1, we repeatedly observed very little impact following glibenclamide treatment and SUR1 knockdown in both cell lines examined. As highlighted above, given we observe a G1 arrest after KATP channel knockdown/inhibition, it would perhaps be unusual to observe changes in the expression of cyclins which drive G2 and M phase progression, respectively.

If activated ERK1/2 and c-Jun is required for URR activity, why are not they detectable in DSO or scrRNA treated HeLa cells (Fig 8A, B)? Why there is no 18 E7 in DMSO treated HeLa cells (Fig. 8A)? Authors also did not explain how inhibition of KATP channel regulates ERK phosphorylation in cervical cancer cell lines. There is no data from additional cervical cancer cell lines or HSIL mimicking W12E.

As mentioned above and as referred to in the figure legend, the experiment presented in Figure 8A was performed under serum starved conditions. Thus, the levels of phosphorylated ERK1/2 and cJun are much reduced in the unstimulated, DMSO-treated cells. Importantly, cJun/AP-1 is capable of activating its own expression, explaining the low total protein level of cJun in this sample. Further, as has widely been reported, MAPK signalling and AP-1 activity are critical drivers of HPV URR-driven transcription (e.g., see: Morgan et al. (2021) Cell Death Differ. 28(5):1669-87), so the reduced signalling activity resulting from serum starvation will consequently lower 18E7 expression. Shorter exposure times were selected for the pERK1/2, p-cJun and cJun western blots in Figure 8B to highlight the dramatic increase in pathway activation following KATP channel overexpression and for consistency with Figure 8A. We apologise for any confusion caused but do not believe that the potential issue raised here significantly alters any of the conclusions drawn.

We believe that an explanation of the mechanism by which KATP channel activity contributes to the activation of ERK1/2 signalling in HPV+ cervical cancer cells is beyond the scope of this initial publication. In the course of acquiring the data presented here, we aimed to provide some evidence of how KATP channels regulate proliferation in these cells, and therefore decided to investigate what impact they had on host cell signalling pathways known to be critical for proliferation. This led to us identifying enhanced ERK1/2 activity in response to KATP channel stimulation/overexpression. We agree that the critical next step will be to elucidate how channels promote ERK1/2 signalling, but this would require a significant body of work and as such would warrant being in a standalone or follow-up publication.

Throughout the manuscript, we endeavoured to validate our discoveries in the HPV18+ HeLa cell line in an additional cervical cancer cell line (HPV16+ SiHa cells). We provide evidence that the requirement for KATP channel activity is shared by both cell lines, and the impacts on oncoprotein expression, proliferation and cell cycle progression are highly concordant between the two cell lines. Thus, it is reasonable to assume that the mechanism by which KATP channels drive proliferation and HPV URR activity, elucidated in Figure 8 using HeLa cells, will be common to other HPV+ cervical cancer cell lines. Given the wealth of evidence supporting our proposal in Figure 8, and the highly concordant data presented earlier in the manuscript, we do not believe repeating all of the experiments in Figure 8 in a HPV16+ cell line is warranted. Similarly, as this manuscript focusses on HPV+ cancer, we believe that a study of the activation of MAPK/AP-1 signalling by KATP channels in HSIL mimicking W12E cells is beyond the scope of this paper and should constitute part of a standalone manuscript.

Minor Comments: In introduction, the authors mentioned that high risk HPV E6 and E7 deregulate cell cycle in host cells, and current limitations in cervical cancer treatments. Then they introduced importance of K+ ion channels in cell cycle regulation by sighting published literature not related to HPV, immediately followed by their proposed study on role of K+ATP channels in HPV infection. However, authors did not sufficiently clarify the rational of taking up a study on K+ channels in the context of HPV infection or E6 and E7 expression. If K+ATP channel proteins are elevated by E7, it is highly likely that there are some prior information on status of these transporters in the published literature or data from RNAseq analyses.

As the reviewer acknowledges, we described in the introduction the importance of K+ channels in regulating cell proliferation and the absence of effective HPV-specific therapies. We also stated that “ion channels may represent ideal candidates for … novel therapies given the abundance of licensed and clinically available drugs targeting the complexes which could be repurposed…”. Thus, the rationale for studying K+ channels in HPV+ cancers was to see if any of the available inhibitors of K+ channels could potentially be repurposed to be used in treating HPV-driven cervical cancer. We believe this logic is explained with sufficient clarity in the original draft.

Regarding prior analysis of KATP channel expression, in Figure 2H of the manuscript we analyse a publically available microarray dataset, which provides mRNA expression data for 128 cervical tissue specimens (24 normal, 14 CIN1 lesions, 22 CIN2 lesions, 40 CIN3 lesions, and 28 cancers specimens). ABCC8 expression was found to be significantly higher in the CIN3 and cervical cancer specimens, compared to normal samples. Details of the dataset used are provided in the ‘Materials and Methods’ section.

The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

Organotypic raft culture data was included to demonstrate increased SUR1 expression in primary keratinocytes containing HR-HPV to show broader upregulation of the host factor and strengthen data from cell lines and clinical samples. Our data shows an upregulation of SUR1 in the HPV-containing rafts compared to controls. However, raft culture is a highly complex, time-consuming and expensive technique to perform. Therefore, whilst we agree, in principal, that being able to more closely correlate the increase in SUR1 protein levels to specific layers of the epithelium (e.g. via H&E staining and for markers of E6/E7 activity) would be of value, we would not be able to perform these assays within a reasonable time frame. Moreover, we feel that it would not add significant new knowledge to the study of SUR1 in the context of HPV-driven cancers.

There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

As this manuscript primarily focusses on HPV+ cancer rather than HR-HPV infection, we believe an assessment of the role of KATP channels on HPV episome maintenance and/or genome replication to be beyond the scope of this study.

Reviewer #2 (Significance (Required)):____ (1) General Assessment: Strengths and limitations This study identified KATP channel components as novel regulators of transcriptional activity of high-risk HPV-18 URR through ERK1/2-c-Jun/AP1 pathway. Authors revealed that HPV E7 regulates expression of ABCC8, the gene for channel component SUR1 protein.

There are important limitations. (1) Lack of any information about homeostatic regulation of SUR1 in HPV infection, (2) Lack of sufficient evidence about potential confounding cytotoxic effects of SUR1 inhibition or E7 down regulation on cervical cancer cells. (3) Immunoblot experiments are not consistent. (4) There is no mechanism of how E7 regulates ABCC8 transcription. (5) What is the mechanism for SUR1 regulate cell cycle in HPV+ cells? (6) There is no mention of effect of SUR1 on cell cycle regulators p53 and pRB, which are direct targets of HPV E6 and E7 proteins. (7) There is no evidence for the role of Kir6.2 /SUR1 in the regulation of HPV-16 URR, which causes most of HPV-attributed cancers. (8) Authors did not analyze spatial relationship between HPV E6 and E7 activity and expression of SUR1 protein in raft cultures of human foreskin keratinocytes with or without E6E7 expression and in cervical cancer tissue.

We thank the reviewer for summarising in a concise manner the areas of this manuscript they believe could be improved. Our responses to points 1-6 and point 8 have been detailed above. Regarding (7), we present data showing that KATP channel inhibition/knockdown negatively affects E6 and E7 expression in HPV16+ SiHa cells (Figure 1D-E, Figure 3D and F, Supp Figure 3C-D). Furthermore, we present evidence that SUR1 knockdown in SiHa cells correlates with a reduction in HPV16 URR-driven luciferase activity (Figure 3H). We therefore believe that this issue was adequately addressed in the original manuscript.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ __Minor comments:

Knockout SUR1 stable cell lines and knockout HPV E7 stable cell lines should be established to test all the related data.

We have performed experiments using two well characterised inhibitors of KATP channels (glibenclamide and tolbutamide) and both siRNA- and shRNA-mediated knockdown of SUR1, all of which result in similar reductions in HPV E6/E7 expression. Further, the reductions in proliferation observed in HPV+ cell lines following glibenclamide treatment or siRNA/shRNA knockdown of SUR1 are also highly concordant. Thus, we do not believe the establishment of knock__out__ cell lines, using e.g. CRISPR/Cas9 technology, would significantly enhance the manuscript, particularly given the time and expense involved in this.

As noted by Reviewer #1 following cross-consultation, the establishment of E7 knockout cells lines is “unlikely to be possible because the cells require E7 for survival”. It has been previously demonstrated that in the absence of E7 expression, HeLa cells cease to proliferate and undergo senescence within 10 days (see: DeFelippis et al. (2003) J.Virol 77(2): 1551–1563). We therefore agree with Reviewer #1 and believe that, unfortunately, we would be unable to carry out the suggested experiment.

Tumor weights of the in vivo experiment should be indicated.

Tumour weights were not collected following the conclusion of the in vivo experiment, so we are unable to provide this information. The experiment was designed such that animals would be sacrificed upon the tumours reaching a set measurement (15 mm in either direction), rather than concluding the experiment at a set end point. Therefore, many of the tumours would have been of similar weight upon sacrifice, but critically the SUR1-depleted tumours took significantly longer to reach that size. We therefore believe that, given the experimental set-up, adding the tumour weights would not add significant value, even if we were able to provide this information.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

Authors present the evidence that HPV can target ATP-sensitive potassium ion (KATP) channels of a host to promote cervical carcinogenesis. They indicate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression by using activators and inhibitors of KATP channels. Furthermore, they verified SUR1 was upregulated in both HPV+ cervical cancer cells and in clinical samples in a manner dependent on the E7 oncoprotein. Knockdown of SUR1 or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. They propose that tumorgenesis effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. Overall, It is an interesting research to unveil the mechanism how HPV promote cervical carcinogenesis through ATP-sensitive potassium ion channels. However，some major concerns should be addressed.

Major comments:

• Are the key conclusions convincing?

I think the key conclusions are convincing. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

NO - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

NO - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. - Are the data and the methods presented in such a way that they can be reproduced?

Yes - Are the experiments adequately replicated and statistical analysis adequate?

The experiments are adequately replicated and statistical analysis

Minor comments:

• Specific experimental issues that are easily addressable.

Yes - Are prior studies referenced appropriately?

Yes - Are the text and figures clear and accurate?

yes - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Knockout SUR1 stable cell lines and knockout HPV E7 stable cell lines should be established to test all the related data.

Tumor weights of the in vivo experiment should be indicated.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The author explain for the first time that HPV can upregulate host ATP-sensitive potassium ion channels, consequently activate MAPK/AP-1 signalling contribute to cervical carcinogenesis. This is a new mechanism how HPV cause cervical carcinogenesis. However, the MAPK/AP-1 signalling contributes to carcinogenesis is well known. The technical in the experiment is commonly used. - Place the work in the context of the existing literature (provide references, where appropriate).

HPV can promote cervical carcinogenesis through different pathway including MAPK/AP-1(1: Wang M, Qiao X, Cooper T, Pan W, Liu L, Hayball J, Lin J, Cui X, Zhou Y,Zhang S, Zou Y, Zhang R, Wang X. HPV E7-mediated NCAPH ectopic expression regulates the carcinogenesis of cervical carcinoma via PI3K/AKT/SGK pathway. Cell Death Dis. 2020 Dec 11;11(12):1049. doi: 10.1038/s41419-020-03244-9. PMID:33311486; PMCID: PMC7732835. 2. Singh T, Chhokar A, Thakur K, Aggarwal N, Pragya P, Yadav J, Tripathi T, Jadli M, Bhat A, Gupta P, Khurana A, Chandra Bharti A. Targeting Aberrant Expression of STAT3 and AP-1 Oncogenic Transcription Factors and HPV Oncoproteins in Cervical Cancer by Berberis aquifolium. Front Pharmacol.2021 Oct 28;12:757414. doi: 10.3389/fphar.2021.757414. PMID: 34776976; PMCID: PMC8580881.). However, the detail mechanism is still elusive. Here, authors indicated E7 can upregulate SUR1, one component of ATP-sensitive potassium ion channels and activate MAPK/AP-1. SUR1 can also upregulat E7 levels. They set up a positive feedback loop to contribute cervical cancer. - State what audience might be interested in and influenced by the reported findings.

cervical carcinogenesis researchers and cancer drug researches. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

I familiar with cancer related signaling pathway and cancer chemoprevention research. Especially MAPK signaling pathway and related drugs.

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Referee #2

Evidence, reproducibility and clarity

Summary: Authors explored the role of k+ channel transporters in HPV induced cervical cancers. They used several inhibitors and gene knockdowns in biochemical and cell biology experiments to show that SUR1 and Kir6.2 components of K+ ATP channel, via activating pERK1/2 and c-Jun/AP1, up-regulate HPV URR promoter mediated expression of E6 and E7 proteins. They showed that SUR1 knockdown inhibited growth of HeLa cells in vivo.

Major Comments:

Overall, the authors performed many experiments to reveal an interesting and novel mechanism. (1) SUR1 expression and activity is necessary for HPV16 and-18 E6 and E7 expression. (2) HPV-16/18 E7 upregulates expression of ABCC8/SUR1 transcription. (3) SUR1 containing K+ATP channel then phosphorylates ERK. (4) Activated ERK then phosphorylates JUN/AP1. (5) Next, activated JUN/AP1 promotes E7 or E6E7 expression from HPV URR. However, in this cyclic feedforward regulation of these genes there is no control mechanism. Then how is homeostasis maintained in HPV infected lesions?

E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

Authors did not explain how HPV E7 would upregulate ABCC8 transcription or elevate SUR1 protein (Figure 4). Depletion of E7 is known to produce lethal effect in cervical cancer cell lines. No experiment was done to assess cytotoxicity. Hence it is not clear from the available evidence if the SUR1 is reduced by direct E7 mediated event or indirectly by general cytotoxicity induced by E7 knock down.

The increase of G1 population, determined by flow cytometry, of HeLa cells treated with Glib or SUR1 siRNA is relative to controls appears to be small and not supported by similar study on other HPV+ or HPV_ vervical cancer cell lines. Importantly the mechanism of this increased G1 in HeLa cell line is not clear. The immunoblot data about the role of cyclins are not sufficient.

What is the physiological effect of cyclin D1 in the context of HR-HPV infection (Figure 7)? In the event of HPV E7 mediated pRB degradation in cervical cancer cell lines, the inactivation of pRB by cyclin D1 does not appear to be physiologically relevant, may not account for difference in growth. It is known in literature that Cyclins A2 and B1 are often elevated by E7 activity. If SUR1 siRNA reduces E7-transcription and protein levels as shown in earlier results, why cyclinB1 and A2 protein level did not change?

Authors did not analyze expression level and role of p53, pRB proteins, the direct targets of E6 and E7 proteins, on cell cycle regulation following SUR1 siRNA or Glibenclamide-treatment in cervical cancer cell lines.

If activated ERK1/2 and c-Jun is required for URR activity, why are not they detectable in DSO or scrRNA treated HeLa cells (Fig 8A, B)? Why there is no 18 E7 in DMSO treated HeLa cells (Fig. 8A)? Authors also did not explain how inhibition of KATP channel regulates ERK phosphorylation in cervical cancer cell lines. There is no data from additional cervical cancer cell lines or HSIL mimicking W12E.

Minor Comments:

In introduction, the authors mentioned that high risk HPV E6 and E7 deregulate cell cycle in host cells, and current limitations in cervical cancer treatments. Then they introduced importance of K+ ion channels in cell cycle regulation by sighting published literature not related to HPV, immediately followed by their proposed study on role of K+ATP channels in HPV infection. However, authors did not sufficiently clarify the rational of taking up a study on K+ channels in the context of HPV infection or E6 and E7 expression. If K+ATP channel proteins are elevated by E7, it is highly likely that there are some prior information on status of these transporters in the published literature or data from RNAseq analyses. They did not provide physiological functions of K+ATP channel. I consider this information should be important part of the introduction.

The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

Additional immunofluorescence or histological analysis is necessary to assess the potential cytotoxic effects of E7 siRNA, SUR1 siRNA or KATP inhibitors (Glibenclamide) in cervical cancer cell lines

There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

Significance

(1) General Assessment: Strengths and limitations

This study identified KATP channel components as novel regulators of transcriptional activity of high-risk HPV-18 URR through ERK1/2-c-Jun/AP1 pathway. Authors revealed that HPV E7 regulates expression of ABCC8, the gene for channel component SUR1 protein.

There are important limitations. (1) Lack of any information about homeostatic regulation of SUR1 in HPV infection, (2) Lack of sufficient evidence about potential confounding cytotoxic effects of SUR1 inhibition or E7 down regulation on cervical cancer cells. (3) Immunoblot experiments are not consistent. (4) There is no mechanism of how E7 regulates ABCC8 transcription. (5) What is the mechanism for SUR1 regulate cell cycle in HPV+ cells? (6) There is no mention of effect of SUR1 on cell cycle regulators p53 and pRB, which are direct targets of HPV E6 and E7 proteins. (7) There is no evidence for the role of Kir6.2 /SUR1 in the regulation of HPV-16 URR, which causes most of HPV-attributed cancers. (8) Authors did not analyze spatial relationship between HPV E6 and E7 activity and expression of SUR1 protein in raft cultures of human foreskin keratinocytes with or without E6E7 expression and in cervical cancer tissue.

(2) Advance: This study identifies SUR1/Kir6.2 as new targets to intervene HPV-18 URR activity and demonstrates potential to inhibit growth of cervical cancer tumors using HeLa xenograft model. This study did not develop any new methodology, novel mutation or model system.

(3) Audience: This study is aimed at basic scientists involved in the field of HPV research.

(4) Describe your expertise. I have long experience in HPV research. I study regulation of HR-HPV18 life cycle in 3D organotypic raft cultures of HPV-18 infected neonatal foreskin keratinocytes. A major part of my research is focused on identification of novel therapeutics against cervical cancers using in vitro 3D organoids and in vivo PDX models.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript by Scarth and colleagues, the authors investigate the relationship between ATP-sensitive potassium ion channels (KATP) and the viability and growth of certain HPV-positive cancer cell lines. In a series of detailed and carefully conducted experiments, they determine that there is a correlation between KATP channel activity and levels of certain HPV E6 and E7 RNA and protein. KATP channels are active in HeLa cells. In HeLa and SiHa cells, inhibiting KATP activity decreases HPV oncoprotein levels. HPV-positive status in the cell lines examined is found to be associated with an upregulation of the ABCC8 gene, which encodes the SUR1 KATP subunit, and some of the data supports that SUR1 protein levels increase with cervical cancer CIN grade. Depleting SUR1 with shRNA or siRNA reduces KATP activity and decreases HPV E6 and E7 levels in HeLa and SiHa cells. The opposite is also true; depleting ABCC8 reduces the levels of HPV E6 and E7 transcripts. The effect on ABCC8 appears to be due mainly to the effects of HPV E7. Decreased KATP activity is associated with decreased growth of HeLa and SiHa cells in monolayer and in anchorage independent growth assays, perhaps not surprising given that E6/E7 levels are reduced in the cells under the treatment conditions. SUR1 overexpression itself promotes cell growth even in the absence of HPV oncoproteins. The growth defect upon KATP inhibition or siSUR1 is associated with some modest cell cycle dysregulation and, impressively, with reduced tumor growth in a mouse model. Finally, the authors present evidence that increased KATP activity is associated with increased recruitment of the transcription factor AP-1 to the HPV18 promoter and enhancer.

Overall, the data are of high quality and the individual results are consistent with each other and are convincing. However, the authors have understandably focused on two HPV-positive cancer cell lines (affected by modulating KATP levels) and one HPV-negative cancer cell line (which is not affected in the same way). The ability to extrapolate to conclusions about cervical or HPV-positive cancers in general is therefore limited and many of the authors' statements should be tempered to reflect the experiments they have conducted.

Addressing the following major points would help to strengthen the impact of the work:

1. The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?
2. There is a major concern regarding whether SUR1 protein is produced at a biologically relevant level in SiHa and HeLa cells, in which most of the experiments in the paper were conducted. Protein levels are assessed in Fig 2 by immunostaining in raft cultures and in a cervical cancer tissue microarray. However, protein levels are otherwise not examined, especially in SiHa and HeLa cells. Is SUR1 protein produced in these cells? Are its levels reduced by the knockdown approaches? The fold change RNA data presented in figure 2A does not convincingly address this question, since even an 8-fold increase of ABCC8 mRNA over a low background level might not have biological significance. It would be very helpful to measure SUR1 protein in several of the experiments in HeLa and SiHa cells.
3. The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.
4. Finally, since many of the experiments rely on knockdown approaches that show similar readouts, a rescue experiment (restore sh or si-resistant SUR1 and assess the impact on the phenotype) would confirm that the effects being observed are due to changes in SUR1 levels and not to off-target effects.

It is recognized that some of these experiments would be lengthy and technically challenging to perform. Measuring cell viability and SUR1 protein levels in SiHa and HeLa cells should be relatively straightforward. The experiments to address off-target effects (rescue experiment, deconvolving siRNA pool) are more involved. If it is not possible to complete such experiments, the possibility of off-target effects should be discussed in the text.

Minor comments:

The text and figures are clear and statistics are appropriate. The authors should include at what time point post siRNA transfection the experiments were conducted.

Referees cross-commenting

I note several areas of common feedback among the reviews. Several reviewers commented on the large number of experiments and that the work is of interest to researchers working on HPV and cancer therapeutics. Several reviewers shared concerns about cell viability upon HPV oncoprotein knockdown and about toxicity in various experiments. Several reviewers also raised concerns about the validation of SUR1 protein levels in several experiments. These concerns seem to me to be critical to address to strengthen the manuscript. I note that Reviewer #3's suggestion of making E7 knockout cells (presumably in HPV+ cancer cell lines) is unlikely to be possible because the cells require E7 for survival.

Significance

The work connects the biology of certain cervical cancer cell lines to KATP channels. It will be of interest to HPV researchers and to cancer researchers whose interests involve KATP signaling. As a reviewer, I have expertise in HPV biology but not in KATP signaling.

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Reply to the reviewers

Manuscript number: RC-2022-01673

Corresponding author(s): Eric Shoubridge

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This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

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

*In this MS, Scheuttpelz et al demonstrate that SLC25A46, a novel member of the mitochondrial carrier protein family localized to the outer-mitochondrial membrane, is an important regulator of mitochondrial dynamics. They show that knockout of SLC25A46 results in mitochondrial fragmentation, whereas over-expression of WT SLC25A46 or pathogenic variants/mutants of SLC25A46 results in mitochondria hyperfusion. SLC25A46 might affect fusion/fission directly since it is localized to both mitochondrial fusion and fission sites. Moreover, its loss/expression of variants alters the levels of the high molecular weight complexes of MFN2 and alters the levels of the long/short forms of OPA1. In addition, Scheuttpelz et al show that loss of SLC25A46 results in changes in the mitochondrial lipid profile, suggesting that SLC25A46 might regulate mitochondrial dynamics via regulation of mitochondrial lipid metabolism. Thus, the findings described are novel and exciting, however it remains poorly understood how SLC25A46 localization to fusion/fission sites is related to mitochondrial fusion/fission, and how are these results related to its effect on the MFN2/OPA1 complexes/forms, and to its possible role in regulating lipid metabolism. *

*Specific comments: *

1. *Are the DRP1 and the DRP1-receptor native complexes (appearing in BN-PAGE) altered in the SLC25A46 KO/+pathogenic variants cells? * We have tried to visualize the DRP-1 receptor complexes (MID49, MID51, MFF) on BN-PAGE gels without success. The Western blot in (a) below shows that the steady-state levels of all three receptors are similar under all conditions we tested, but the same antibodies used in this blot did not detect the native complexes on BN-PAGE gels. To our knowledge this has not been done in the literature. We previously reported (Janer et al, 2016) that DRP1 recruitment in patient fibroblasts (T142I) was only slightly reduced and that its oligomerization state (after crosslinking analysis) was slightly increased in the patient cells, which would not explain the mitochondrial hyperfusion.

*Do the pathogenic variants of SLC25A46 localize only to mitochondria? Do they fold similar to the WT protein (i.e., similar prot K cleavage products)? Are they loss- or gain-of-function variants/mutants? *

We previously provided images of all pathogenic variants in Supplementary Figure 1 by decorating with an SLC25A46 antibody; however the low steady-state levels of all but the R257Q variant make visualization difficult. Supplementary Figure 3d shows the R257Q variant with an analysis of its suborganellar localization. We performed a PK assay of R257Q (the most abundant pathogenic variant) and it behaves as the wild-type protein (rescued in knock-out background and in the control cell line) and as the outer membrane protein MFN2. We have now performed an alkaline carbonate extraction assays showing that all pathogenic variants (T142I, R257Q and E335D) are integral membrane proteins. (results shown below)

All described SLC25A46 mutations are loss-of-function biallelic missense, STOP or frameshift mutations, and where it has been investigated, all are associated with reduced steady-state levels of SLC25A46 protein compared to controls. The level of residual SLC25A46 protein correlates with disease severity Abrams et al. (2018).

Proteinase K Assay and Alkaline Carbonate Extraction show an integral insertion of SLC25A46 and its pathogenic variants into the outer membrane.

a) Proteinase K digestion assay of mitochondria from control fibroblasts or SLC25A46 knock-out fibroblasts with reintroduced wild-type protein (+wt) of SLC25A46 or the pathogenic variant R257Q. Mitochondria were exposed to an increasing concentration of proteinase K to determine the submitochondrial localization of SLC25A46. SLC25A46 and its pathogenic variants behave as outer membrane proteins. MFN2 was used as a control for an outer membrane protein, AIF for protein present in the inter‐membrane space, and SCO1 for an inner membrane protein. b) Alkaline carbonate extraction of mitochondria from control fibroblasts or SLC25A46 knock-out fibroblasts with reintroduced wild-type protein (+wt) of SLC25A46 or the pathogenic variants (+T142I, +R257Q, +E335D) . Immunoblot analysis shows that all SLC25A46 variants behave as integral membrane proteins. PRDX3 (soluble mitochondrial matrix protein) and MFN2 (integral outer membrane protein) were used as controls.

*The BN-PAGE results presented in Fig 5 appear without molecular weight markers, and thus the sizes of the complexes are not known. Why did the authors conclude that the bands that appear in the MFN1, MFN2, and OPA1 blots represent monomers and oligomers of these proteins (Fig 5b)? Is it possible that all/part of these immune-reactive bands represent complexes with other proteins and not monomers and/or homo-oligomers? How does SLC25A46 affect the complex state of these proteins if it does not associate with them in the native state, as seen in Fig 5d? *

We added a molecular weight ladder in Figure 5b which was confirmed using the known molecular weights the complexes of the oxidative phosphorylation complexes.

*Fig 5b (MFN2 blot): SLC25A46 KO cells expressing each of the pathogenic variants/mutants of SLC25A46 show different levels of the MFN2-immuoreactive higher molecular weight band (MFN2-HMWB; last three lanes), however all three cell lines show mitochondria hyperfusion. Moreover, the intensity of the MFN2-HMWB in two of these mutant lines (+T142I and +E335D) is similar to the intensity of the band that appears in the SLC25A46 KO cells, cells which show fragmented mitochondria. Thus, there is not a clear correlation between the state of SLC25A46, the levels of the MFN2-HMWB, and the mitochondrial morphology. *

The reviewer is correct and in fact we discusssed this point in the fourth paragraph of the discussion part in our paper: “The oligomerization of both MFN2 and OPA1 was altered by the loss of SLC25A46 function. High molecular weight oligomers of MFN2 were reduced in the null cell line and in the presence of all three pathogenic variants, a reduction that correlated with the steady-state level of residual SLC25A46 protein. Thus, rather unexpectedly MFN2 oligomerization did not correlate with mitochondrial morphology in our model.” It thus appears that the oligermerization state of MFN2 is not the determining factor for the observed changes in mitochondrial morphology.

*The authors' interpretations of the results presented in Fig 5d, arguing that there is a correlation between the appearances of the short/long forms of OPA1 and the fusion/fission state of the different cells, are not convincing. BN-PAGE results can vary between experiments, and thus need to be repeated and accompanied by densitometry analyses, especially in cases where the intensity of the bands (short and long forms of OPA1) seem largely similar in the single experiment presented. *

We have now performed additional two-dimensional electrophoresis (BN-PAGE/SDS-PAGE) analyses and have quantified the results. (a) Mitochondria from control, knock-out, re-expression of wt-SLC25A46 and the pathogenic variant p.T142I were run on a BN-PAGE with additional SDS gel-electrophoreses and immunoblotted against OPA1. (b) Quantification of the high molecular weight complexes (>600 kDa) of OPA1 (indicated in the green boxes in (a) relative to the total signal. (c) Quantification of the relative proportions of the long vs short forms of OPA1 forms in the high molecular weight complexes (>200 kDa) as indicated in the example (d) showing the longer forms of the higher complexes in the yellow box and the shorter forms in the purple box.

OPA1 forms high molecular oligomeric complexes that are altered in SLC25A46 loss of function cells

*Reviewer #2 (Significance (Required)): *

*The manuscript "SLC25A46 localizes to sites of mitochondrial fission and fusion and loss of function variants alter the oligomerization states of MFN2 and OPA1" partially characterizes the outer mitochondrial membrane protein SLC25A46, finding a localization to the tips and branching points of mitochondria and an effect on both mitochondrial internal structure and mitochondrial network dynamics in deletions and expression of specific mutants. The localization was conducted both with tagged protein and antibodies, which is appreciated, as tagging and overexpression can often alter localization of mitochondrial proteins. Interestingly, disease variants have an opposite effect as the deletion in mitochondria network behavior, with fragmented mitochondria in deletion strains and elongated or fused mitochondria in the mutant strains. The paper also finds alteration on membrane composition, and postulates a function in lipid exchange. While the paper falls short of a full functional characterization, the results are reasonable, internally consistent, and promising for future follow-ups. *

*Altered protein expression levels for the disease variant proteins is somewhat of a concern regarding the results, as it can be difficult to parse what cellular effects are due to altered protein activity versus altered protein levels, however this protein expression effect is consistent with previous literature and is likely unavoidable for this investigation. *

*Overall, the characterization of SLC25A46's localization, interactions, and effects on protein and mitochondrial structural/network organization suggests a function in mitochondrial OMM contact sites and that loss or mutation of this protein results in significant stress to the mitochondria with downstream effects. *

*Minor comments: *

*- What type of fibroblasts were used and was any subject information worth mentioning? I did not find this mentioned anywhere. *

We added an explanation in the Materials and Methods: “Fibroblasts were obtained from a cell bank located in the Montreal Children’s Hospital and the cell line we used was from a female healthy subject, 58 years old.”

*- For the confocal and STED microscopy use, what laser power was used for each excitation? More detail on the settings used for imaging with the microscopes would be help for experimental reproducibility. *

We have added to the Materials and Methods: For confocal microscopy “A laser power of 10 (for i.e. anti-PRDX3, MitoTracker, anti-OPA1) or 20% (anti-SLC25A46, SLC25A46-GFP) with a dwell time of 100 - 500 μs was used, depending on the strength of the antibody.” For the STED microscopy, we added: “A laser power of 90% was used for the confocal lasers and a laser power of 100% was used for the STED laser with dwell times of 5 μs and 20 μs, respectively.”

- Figure 7 C - the bar graph is very squished; one can barely see the levels of the small bars.

We have modified Figure 7C to make the results more visible.

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Referee #2

Evidence, reproducibility and clarity

See below my comments

Significance

The manuscript "SLC25A46 localizes to sites of mitochondrial fission and fusion and loss of function variants alter the oligomerization states of MFN2 and OPA1" partially characterizes the outer mitochondrial membrane protein SLC25A46, finding a localization to the tips and branching points of mitochondria and an effect on both mitochondrial internal structure and mitochondrial network dynamics in deletions and expression of specific mutants. The localization was conducted both with tagged protein and antibodies, which is appreciated, as tagging and overexpression can often alter localization of mitochondrial proteins. Interestingly, disease variants have an opposite effect as the deletion in mitochondria network behavior, with fragmented mitochondria in deletion strains and elongated or fused mitochondria in the mutant strains. The paper also finds alteration on membrane composition, and postulates a function in lipid exchange. While the paper falls short of a full functional characterization, the results are reasonable, internally consistent, and promising for future follow-ups.

Altered protein expression levels for the disease variant proteins is somewhat of a concern regarding the results, as it can be difficult to parse what cellular effects are due to altered protein activity versus altered protein levels, however this protein expression effect is consistent with previous literature and is likely unavoidable for this investigation.

Overall, the characterization of SLC25A46's localization, interactions, and effects on protein and mitochondrial structural/network organization suggests a function in mitochondrial OMM contact sites and that loss or mutation of this protein results in significant stress to the mitochondria with downstream effects.

Minor comments:

• What type of fibroblasts were used and was any subject information worth mentioning? I did not find this mentioned anywhere.
• For the confocal and STED microscopy use, what laser power was used for each excitation? More detail on the settings used for imaging with the microscopes would be help for experimental reproducibility.
• Figure 7 C - the bar graph is very squished; one can barely see the levels of the small bars.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this MS, Scheuttpelz et al demonstrate that SLC25A46, a novel member of the mitochondrial carrier protein family localized to the outer-mitochondrial membrane, is an important regulator of mitochondrial dynamics. They show that knockout of SLC25A46 results in mitochondrial fragmentation, whereas over-expression of WT SLC25A46 or pathogenic variants/mutants of SLC25A46 results in mitochondria hyperfusion. SLC25A46 might affect fusion/fission directly since it is localized to both mitochondrial fusion and fission sites. Moreover, its loss/expression of variants alters the levels of the high molecular weight complexes of MFN2 and alters the levels of the long/short forms of OPA1. In addition, Scheuttpelz et al show that loss of SLC25A46 results in changes in the mitochondrial lipid profile, suggesting that SLC25A46 might regulate mitochondrial dynamics via regulation of mitochondrial lipid metabolism. Thus, the findings described are novel and exciting, however it remains poorly understood how SLC25A46 localization to fusion/fission sites is related to mitochondrial fusion/fission, and how are these results related to its effect on the MFN2/OPA1 complexes/forms, and to its possible role in regulating lipid metabolism.

Specific comments:

1. Are the DRP1 and the DRP1-receptor native complexes (appearing in BN-PAGE) altered in the SLC25A46 KO/+pathogenic variants cells?
2. Do the pathogenic variants of SLC25A46 localize only to mitochondria? Do they fold similar to the WT protein (i.e., similar prot K cleavage products)? Are they loss- or gain-of-function variants/mutants?
3. The BN-PAGE results presented in Fig 5 appear without molecular weight markers, and thus the sizes of the complexes are not known. Why did the authors conclude that the bands that appear in the MFN1, MFN2, and OPA1 blots represent monomers and oligomers of these proteins (Fig 5b)? Is it possible that all/part of these immune-reactive bands represent complexes with other proteins and not monomers and/or homo-oligomers? How does SLC25A46 affect the complex state of these proteins if it does not associate with them in the native state, as seen in Fig 5d?
4. Fig 5b (MFN2 blot): SLC25A46 KO cells expressing each of the pathogenic variants/mutants of SLC25A46 show different levels of the MFN2-immuoreactive higher molecular weight band (MFN2-HMWB; last three lanes), however all three cell lines show mitochondria hyperfusion. Moreover, the intensity of the MFN2-HMWB in two of these mutant lines (+T142I and +E335D) is similar to the intensity of the band that appears in the SLC25A46 KO cells, cells which show fragmented mitochondria. Thus, there is not a clear correlation between the state of SLC25A46, the levels of the MFN2-HMWB, and the mitochondrial morphology.
5. The authors' interpretations of the results presented in Fig 5d, arguing that there is a correlation between the appearances of the short/long forms of OPA1 and the fusion/fission state of the different cells, are not convincing. BN-PAGE results can vary between experiments, and thus need to be repeated and accompanied by densitometry analyses, especially in cases where the intensity of the bands (short and long forms of OPA1) seem largely similar in the single experiment presented.

Significance

See above

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Reply to the reviewers

We thank the reviewers for thorough reading and for providing useful suggestions to improve our manuscript. We find two major issues indicated by the reviewers.

1. Lack of pathophysiological relevance to attract a broader readership – to address, we have stained brain slices of PD patient’s with p129-Syn and Lamin B1 antibodies. Microscopy images show extensive lamina damages in the patient brain slices which contain p129-Syn positive inclusions. These images are now included in the current revision of the manuscript as 6C-D. We think that these results in the pathologically relevant systems will now establish a connection between lamina defects with neurodegeneration in PD and will be attractive for a broader audience.

Experimental issues as indicated by major and minor points – majority of the points have been addressed in the current revision attached herewith. Given opportunity to submit a full revision, we shall incorporate more experiments to address all the points in the final revised manuscript.

Point by point response to reviewer’s concerns:

Reviewer 1

R1: The work by Mansuri and collaborators reports that LB-like filamentous inclusions of α-Synuclein are able to associate with and perturb the nuclear lamina due to an unbalanced mechanical tension between cytoskeleton and nucleoskeleton. Consequently, lamina-injuries are proposed as a major driver of proteostasis sensitivity in cells with LB-like Syn-IBs.

It is a complex work, in which a range of different cellular, biochemical and molecular techniques have been used. Readers of the paper (including the undersigned) will be wondering if a similar behaviour occurs in pathological systems, such as iPSC derived dopaminergic neurons arising from patients carrying the synuclein pathological mutations reported in this work.

Response: We thank the reviewer for bringing out the lack of pathophysiological relevance in our manuscript. To address, we imaged post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our experiments clearly show extensive lamina deformities in the patient brain (Fig. 6C-D) and connects with neurodegeneration in a pathological system.

Major points

R1: Authors should explain why there is a so high amount of p129-Syn in unseeded neurons (Fig. 1Ai, Fig. S1Bi): "p129-Syn was distributed throughout the neuron cell body and projections including light staining in the nucleus", as its accumulation is typical of PD-like α-syn aggregates. Similarly, unseeded neurons labeled with p129-Syn in Fig. 1Ai, Fig. 1Bi, and Fig. S1Bi and Fig. S1Ci are very different each other. Why? As neurons are unseeded, the pathological signature of PD-like α-syn aggregates should be very low or absent in all cases.

Response: We agree with the reviewer that very low amount of p129-Syn should be present in unseeded neurons. We standardized microscopy parameters using fields that contained neurons with both large LB-like perinuclear IBs and smaller peripheral Syn-filaments. We used Leica SP8 confocal microscope. Argon laser power was kept constant at 30% of full potential while Smart Gain was titrated to visualize the smaller filaments. For example, the smaller filaments were not clearly visible in Annexure Figure 1Ai when Smart gain was 690V. Smaller filaments were prominent when the Smart Gain was increased to 848V (Annexure Figure 1Aii, included with the revision plan attached herewith). We also observed light intra-nuclear staining of p129-Syn at 848V Smart Gain when we zoomed the arrow indicated nucleus in Fig. 1Aii shown below as Annexure Figure 1Aiii. Accordingly, we used Smart Gain: 650-850V in all the images presented in the manuscript. Brightness and contrast are now adjusted for all the images prepared for the revised manuscript for the optimum view of the immunostaining. All the raw image files will be submitted to https://www.ebi.ac.uk/biostudies in due course.

In order to rule out imaging artefacts at the higher Smart Gain (650V – 850V), we performed a control experiment without adding primary antibody against p129-Syn during immunostaining. Secondary antibodies were added and the Smart Gain was ~950-1000V during imaging. The light staining of p129-Syn as visible in Fig. 1Ai and 1Bi in the revised manuscript were not visible in this experiment (Annexure Figure 1B).

A table indicating the Smart Gain for all the images is included in the revised manuscript as__ Methods Table S5 - Laser Intensity.__

Reviewer 1 has also pointed out the difference in staining of p129-Syn in Fig. 1Ai and Fig. 1Bi. For Fig.1Ai, Rabbit monoclonal (p129-Syn (MJF-R13 (8-8), epitope: phosphoserine 129, cat# ab168381), and for Fig. 1Bi Mouse monoclonal (P-syn/81A, epitope: phosphoserine 129, cat# ab184674) were used. This information is now included in the figure legends. The difference in the staining pattern is due to the use of the different primary and secondary antibodies.

Lastly, we want to emphasize that the staining pattern seen in unseeded neurons () are not the typical PD-like Syn-aggregates but the soluble p129-Syn that is yet to be incorporated into the amyloid-filaments. p129-Syn ((antibody MJF-R13 (8-8)) staining pattern in 1Ai is continuous in the projections and light dotted in the periphery and inside nucleus. These dots also accumulate on the Microtubule Organizing Centre (MTOC) indicating the presence of aggresome-like inclusion bodies in the neurons. The staining pattern in 1Bi (antibody P-syn/81A) is dotted throughout. In both the cases, the continuous or dotted staining were not observed after seeding. The continuous staining at the projections seen in 1Ai is broken into smaller filaments in 1Aii (indicated by arrowheads). The broken filaments are much more increased in number and length in Fig 1Bii and the staining-intensity prominently increased. Accumulation of multiple larger filaments into perinuclear LBs is typical PD-like (Fig. 1Bii, yellow arrowhead).

The continuous staining and the broken staining patterns at the projections are also visible in the zoomed out MIP images presented in S1Bi and ii, respectively. The increase in fluorescence intensity of p129-Syn staining is prominent between S1Ci and ii indicating accumulation of p129-Syn in the form of large amyloid filaments in seeded neurons.

We now discuss the staining patterns in the revised manuscript. Please see pages 4-7.

R1: Authors should try to perform a more accurate quantification of the various colocalizations reported along the manuscript, i.e. by reporting the Pearson correlation coefficient or the Mander's overlap coefficient.

Response:As suggested by the reviewers, Pearson’s co-localization coefficient values have been added separately for all figured showing co-localization in Supplementary note: Colocalization figures and table.

Minor points

R1: In Fig. S1B the red fluorescent signal arising from γ-tubulin staining is not visible in the merged picture.

Response: Fig. S1B are the zoomed out MIP images of Fig. 1A. γ-Tubulin stains centrosome as tiny dots at the perinucleus in one of the z-sections of the MIP. To visualize these tiny dots in the MIP images, we have 1) optimized the brightness contrast of the MIP images and 2) provided a separate channel for γ-tubulin (arrowheads). These corrections are included in the revised version.

R1: Page 6: results of Fig. S1D-E should be explained properly (CALNEXIND and CMX-Ros staining).

Response:As suggested, we revised this part in Page 7.

R1: Fig. 2A: the indication of SNCA in western blotting is not proper, as in this experiment you evaluated the protein level, so it is better to report "α-syn";

Response:We agree with the reviewer. SNCA in western blots has been changed to α-Syn all the figures and figure legends.

R1: Fig. S2B: there is great variability in the number of SNCA(A53T)- EGFP and SNCA(DM)-EGFP cells with IBs during the course of PFF-incubation, so that authors did not reveal any significant difference. I think it is not completely correct to emphasize this data at page 9, lanes 12-13;

Response:We agree with the reviewer that the difference in number of SNCA(A53T)-EGFP and SNCA(DM)-EGFP cells with IBs was not statistically significant. Yet, we always observed aggressive biogenesis LB-like IBs in SNCA(DM)-EGFP cells. The statement in the manuscript is now corrected as per the reviewer’s suggestion (Page 9).

__R1:__Did authors reveal any cytotoxicity upon Congo Red treatment at the indicated concentrations (Fig. S2G)?

Response: Previously, Congo Red incubation was found to be non-toxic for neuronal cells even at 350 µM (PMID: 7991613). We have now performed MTT assay after Congo red treatment in our cells. The graph is now included as S2H. We did not observe any difference in cell viability even after treating the cells with the highest dose (100 µM) used in the experiment.

R1: I have concerns about the percentages reported in Fig. S2G: the percentage of cells with filaments in the absence of Congo Red is apparently too low as compared to the previously reported percentages.

Response:The reviewer is right. Number of Syn-filament containing cells varies between experiments because of ‘age’ of the recombinant amyloid seeds, different batches of seed preparation etc. We are repeating this experiment to increase the biological N. Results will be included and discussed in the final revised version

R1: Fig. S2G: I also believe that authors should report representative images of cells treated with Congo Red, in which Syn-filament biogenesis is prevented;

Response:As instructed by reviewer, the images are included in Fig. S2G.

R1: Fig. 2Eiii: The stick arrowhead seems to indicate a separate blob that is not so red: authors should consider to show separated channels and not only the merged picture (as in Fig. S3).

Response:We agree with the reviewer that the blob is not so red. We could not accommodate the separate channels in the main figure because of space constraint. Therefore, we presented the separate channels in Fig. S3A. Now we are including the stick arrowhead also at Fig. S3A.

R1:Page 10: authors should explain why they performed the LC3 staining;

Response:Previous reports indicated association of LC3B with α-Synuclein inclusions in neurons (PMID: 21412173, 31375560). Therefore, we also stained our cells with LC3 antibody. The references are now incorporated in Page 10.

R1: Why in Fig.2i, SNCA(DM) the ubiquitin signal is pink and not red?

Response:The blue of the DAPI is slightly overlapping with the ubiquitin staining at the aggresomes as these bodies are perinuclear making it appear pink. Separate channels are provided in Fig. S3E.

R1: Fig. 3, western blotting: as I previously reported, I think it would be better to write "total α-syn" instead of SNCA. Fig. 3D: is should be useful to explain properly the content of the soluble and insoluble fractions.

Response:We agree with the reviewer. SNCA in western blots has been changed to α-Syn all the figures and figure legends.

R1: Explain in the legend of Fig. 4 what is h2b tdTOMATO

Response:We thank the reviewer for pointing out the lack of information. This is now included with a reference in the revised manuscript.

Significance

R1: Overall this is interesting to read, a lot of data are presented, demonstrating a new potential phenomena that would be important to a specialized audience in the field of synuclein misfolding, aggregation and cellular toxicity.

Response: We have now included immunofluorescence images of post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our experiments clearly show lamina deformities in patient brain (Fig. 6D). We think that these experiments will highlight the pathophysiological relevance of the manuscript to make it appropriate for a wider audience.

Reviewer 2

__R2:__The present paper titled "Nuclear-injuries by aberrant dynein-forces defeat proteostatic purposes of Lewy Body-like Inclusions" provides an in details and compelling study about the formation of aggregates of SNCA in presence of PFFs, which other proteins play a role in the formation of this inclusions, and which pathways are the major players. They study provides many well-done experiments to highlight the composition and the process formation of these aggregates. unfortunately I think the study is lacking in connecting these events with neurodegeneration. how do all the pathways study impact viability and functionality of neurons and other disease relevant cells like astrocytes and microglia? it is thus a work which mainly focuses on the pathways leading to the formation of inclusions leaving untouched the question of how this might impact the disease. This does not take away the value of the findings but it should be taken in consideration when deciding which journal to submit.

Response:We thank the reviewer for the encouraging words and also for bringing out the lack of pathophysiological relevance in our manuscript. To address, we have performed immunofluorescence experiments with post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our results show extensive lamina deformities in patient brain (Fig. 6C-D) connecting neurodegeneration in PD with lamina injuries.

Further, although we found that LB-containing primary neurons and Hek293T cells do not show any loss in cell viability as estimated by LDH and MTT assays respectively (Fig 4A-B), they show sensitivity to additional stresses. LB-like IB containing Hek293T cells were unable to trigger stress response pathways and were vulnerable to heat stress. These results were already included in the earlier version of the manuscript (Fig. 4H-I). We now estimated sensitivity of neurons in presence of additional stress. We have subjected LB-containing neurons and control neurons to heat stress and estimated induction of Hsp chaperones by western blot and quantitative mass spectrometry. Preliminary results (included herewith) indicate that Hsp-upregulation is defective in neurons with LB-like IBs. These results are now included as Figure 4J-M in the attached revised manuscript. Repeat experiments with quantitative mass spectrometry will be included in the final revision.

R2: I have a few suggestion for each figure which will not take much time, energies or expenses but that would overall make the paper easier to read and digest.

R2::Fig 1: quantification of aggregates dimension, number and colocalization score with p62 (Pearson)

Response:Co-localization score with p62 is included in the current revision (Supplementary note: Colocalization figures and table). Quantification of aggregate dimension, number etc. in neurons have been already documented by Mahul-Mellier et al. (PMID: 32075919). We are following the same protocol and therefore did not repeat the counting for neurons. However, if the reviewer thinks that its mandatory, we shall do that and include with full revision.

__R2:__Fig 2: aesthetic comment: the way to read the figure should be consistent throughout the figure. they should be assembled either all in vertical or all in horizontal.

Response:We tried. We find the current organization is the best fit to accommodate all panels.

R2: Fig 3: 3E better to put an image without nocodazole to visualize the difference

Response:The control image is now added in Fig. 3E.

R2: 3D probe WB also for SNCA

Response:Sorry for the confusion. The western blots in 3D are probed for both total Synuclein and p129-Syn. As suggested by the first reviewer, we have also changed SNCA to α-Syn which indicates the total Synuclein protein level.

R2: 3K this WB needs quantification to backup the statement made

Response:We are repeating this experiment. Results will be included and discussed in the final revised version.

R2: 3I check the - and + for PFF and doxy. I believe they are wrong

Response:We have rearranged the figure. The scheme in Fig. 3I (now Fig. 3H) is correct but we have made it simpler to avoid confusion.

R2: Fig 4: missing IF of peri nuclear IBs with HS

Response:The images are now included as Fig. S4E and discussed in page 19.

R2: Fig 5: quantification of H2BTdTom exit from the nucleus

Response:We have performed this experiment as a supporting evidence of the nuclear damage in presence of LB-like IBs. We have quantified the damages in Fig. 5A and D. We have also performed quantitative mass spectrometry to show nuclear entry of associated organelle proteins (Fig. S5G). We think, quantifying the H2BTdTom exit will not be a significant value addition to the manuscript.

R2: Fig 6: some neurons with large PFF seems very unhealthy. is it possible to quantify neuronal viability may not with MTT which is not suited for single cells analysis?

Response:The reviewer correctly pointed out that neurons with large LB-like IBs seemed unhealthy which was confirmed by ƴH2AX staining indicative of extensive DNA damage in Fig. 6B.

R2: maybe it would be nice to have a WB with soluble and insoluble SNCA and p129 with ciliobrevin D with and without PFF. Ciliobrevin D might also impact degradative systems as demonstrated by the EHNA compound (PMCID: PMC5584856).

Response:We have performed the dynein experiments to figure out the role of cytoskeleton-nucleoskeleton tension in the lamina injuries in LB-like inclusion containing cells. However, we think that the reviewer has correctly pointed out that dynein may have a direct role in degrading Synuclein by either autophagy or proteasome. Given the results of the suggested experiments are not going to change the final conclusion of the manuscript, we propose to limit ourselves in discussing this possibility and citing the paper in the current revised version of the manuscript (page 29).

Significance

R2: As already stated above, the experiments are correctly performed and the evidence are well-presented and demonstrated. the realm that this paper falls into is not though neuroscience. The aim of this paper is to study the formation of inclusions regardless of their impact on disease-relevant cell type functions. the presented experiments are numerous and even though the message is pretty clear some figure might be too crowded to correctly convey the message (see fig 3). some of these findings even tough with much less details were already suggested by other papers (PMCID: PMC5584856) in which the importance of the dynein was studied in the context of the communication between autophagy and proteasome. I think adding this angle with few experiments might add a little bit more relevance but it is also true that this paper has already a lot of data.

Response:Thank you very much for the encouraging comments

R2: the type of audience for this paper I think is a very specialized audience which is interested in molecular mechanisms of inclusions formation and protein-protein interaction. as a final statement the paper is beautifully done and is relevant but it lacks the translational angle.

Response:We again thank the reviewer for reminding the lack of pathophysiological relevance. We have now included microscopic images of brain slices of PD patients with extensive lamina defects (Fig. 6D) and think this will attract a broader audience.

R2: my field of expertise is neuroscience. I have expertise in bimolecular techniques as well as cellular techniques to study neurodegenerative diseases

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Referee #2

Evidence, reproducibility and clarity

The present paper titled "Nuclear-injuries by aberrant dynein-forces defeat proteostatic purposes of Lewy Body-like Inclusions" provides an in details and compelling study about the formation of aggregates of SNCA in presence of PFFs, which other proteins play a role in the formation of this inclusions, and which pathways are the major players. They study provides many well-done experiments to highlight the composition and the process formation of these aggregates. unfortunately I think the study is lacking in connecting these events with neurodegeneration. how do all the pathways study impact viability and functionality of neurons and other disease relevant cells like astrocytes and microglia? it is thus a work which mainly focuses on the pathways leading to the formation of inclusions leaving untouched the question of how this might impact the disease. This does not take away the value of the findings but it should be taken in consideration when deciding which journal to submit.

I have a few suggestion for each figure which will not take much time, energies or expenses but that would overall make the paper easier to read and digest.

Fig 1: quantification of aggregates dimension, number and colocalization score with p62 (Pearson)

Fig 2: aesthetic comment: the way to read the figure should be consistent throughout the figure. they should be assembled either all in vertical or all in horizontal.

Fig 3: 3E better to put an image without nocodazole to visualize the difference 3D probe WB also for SNCA 3K this WB needs quantification to backup the statement made 3I check the - and + for PFF and doxy. I believe they are wrong

Fig 4:missing IF of peri nuclear IBs with HS

Fig 5: quantification of H2BTdTom exit from the nucleus

Fig 6: some neurons with large PFF seems very unhealthy. is it possible to quantify neuronal viability may not with MTT which is not suited for single cells analysis

Fig 7: maybe it would be nice to have a WB with soluble and insoluble SNCA and p129 with ciliobrevin D with and without PFF. Ciliobrevin D might also impact degradative systems as demonstrated by the EHNA compound (PMCID: PMC5584856).

Significance

As already stated above, the experiments are correctly performed and the evidence are well-presented and demonstrated. the realm that this paper falls into is not though neuroscience. The aim of this paper is to study the formation of inclusions regardless of their impact on disease-relevant cell type functions. the presented experiments are numerous and even though the message is pretty clear some figure might be too crowded to correctly convey the message (see fig 3). some of these findings even tough with much less details were already suggested by other papers (PMCID: PMC5584856) in which the importance of the dynein was studied in the context of the communication between autophagy and proteasome. I think adding this angle with few experiments might add a little bit more relevance but it is also true that this paper has already a lot of data.

the type of audience for this paper I think is a very specialized audience which is interested in molecular mechanisms of inclusions formation and protein-protein interaction. as a final statement the paper is beautifully done and is relevant but it lacks the translational angle.

my field of expertise is neuroscience. I have expertise in bimolecular techniques as well as cellular techniques to study neurodegenerative diseases

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Referee #1

Evidence, reproducibility and clarity

The work by Mansuri and collaborators reports that LB-like filamentous inclusions of α-Synuclein are able to associate with and perturb the nuclear lamina due to an unbalanced mechanical tension between cytoskeleton and nucleoskeleton. Consequently, lamina-injuries are proposed as a major driver of proteostasis sensitivity in cells with LB-like Syn-IBs. It is a complex work, in which a range of different cellular, biochemical and molecular techniques have been used. Readers of the paper (including the undersigned) will be wondering if a similar behaviour occurs in pathological systems, such as iPSC derived dopaminergic neurons arising from patients carrying the synuclein pathological mutations reported in thins work.

There are some concerns that should be addressed by the authors.

Major points

• Authors should explain why there is a so high amount of p129-Syn in unseeded neurons (Fig. 1Ai, Fig. S1Bi): "p129-Syn was distributed throughout the neuron cell body and projections including light staining in the nucleus", as its accumulation is typical of PD-like α-syn aggregates. Similarly, unseeded neurons labelled with p129-Syn in Fig. 1Ai, Fig. 1Bi, and Fig. S1Bi and Fig. S1Ci are very different each other. Why? As neurons are unseeded, the pathological signature of PD-like α-syn aggregates should be very low or absent in all cases.
• Authors should try to perform a more accurate quantification of the various colocalizations reported along the manuscript, i.e. by reporting the Pearson correlation coefficient or the Mander's overlap coefficient. Minor points
• In Fig. S1B the red fluorescent signal arising from γ-tubulin staining is not visible in the merged picture.
• Page 6: results of Fig. S1D-E should be explained properly (CALNEXIND and CMX-Ros staining).
• Fig. 2A: the indication of SNCA in western blotting is not proper, as in this experiment you evaluated the protein level, so it is better to report "α-syn";
• Fig. S2B: there is a great variability in the number of SNCA(A53T)- EGFP and SNCA(DM)-EGFP cells with IBs during the course of PFF-incubation, so that authors did not reveal any significant difference. I think it is not completely correct to emphasize this data at page 9, lanes 12-13;
• Did authors reveal any cytotoxicity upon Congo Red treatment at the indicated concentrations (Fig. S2G)?
• I have concerns about the percentages reported in Fig. S2G: the percentage of cells with filaments in the absence of Congo Red is apparently too low as compared to the previously reported percentages.
• Fig. S2G: I also believe that authors should report representative images of cells treated with Congo Red, in which Syn-filament biogenesis is prevented;
• Fig. 2Eiii: The stick arrowhead seems to indicate a separate blob that is not so red: authors should consider to show separated channels and not only the merged picture (as in Fig. S3).
• Page 10: authors should explain why they performed the LC3 staining;
• Why in Fig.2i, SNCA(DM) the ubiquitin signal is pink and not red?
• Fig. 3, western blotting: as I previously reported, I think it would be better to write "total α-syn" instead of SNCA.
• Fig. 3D: is should be useful to explain properly the content of the soluble and insoluble fractions.
• Explain in the legend of Fig. 4 what is h2b tdTOMATO.

Significance

Overall this is interesting to read, a lot of data are presented, demonstrating a new potential phenomena that would be important to a specialized audience in the field of synuclein misfolding, aggregation and cellular toxicity.

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Reply to the reviewers

The authors do not wish to provide a response at this time.

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Referee #3

Evidence, reproducibility and clarity

see the "Significance" section.

Significance

This manuscript reports the role and mechanism for AF10 in inhibition of mouse somatic cell reprogramming. It is known that DOT1L inhibits somatic cell reprogramming. In this study, a number of known DOT1L-interacting proteins were examined for their role in this process. They found that only AF10 (MLLT10) plays a similar role in somatic reprogramming, i.e., deletion of AF10 promotes reprogramming of somatic cells into iPS cells. Experiments in combination with DOT1L inhibitors showed that AF10 functioned in the same pathway as DOT1L. Reprogramming with AF10 mutants revealed that the AF10-DOT1L interaction but not the binding of AF10 to unmodified H3K27 is critical for reprogramming and somatic cell identity. ChIP-seq showed that AF10 deletion caused an ESC-like pattern of H3K79me1 at house-keeping genes. The data supported the conclusions. It is well-written. This study provided mechanistic insights into the role of DOT1L-AF10 in maintaining somatic cell identity and inhibiting somatic cell reprogramming.

Major:

This study is very similar to the following publication as cited:

Deniz Uğurlu-Çimen, Deniz Odluyurt, Kenan Sevinç, Nazlı Ezgi Özkan-Küçük, Burcu Özçimen, Deniz Demirtaş, Eray Enüstün, Can Aztekin, Martin Philpott, Udo Oppermann, Nurhan Özlü, Tamer T. Önder. (2021). AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation. Epigenetics Chromatin 14, 32. https://doi.org/10.1186/s13072-021-00406-7.

Both manuscripts were deposited in BioRxiv in December 2020. Clearly these were two independent studies. The methodology and conclusions are very similar.

Minor:

Fig. 1B. The Axis labels are too small.

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Referee #2

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors investigated the role of AF10, a subunit of DOT1L histone-methyl transferase complex for writing H3K79me1-2-3 marks, in cellular reprogramming. Using siRNA-mediated knockdown and chemical inhibitors, the authors show that AF10, and DOT1L as a whole, are inhibitory to reprogramming of mouse embryonic fibroblast cells (MEF) to induced pluripotent cells (iPSC), suggesting that AF10 plays an important role in determination and changes in cell lineages. The authors also show that this effect of AF10 is not transcription mediated. Based on their ChIP experiments of H3K79me1,2,3 and RNA Pol II, the authors claim that the effect of AF10 is mediated by "changes in epigenome circuitry".

Major comments

1. The claim that AF10 and DOT1L inhibits reprogramming of MEF to iPSC is largely supported by authors' experiments. Mostly, the authors used expression levels of NANOG as a mark for pluripotency. While it is a well-documented mark, an orthogonal mark (such as colony morphology, embroid bodies, etc.) will increase the rigor and confidence. This is especially important in the context of testing something like DOT1L complex which plays important role in transcription.
2. The data presented here largely supports the claim that AF10-mediated effect is not through transcription.
3. The authors final model ¬- "negative feedback by RNA-PolII recruited DOT1L leading to ESC-like state" - is not supported by the data presented here.
   • For example, at line 295, the authors say that H3K79me1 pattern in ΔAF10 "resembles the H3K79me1 found in ESCs which are much more TSS-enriched for this modification compared to MEFs." However, the data in 5H show that the pattern in ESC matches more with AF10 fl than ΔAF10.
   • At line, 299, "given that AF10 deleted cells retain H3K79 methylation..". This statement highly contradicts data in 4B, 4C, 5G and 5H where it is shown that deletion of AF10 leads to substantial loss of H3K79me1,2.
   • While the authors showed there are changes in H3K79 methylation pattern upon AF10 deletion, its link to changes in iPSC reprogramming is not shown. The Pol II occupancy data, shown for WT MEFs and ESC, do not support any of part of this claim. Even further, there is no evidence for changes in Pol II occupancy levels upon AF10 deletion.
4. How do authors reconcile that there is increased expression of AF10 in pluripotent cells (Fig. 1A and 1B) although it inhibits pluripotency?
5. Line 341, "We do not find any evidence that H3K79me2 opposes spreading of H3K27me3 in reprogramming to iPSCs" seems to be an over-interpretation. The experiment just shows that inhibition of PRC does not change global H3K79me2 levels. A direct role of H3K79me2 on H3K27me3 is not tested here.
6. Fig S1D shows that deletion of AF10 can have additional effect to inhibition of DOT1L. This is in contrast to most of the main figures, especially, fig 1E. Some comment about this discrepancy is warranted.

Minor comments

1. It might help the reader if authors put a schematic of reprogramming regimen for Fig. 1A.
2. At line 146, the authors inference " ΔAF10 is estimated to contribute about 40% of the DOT1Li phenotype in reprogramming" is not clear. It may help the reader the reader if more information is provided for their analyses and interpretation.
3. Line 324, a typo: it should be "AF10"
4. Line 456, It might be better for readers if the authors report whether and how RT-qPCR was normalized to housekeeping genes etc.
5. Line 582, It is not clear at what step human cells were spike in. Also the type of human cells should also be reported.
6. At many places (e.g. Fig 1E, Fig S3D) authors seem to have used multiple t-tests. Please consider using something like ANOVA to avoid multiple t-test error.
7. Fig 1E. It is commendable that authors show factor independent reprogramming. It will be helpful for readers if authors show number of days for OSKM-dependent and OSKM-independent growth in the schematic.
8. Fig S1C is not clear as such. Please add more information in the figure or legends.

Referees cross-commenting

With regards to reviewer1's comments: I particularly agree with major points 1 and 2 that authors' current model regarding feedback regulation needs more evidence. The technical concerns regarding ChIP normalization, esp. point 5, are also well-warranted.

With regards to rev3's comments: The major concern about another similar study is well-warranted. The authors may want to explicate compare and contrast their key inferences with the other study.

Significance

The present work provides good evidence that AF10-mediated H3K79me can contribute to cellular reprogramming independent of steady-state mRNA levels. However, I think that the manuscript falls short of providing the basis for it. The claim that it is through subtle changes in H3K79me patterns seems nebulous and unsupported by the data presented here. If the manuscript finds the mechanistic basis for AF10's role in cellular reprogramming, it will be of interest to readers in general epigenetics as well as clinical fields that use histone methyl transferase inhibitors for treating leukemia.

I am not an expert in the field of cellular reprogramming; so, I may not be able to judge the merits or caveats of authors' reprogramming methods and analyses.

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Referee #1

Evidence, reproducibility and clarity

Summary

Inactivation of the histone methyltransferase DOT1L increases the efficiency of reprogramming somatic cells to included pluripotent stem cells. Recent studies have shown that loss of the DOT1L-interacting protein AF10 or disruption of the DOT1L-interacting domain (OMLZ) of AF10 phenocopies DOT1L inhibition in human cells. Here, Wille et al use a transgenic reprogrammable mouse model to study the role of AF10 in reprogramming of mouse somatic cells. Using a conditional AF10 deletion allele, loss of AF10 was found to partially phenocopy inhibition of DOT1L and evidence is provided that AF10 and DOT1L act in the same pathway. Elegant rescue experiments showed that loss the OMLZ domain is sufficient to abolish the programming-barrier function of AF10. In contrast to human cells, AF10 deletion had a minimal impact on mRNA expression during reprogramming. Analysis of H3K79me1/2 patterns showed that AF10 loss leads reduced H3K79me1/2 levels across the genome, and a redistribution of H3K79me1 at highly expressed genes from a peak in gene bodies to a peak downstream of the TSS. This pattern is similar to that seen in ESCs, in which DOT1L activity and overall methylation levels are lower compared to MEFs. These findings provide evidence for the model that the DOT1L-AF10 interaction is critical for efficient H3K79 methylation and for posing a reprogramming barrier. In the absence of AF10, DOT1L can still methylate histones at highly expressed genes, presumably due to interactions with RNA Pol II and transcription elongation factors, but its overall activity is reduced.

Major points

1. The quantitative ChIP-seq analyses of H3K79me1 and H3K79me2 in control and AF10 knock-out cells reveal interesting patterns. In MEFs, H3K79me2 peaks at TSSs and H3K79me1 more in gene bodies, consistent with high DOT1L activity and conversion of H3K79me1 to H3K79me2 at TSSs. In ESCs, in which the nuclear DOT1L activity is much lower, H3K79me2 levels at the TSS are lower and H3KK79me1 levels at the TSS higher. AF10 loss during programming leads to a pattern similar to that found in ESCs. The authors suggest that 'deletion of AF10 is likely to enhance reprogramming by making the epigenome more ESC-like at predominantly housekeeping genes' (and Fig 6). This is an interesting hypothesis. However, the data is also consistent with an alternative and more-simple model that AF10 is needed to boost the catalytic activity of DOT1L and that partial loss of DOT1L activity upon loss of Af10 is sufficient to promote reprogramming. The latter model is supported by the observation that DOT1Li has dose-dependent effects and that loss of AF10 enhances reprogramming in combination with a range of DOT1Li concentrations and thus at a range of H3K79me1/2 levels (Figure S1). It would be useful to discuss different models side by side.
2. In this context, the role of housekeeping genes also deserves attention. Line 276: 'Thus, the effect of AF10 deletion on promoting pluripotency occurs on genes that are commonly H3K79 methylated across cell types and not at specific lineage genes'. There is indeed a difference between highly and more lowly expressed genes but the causal relationship and role of housekeeping genes require further study. The data presented in this paper do not demonstrate that AF10 deletion affects pluripotency via genes that are commonly methylated by DOT1L. Therefore, without additional data, it seems too early to propose models of transcriptional feedback for biosynthetic/housekeeping genes (Discussion).
3. For the ChIP-seq studies, a spike-in method is used to detect and take into account global differences in histone methylation. This method is based on the ChIP-Rx protocol of Orlando et al (2014). In the Orlando study, Drosophila chromatin was used for spike with the rationale that there is little homology between human/mouse and fly genome sequence, leading to minimal mapping of spike-in fly genome reads to the human/mouse genome. Here, the authors use mouse chromatin with a human chromatin spike-in. In the analysis method described (first mapping to the mouse genome and then aligning unmapped reads to the human genome), this potentially leads to mapping of human spike-in reads to the mouse genome. Even though the human spike in is only 1/53th of the total sample, the authors should adjust their ChIP analysis to avoid this issue. One possible solution is to generate a combined human-mouse reference genome, map unique reads, and then calculate the fraction of reads mapped to human and mouse. Alternatively, non-unique regions can be blacklisted.
4. Related to the previous point, it is not clear to me how the scaling factor is calculated based on the numbers of Table. 1. The numbers given for the scaling factors do not seem to relate to the ratio of mouse/human reads. The authors should explain the scaling factor in more detail.
5. H3K79me enrichment is calculated per gene body, normalized per kilobase of gene length. The authors should consider alternative metrics. While the method used is suitable for histone modifications that occur across the gene body, it might be less suitable or relevant for H3K79me, which predominantly occurs at the 5' end of transcribed gene bodies until it reaches internal exons (DOI 10.1038/nsmb.1924). Based on this distribution, normalizing per kilobase of gene length will lead to artificial lower enrichment scores for longer genes. Given the predominant localization of H3K79me at the 5' end of genes bodies, it seems more meaningful to calculate H3K79me enrichment in this region only instead of normalize per gene length.
6. The label of Figure 1B is hard to read and the cell dots are hard to distinguish. Please increase font size and resolution. In this panel it is not clear to me whether the color indicates (graded) expression level or a more binary detection of transcripts? If the latter is the case, the signal (detection of a transcript in a single cell) might depend on the expression level of a transcript and the sequencing depth of each sample. Because of this uncertainty, it seems premature to speculate, based on single cell RNA-seq data, about variation in DOT1L complex formation. The authors should discuss this and take this into account in the analysis and representation of the data, or remove the panel and panel S1A.
7. Several figure panels have very small fonts. Some of the text is not readable. The authors should increase the font size of these panels. Some of the legends are incomplete. Please explain all the abbreviations used in the legends.

Minor points

Figure 1D. Please explain the abbreviations in the legend.

Figure 1E and 3F. Please explain the statistical test in the legend: e.g. tested against ff control. If the data was normalized to deltaAF10+DOT1Li, how was this condition taken along in the statistical test?

Figure 1F. The line can be drawn this way across the datapoints but whether or not this is evidence for a linear relationship is not clear because the data points do not all follow the trend. More importantly, the added value of this analysis is not obvious. Clearly, a higher fraction of Af10-deleted cells is expected to lead to a higher fraction of cells with a programming phenotype associated with Af10 loss. I suggest that this panel is removed but that the relevant notion that near complete of Af10 loss contributes about 40% of the DOT1Li phenotype is maintained.

Figure 3B/D. The pairing of the figure panels can lead to confusion. Empty vector refers to fl cells (black bar) as well as deltaAF10 cells (set to 100% and used as a reference; please add a dashed line at 100% with deltaAF10 label like in Fig. S3B), while the other constructs refer to deltaAF10 cells. To avoid confusion it would help to separate panel B and D and in panel D more clearly separate the fl cells from the deltaAF10 cells.

Figure 3-4. Tubulin is used as a loading control for H3K79me1/2. A pan-histone H3 would be a more unambiguous control.

Figure 4D. This panel shows changes in H3K79me1/2 ChIP-seq in DOT1Li treated vs control. Was this data normalized by the spike-in method? The samples are not mentioned in Table 1. The same question applies to the ESC vs MEF comparison.

Figure 5B. It is not clear to what section of the bars the percentages next to the bars refer to.

Figure 5D. Overlap of gene should be overlap of genes

Figure 5G-H. Please explain the percentages in the legend.

Figure S1B. Please explain the error bars and number of replicates.

Figure S1D. The error bars refer to technical replicates. The authors should show biological replicates.

Figure S2B. I could find a discussion in the text of the enriched motif of Cluster 7.

Figure S3A. The axis labels are not readable. Please explain the two axes and the rationale for using this gate.

Figure S3B. The authors should use SD instead of SEM.

Significance

This study builds on a growing body of work on the role of DOT1L in reprogramming of somatic cells. Recent studies point to a connection with the DOT1L-binding protein AF10 but the mechanisms, especially at the level of the epigenome, remained unclear. In general, very little is known about how DOT1L, its partners, and the methylation it deposits affect gene expression and cell fate.

This study confirms that in mouse cells, similar to human cells, the DOT1L-interaction domain is involved in the reprogramming function of AF10. Importantly, in contrast to human cells, in the mouse model AF10 loss has minimal effect on gene expression, suggesting that alternative mechanisms must be involved. Focusing on the epigenome, and using a quantitative ChIP approach, the authors describe how H3K79me1 and H3K79me2 are affected by loss of AF10 and how this relates to gene expression and occupancy of RNA PolII. Although the precise mechanisms remain to be elucidated, the results provide an important basis for identifying the relevant molecular changes at the epigenome.

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Reply to the reviewers

Manuscript number: RC-2022-01682

Corresponding author(s): Peter Keyel

1. General Statements

We thank the reviewers for their thorough and critical analysis of our manuscript. We have addressed most of the concerns and questions with our revised version. To address the remaining concerns, we plan to perform two lines of experiments— aerolysin sensitivity of dysferlin null C2C12 muscle cells and aerolysin sensitivity of ESCRT-impaired cells. When these experiments are complete, we believe the revised contribution will provides important novel insights into membrane repair that will appeal to a broad audience.

Reviewer comments below are in italics.

Description of the planned revisions

Reviewer 1

Major

In order to show that patch repair is indeed protecting cells against aerolysin, the authors should disrupt patch repair of the cells under study and observe and increased toxicity.

Reviewer 2

Major

*1. The effect of dysferlin overexpression does not indicate that patch repair is a protective mechanism or that dysferlin plays a significant role in aerolysin resistance. The authors should knock out dysferlin and assess cell resistance to lysis. *

Reviewer 3

Significance

The work presents a foundation to further investigate into the mechanism of aerolysin function, following the discovery of the role of extracellular Ca2+ in its activity. As aforementioned, the role of dysferlin in resisting aerolysin also has potential, but the limitations of this work were discussed including the absence of performing a dysferlin knockout, although performing this experiment may help to strengthen the current finding.

We agree with all 3 reviewers that a dysferlin knockout will complement our gain-of-function studies and this will strengthen the manuscript. We plan to challenge C2C12 myocytes that express control shRNA or dysferlin shRNA with toxin and determine their sensitivity.

We chose this system instead of targeting a patch repair protein in HeLa cells for 3 reasons. First, it will provide the corresponding loss-of-function experiment to match the gain-of-function experiments we have already done. Second, other patch repair proteins work redundantly with other proteins, complicating their knockdown and/or their disruption may interfere with lipid/protein transport. Finally, dysferlin null C2C12 cells are commercially available, so other groups will have an easier time replicating our results.

Reviewer 1

Significance

*and in the statement that a cellular process that has been artificially introduced in the experimental system is the cellular protection mechanism against aerolysin attack. In order to prove that this process is a bona fide protection mechanism, the authors should show that it is present without the need of overexpressing a protein that is not expressed at all either in the used cell line (HeLa), or in the natural cellular target of aerolysin (epithelial cells). The significance of the proposed protection mechanism is therefore questionable. *

We plan to address this concern by using C2C12 muscle cells that have and do not have dysferlin. Muscle cells are natural cellular targets of Aeromonas during necrotizing soft-tissue infections.

Reviewer 2

Major

*2. ESCRT complex was shown to play a role in plasma membrane repair following mechanical damage or perforin treatment of cells (Jimenez 2014, and Ritter, 2022). Whether ESCRT is important in aerolysin pore repair can be assessed by knocking out the Chmp4b gene or overexpressing dominant-negative mutant of VPS4a, E228Q. *

We plan to use a previously characterized (Lin 2005 PMID: 15632132) inducible system (TRex cells) to express the dominant negative VPS4b E235Q in cells. We plan to pulse cells for 2 h with 1 ug/mL doxycycline one day prior to the assay. This pulse time and dose strikes a balance between cell death due to non-functional ESCRT, and compromising ESCRT function. Then we will challenge parental cells (TRex) or TRex cells expressing VPS4b E235Q with toxin and measure lysis. We also plan to compare plus/minus doxycycline as a further control. We will also use fluorescent toxins to compare binding across cell types.

One caveat on the ESCRT work is that ESCRT has an essential role in MVB formation, and ESCRT effects might be due to perturbation of protein/lipid flux through this system in addition to their recruitment to the plasma membrane. Even with knockdowns and overexpression, it can be challenging to interpret some of the pleiotropic effects of altering the ESCRT complex. While we do not contest the role for ESCRT in plasma membrane repair, we suspect the role for ESCRT will be more complicated than previously appreciated. Digging deeper into these possibilities beyond our proposed experiment is beyond the scope of this manuscript.

Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer 1

*Major: The authors conclusions contradict established results, which they cite. Yet experimental conditions are not similar in two ways: toxin concentration-wise and toxin treatment duration-wise. *

We agree with the reviewer that there were differences in experimental design between our study and the other cited studies. Due to the cited differences, our results, Gonzalez et al and Larpin et al are not necessarily contradictory on most points. Our conclusions differ from Gonzalez et al in that we do not think K+ efflux drives repair in the first hour, and differ from Larpin et al in that we observe Ca2+ flux after aerolysin challenge. Along with the toxin variables discussed below, we also discussed the potential cell type differences between the studies that may account for the discrepancy. We have now included these additional differences in our manuscript on line 435 for Larpin et al and lines 423-425 for Gonzalez.

Our study set out to do something distinct from the prior studies. The prior studies did not compare the efficacy of distinct membrane repair mechanisms to the same toxin because that was not their study aim. Hence, our goal is not to prove the prior literature wrong, but contribute to a better understanding of the immediate membrane repair events triggered by aerolysin. We argue that the significance of our contribution is this comparative approach to membrane repair, which has not previously been done, and our finding that aerolysin engages distinct, but overlapping mechanisms compared to CDCs. We have updated our significance to better convey our advance, which is explained on lines 99-102, 128, 519-525.

*While we appreciate the efforts of the authors to standardize the concentration of toxins used based on hemolytic units, we note that the concentrations used are very much higher than in the other studies cited. Indeed, based on table 1, materials and methods, and the various experiments, aerolysin has a LC50 of approximately 200 HU/ml, which corresponds to about 2 ug/ml. This is approximately 200x more concentrated than for example in Gonzalez et al 2011 and Larpin et al. 2021. It makes the validity of direct comparison with those studies questionable. *

We agree with the reviewer that the toxin concentrations are different from prior studies. This is why we argue hemolytic activity needs to be reported along with toxin mass.

One potential explanation for this difference is purification method. We do nickel NTA purification from whole bacterial lysates, instead of from the periplasm. It is possible that the most active aerolysin precipitates early or is otherwise lost in our purification process, which accounts for both the lower toxin specific activity and lack of toxin precipitation during trypsin activation that we observe. To control for impurities, we purified two preps of our aerolysin to >90% purity after nickel beads. However, we did not observe a significant change in specific activity or cytotoxic activity. We interpret this finding to suggest there was a trade-off between improved specific activity due to increased purity and loss of specific activity due to toxin inactivation during the extended purification process.

We have included a new figure (Fig S10) showing our toxin purification and activity.

*We noticed that the authors activate pro-aerolysin at high concentration (in the range of 1 to 5 mg/ml) and at room temperature. In our experience, under these concentration, activation leads to immediate oligomerization and massive precipitation. The final concentration of active toxin is thus unknown. *

When we titrated the trypsin to determine the optimal concentration of trypsin to use, we did not observe oligomerization/precipitation (Fig S10B). If there was precipitation of aerolysin after trypsin treatment, we would expect a difference in cytotoxicity between pro-aerolysin and aerolysin treatment. We did not observe significant differences in cytotoxicity between pro-aerolysin and activated aerolysin (see Figs 1-2). Finally, we measured hemolytic activity on trypsin-activated toxin, so any precipitation would be expected to occur prior to assessing hemolytic activity. Thus, we argue our use of hemolytic activity measured after trypsin activation mitigates this risk.

* The authors keep their cells in toxin-containing medium for the whole duration of the experiments, typically 45 minutes. This is in stark contrast with 45 seconds to 3 minutes transient exposure to toxin in Huffman et al 2004. *

We agree this is one of the differences. We also note Huffman et al examined cells at 6 or 28 h later. While we ruled out the impact of MAP kinases on membrane repair occurring within 30 min of toxin challenge, we make no claims about their ability to promote cell survival at later time points. We have clarified these differences in the manuscript (line 461).*

The authors do not report binding and oligomerization assays of the toxins. The only figure showing a western blot (fig. 7) is of low quality and shows unexpected observations. Aerolysin Y221G mutant is expected to bind and oligomerize. Yet, no band is present at about 250 kDa (expected oligomer) or at about 47 kDa (monomer). In addition, in aerolysin lanes (1 and 2) the oligomer is saturated, seems to be covering three lanes, indicating a possible spill-over. *

We performed binding studies in Fig S3C and Fig S5. For Fig 7, in the original blot, the cell lysate is a wider band than the MV band, but there are only two bands, that remained in their respective lanes. We have now included another independent biological replicate of the aerolysin blot as Supplementary Fig S7D which shows clear demarcation between cell lysate and MV pellet. This blot was not included in the main figure because in the process of stripping and reprobing for all of the targets, we lost detection of our penultimate targets. We agree with the reviewer that oligomer bands for the Y221G were very faint, and we expected them to be stronger. In the new blot (Fig S7D), some oligomer can be detected. As a result, we are hesitant to risk over-interpreting these findings.*

Finally, while the patch repair hypothesis is interesting, it is unclear why the authors decided to overexpress dysferlin in cell lines that normally do not express it. Sure, there is a repair phenotype but this phenotype is artificially introduced. Dysferlin is not expressed at all in HeLa cells. *

One challenge with membrane repair is the difficulty perturbing the system due to redundancies. While loss-of-function experiments are important, gain-of-function experiments also add confidence to the system. The simplest way to perform a gain-of-function experiment is to add a well-known patch repair protein to a well-characterized cell line lacking it. Thus, exogenous expression of dysferlin enables us to test the hypothesis that increasing patch repair enhances repair against the toxins.

We have included this rationale now in the manuscript, lines 366-369

*Furthermore, dysferlin is not expressed in epithelial cells, which are the prime target of aerolysin. Why then focus on this protein? *

We chose dysferlin because it is well-characterized as a patch repair protein, whose defect causes Limb-Girdle Muscular Dystrophy 2B and Miyoshi Myopathy. Additionally, setting up this assay enables future work to probe the role of individual dysferlin domains in patch repair.*

Minor: The graphic legends should be boxed out to be clearly separated from the data. In Figure 4A, it is mixed up with the data. *

This has been corrected.*

Some western blots are saturated, e.g. B-actin in figure 4B. Full blots should be provided. *

We have added full western blots as requested as Supplementary Figs S11-12.*

In the methods, aerolysin sublytic dose for HeLa cells is specified at 62 HU/ml. In figure 5C and D, 31 HU/ml kills more than 50% of HeLa cells. This is not compatible. *

Even when controlling by hemolytic activity, and toxin prep, we find some variability in toxin activity between assays. For the live cell experiments, 62 HU/mL remained sublytic despite the higher activity in the flow cytometry assays. We controlled for death in our live cell imaging experiments, by including TO-PRO. This confirmed the toxin was at a sublytic dose in those experiments.

We included a new figure S10C to show the variation in LC50 per assay as a function of toxin specific activity. We have clarified that the sublytic dose was for live cell imaging experiments, lines 640-641.

*Figure 2A and B have quite different LC50 for starting conditions ({plus minus} 200 HU/ml in A, 600-700 HU/ml in B). Why is it so different? Y-axis has a linear scale in A and a logarithmic scale in B. It would make comparison easier to have the same scale in both panels. *

We agree there is variability between assays. We note that toxin doses change vary in other manuscripts that report toxin mass. For example, aerolysin varies by 10-fold (2 – 20 ng/mL) between figures in Gonzalez et al 2011. We interpret this variation as a common challenge for toxin studies. We mitigate this challenge by including controls for each assay so the relative change can be assessed. We provide additional transparency by including Fig S10 to show batch-to-batch variability of both our toxin preps and assays.

We have changed the scale to linear in Fig 2.*

The letters detonating statistically significant groups are sometimes unclear. For example in Figure 1A and B, PFO belongs to group a and b simultaneously. What does this mean? *

Samples that share letters are not statistically distinct from each other. In the example cited, PFO is not statistically significant compared to all other bars with an a and is not statistically significant compared to all other bars with a b. While confusing at first, the alternative is a mess of stars and bars.

This has been explained in lines 981-985.*

In Figure 8, aerolysin hat a LC50 in cells overexpressing GFP-Dysferin of approximately 1700 HU/ml in A and of approximately 400 HU/ml in B. Why is it so different? *

This is due to intra-assay variation. We include controls for each assay to ensure the trend remains consistent.*

In Figure S1, it is unclear what the plots « all events » vs « single cells » mean. *

We have clarified these plots.*

In the discussion, the authors write « First, survival did not correlate with overexpression, which would be expected if dysferlin acted as Ca2+ sink ». What is meant? GFP-dysferlin overexpression does correlate with survival in Figure 1A. *

We meant that the extent of Dysferlin expression did not correlate with survival. If Dysferlin acted as a calcium sink, cells expressing 100x dysferlin levels should be more resistant than cells expressing 1x dysferlin levels. If Dysferlin needs to serve a cellular function, the brightest cells may not be more resistant (or even be less resistant due to aggregates, etc). We checked to see if the brightest Dysf+ cells had better survival than the dimmest Dysf+ cells. They did not. However, all Dysf+ cells had better survival than Dysf- cells.

We have updated the manuscript (lines 496-498) to reflect these changes.

Significance

*General assessment: The study strength lies in the several possible protection mechanisms that are tested. The weaknesses lie in the contradictions of the results reported here with established mechanisms, *

We disagree with the reviewer that findings that contradict previously proposed mechanisms are a weakness for significance. Instead, we argue this is a strength of our study’s significance. Replication of prior studies’ conclusions using distinct experimental conditions is critical for the reproducibility and rigor of the underlying science, and may give new insights into toxin biology. While we acknowledge the differences in approach, these differences narrow the prior mechanisms that may have been assumed to be widely applicable. The finding that they cannot be replicated in our system suggests one or more of the differences between the studies may drive a critical aspect of aerolysin biology. For example, the Ca2+ difference with Larpin et al could be due to a cellular Ca2+ channel present in HeLa cells that is absent in THP.1/U937 cells.

This distinction is expected to spur additional research in the aerolysin field.

* Advance: The study contradicts previously established results but the experimental conditions used here are quite different to those used in the earlier studies, which makes the comparison quite difficult. As such it does not really fill a gap. *

We have rephrased the significance to better convey both the gap our study fills in membrane repair and the advance that it has made. See lines 99-102, 128, 519-525.*

Audience: The study will be of interest of specialized audience. *

Given the emerging broad importance of membrane repair in response to endogenous pore-forming toxins, and the large gaps in the field of membrane repair, we respectfully disagree with the reviewer. We have revised our significance statements to better convey this broad appeal. See lines 99-102, 128, 519-525.

Reviewer 2

Major

*3. I find the optimisation of lysin concentrations and data presentation quite confusing. I eventually understood, what was done, but I feel that the authors should be able to transform the data and plots so these are more accessible to a reader, eg a simple dose/time-response curves would be very helpful in that respect. For example, in Figure S1E, why does aerolysin appear to be less cytotoxic after 24 hrs than after 1 hr. In principle, I would expect to observe an additive effect, i.e. cell death at 1, 3, 6, 12, and 24 hrs should add to 100%; however, if 100% cells die at 500HU/ml, how can more cells die after 24hrs? Or am I missing something in the experimental design/data presentation? *

We agree that presenting the results from cytotoxicity can be challenging. We use LC50 in the main text because it is easiest to understand. However, we provide all dose-response curves underlying those numbers in the supplemental data. We recently published our approach to assays and data analysis (Haram et al PMID: 36373947) to make it easier to understand.

In Fig S1E, each time point is a distinct assay. In contrast to the approach suggested by the reviewer, where we read the plate at different timepoints, we used different replicates to generate the time points. As a result, the % will not add to 100. Instead, we observe that the majority of cell death occurs in the first hour. We have clarified our discussion of Fig S1E, lines 154-155.

At 24 h, it is possible that cell growth interfered with the assay. The plate has a finite surface area. If control cells are confluent near the start of the assay, but toxin-treated cells are not due to cell death by aerolysin, the growth rates may not be equal. Since our focus is on proximal membrane repair events, and not on late signaling events, pursuing this further is beyond the scope of the current manuscript.

*I also wonder whether using haemolytic units is appropriate (it may well be, if justified), given that the toxins used here have various membrane-binding properties. Wouldn't it make more sense to compare the cytotoxicity using nucleated cells? *

We agree with the reviewer on the need for standardization, and do compare cytotoxicity using nucleated cells (HeLa). Our first level of standardization is the use of hemolytic units instead of toxin mass. This normalizes toxin activity to the ability to kill human red blood cells, which are widely accepted as having minimal membrane repair mechanisms. This gives us a baseline activity, and allows us to control for toxin impurities/differences between toxin preps/toxins. We prefer cytotoxicity over membrane binding for our baseline because it is a functional assay.

After this first level of standardization, we compare the cytotoxicity in HeLa cells. This is one reason why the majority of our assays are performed in HeLa cells—we know how they behave at different toxin doses in our hands, the cells are easy to use, and we can standardize assays in the lab. We included HeLa cells as a control in Fig 5 to show the standardization requested by the reviewer. We split Fig 1 up differently to better convey the results.*

1. The authors use "sublytic" concentrations of aerolysin (64HU) throughout most of the paper, but according to Figure S1C, 50% cells died at that concentration after 1hr, suggesting that when the cells were investigated over a shorter period of time, they were already dying - it's almost like the cells had life support turned off, but still being investigated as though they survived aerolysin treatment. This needs to be clarified or reassessed. *

We agree with the reviewer that we did not track cell survival beyond 45 min in our live cell imaging assays. We labeled cells as ‘surviving >45 min’ to acknowledge the fact that these cells could have died at 46, 47, 60, or 600 min after the experiment ended. We focused on time points earlier than 45 min because proximal membrane repair mechanisms are expected to have occurred in that time, and had time to complete. We have updated the manuscript on lines 214-215.

We next considered the reviewer’s excellent point that the cells alive at 30-40 min could be executing a cell death program. If this were the case, then based on our FACS data (Fig S1C), we would predict ~50% of total cells would be dead by 1 h. From Fig 3A, ~35% of the cells died in the first 45 min. From the remaining 65%, we would predict another 15% dying from this programmed cell death pathway, which would be 15/65 = ~25% of the surviving cells. We did not notice 1/4 of the surviving cells behaving distinctly. For example, the large error bars in 3H is due to a range of cell behaviors that we could not easily subgroup. For individual cells (shown in Figs 6 and 7), there is similarly no clear demarcation of 1/4 of the cells. While we see a gap with pro-aerolysin, that is ~1/3 of the cells (not the expected 1/4), and it is not repeated with aerolysin. While we can’t rule out a cell death program contributing to the top or bottom 1/4 of our results, removing the top or bottom 25% of data points would not alter our major conclusions from the live cell imaging. If a programmed cell death pathway that occurs in the 30-90 min range is identified for aerolysin, it would be interesting to see how that pathway changes repair kinetics. However, that would require identification of the death pathway.

*

1. What effect does the addition of 150mM KCl have on the plasma membrane, trafficking/repair - wouldn't the plasma membrane be depolarised? There were a number of papers by John Cidlowski in mid 2000s, where his team explored the effect of potassium supplementation on apoptosis - this may be worth exploring. *

We thank the reviewer for suggesting these interesting papers. We have explored these papers, and our understanding of them is as follows. Franco et al 2008 PMID: 18940791 shows that ferroptosis is independent of high extracellular K+. This contrasts with Fas-dependent apoptosis, which is suppressed by high extracellular K+. This is consistent with the Cidlowski group’s other work (eg Ajiro et al 2008 PMID: 18294629) and Cohen’s group (eg Cain et al 2001 PMID: 11553634) showing that apoptotic DNA degradation performs better at low K+, and extracellular K+ interferes with apoptosis. Similarly, other papers have shown that NLRP3-activated pyroptosis can be blocked by addition of extracellular K+. Depletion of intracellular K+ inhibits endocytosis and other vesicle trafficking pathways.

While these are good papers, they do not directly relate to our K+ findings, which is that blocking K+ efflux via elevated extracellular K+ levels has no impact on aerolysin-mediated killing. Therefore, to stay focused on the repair pathways, we opted not to include these papers to avoid distracting the reader from our key points. *

1. Figure 3 and accompanied text: it would be more informative to show all the data rather than breaking it down to 45 min. In my view, *

We have added histograms to show when individual cells died during the assay as supplemental Fig S3E. We used the three bins for the exact reason articulated by the reviewer—we wanted to consider cells that died fast vs slow differently. However, in order to interpret the data, a cutoff of 5 min was chosen as optimal. While we agree with the reviewer that the 5 min death could be dismissed, we presented the data to avoid questions about why we omitted those data.*

1. I am curious whether EGTA diffuses into the cytosol through aerolysin pores. If so, then unlike BAPTA-am it would affect Ca inside and outside the cell. *

We agree with the reviewer this is an interesting question. While EGTA might diffuse into the cytosol, its binding properties suggest it would be unsuitable to block cytoplasmic Ca2+ transients (see Nakamura 2019 PMID: 31632263). BAPTA binds to Ca2+ ~40x faster than EGTA, which enables it to capture Ca2+ prior to Ca2+-binding proteins. In contrast, EGTA is thought to be too slow to sequester intracellular Ca2+ before Ca2+-binding proteins. While EGTA might perturb Ca2+ close (

*Are the authors confident that in the absence of extracellular calcium (EGTA treatment), aerolysin formed the pores at all? Have they looked, for example, at intracellular Na/K, or have any other evidence of membrane disruption? *

Prior structural studies suggest that Ca2+ is not required for aerolysin pore formation. For example, Iacovache et al (2011) PMC3136475 induce oligomerization with low salt and pH 2+. Cryo-EM from the same group (Iacovache et al 2016 PMID: 27405240), showed pore formation under similar conditions.

In Fig S3, aerolysin kills in the presence of EGTA at higher concentrations, suggesting that it can form pores when EGTA is present. Also, in Fig 2D, we used Tyrode’s buffer, which was made without Ca2+ or EGTA. We added the indicated amounts of Ca2+ in, and observed a reduction in lysis at low [Ca2+]. This argues against EGTA interfering with toxin oligomerization/pore formation because EGTA was not present, and the toxin still failed to kill.

We have updated the manuscript (lines 203-205) to emphasize this point.*

1. Figure 6 (and some other): I find the designation of statistical significance (a-f) quite confusing, as it is unclear which comparisons are statistically different. Looking at Figure S5, there was no difference between the effect of Annexin depletion on the toxicity of the three lysins. *

Samples sharing the same letter are NOT statistically significant. This is done to avoid a mess of stars and bars with multiple comparisons. This has now been explained in lines 981-985.

For Fig 6/ Fig S5 (now S6), there was a statistically significant difference in LC50 between control siRNA and Annexin knockdowns for SLO. We agree that visually the dose-response curve in Fig S6B looks similar. However, we note that the x-axis is a log2 scale, and the control line is distinct over the 250-1000 region. When we calculate the LC50, these differences give different LC50 values. Over multiple reps, these differences were consistent enough to be statistically different.

Significance

*The paper attempts to address an interesting question of aerolysin pore repair, and it is interesting from the perspective of a potential difference between various pore-forming proteins. *

We agree with the reviewer and thank the reviewer for this assessment.*

The study will be potentially interesting to a broad audience of biochemists/cell biologists and microbiologists working in the field of pore-forming proteins/virulence factors. *

We agree with the reviewer and thank the reviewer for this assessment.

Reviewer 3

*Major comments In the first instance, the authors use a method of assaying the specific lytic activity of aerolysin in comparison to a number of different CDCs. Whilst it is acknowledged that these methods have been published in peer-review papers previously (e.g. Ray et al., Toxins, 2018), it would be great to have more information of how the specific activity is derived. Currently there is a convoluted method that makes a number of assumptions such as, but not limited to, 1) the number of dead cells measured in the FACS experiments is proportional to the activity of the different classes of PFPs however the authors do not show how they account for PFPs leading to loss of cells into debris which would involve a total cell count and *

We thank the reviewer for raising these concerns. We tested these assumptions in our previous papers. We compared the FACS assays to other assays that measure total cells (i.e. MTT assay), and found that the FACS assay corresponds with the MTT findings. These findings were published in Keyel et al 2011 PMID: 21693578 and Ray et al 2018.

Loss of countable events to debris is detected in our assay as saturation of cell death at a number under 100%. Since we perform dose-response curves, we can determine when the killing saturates. This is why loss of countable events does not change our ability to accurately calculate LC50.

2) how the inflection or linear point is identified on individual experiments (e.g. Supp. Fig. 1B, 2A, 2B, 3A, 3B to name a few) and how reliable these points are (e.g showing the data points with model sigmoidal (?) curve and corresponding R values).

This had been calculated manually in the prior version of the manuscript. To address the reviewer’s concern and to improve data quality, we reanalyzed all of our data by fitting our dose-response curves to logistic models, and determining the LC50 using that model. An in-depth explanation of our approach was just published in Haram et al PMID: 36373947, which we now cite (line 821). *

Furthermore, the batch-to-batch variability of protein samples presented in table 1 may be an issue where inactive but folded protein can affect the formation of homo-oligomer pores so more effort to reduce the effects of batch variation would be integral to the foundation of this paper. Given that aerolysin has a very different action on cells then this new characterisation should be provided regardless of what has been previously published by the authors on the activity of CDCs on the cells.*

We agree with the reviewer that batch-to-batch variability is a key concern for pore-forming toxins. To address the concern of batch-to-batch variability and toxin purity, we have added Supplemental Fig S10. In Fig S10C, D, we plot the LC50 against specific activity of each toxin prep when used against control cells. We found a statistical difference in LC50 between two of our toxin preps, but not between any of the others. Notably, there was no association between increasing specific activity and LC50.

Furthermore, we tested the impact of impurities on our toxin prep. While we purify most toxins only using His-beads (obtaining ~40% purity) (Fig S10B), we purified two toxin preps to higher purity (>90%) (Fig S10A). We did not observe differences in LC50 between these toxin preps. The specific activity for these toxins did not increase. We interpret that finding to indicate the gain in specific activity for purity was offset by the loss of specific activity due to prolonged toxin purification.*

• Can the authors provide the raw data for the total FACS observations (scatterplot for all events) and show that there is no significant loss of cells? Or at least there is accountability of the cells? *

Our stop conditions were to collect at least 10,000 gated events instead of running for a set period of time/set volume to determine cell density. We provide example scatterplots in Fig S1A.

* - Can the authors provide more information about how the linear regression on Supp. Fig. 1B and other experiments showing the model sigmoidal curve performed such that this work is more reproducible? *

We agree with the reviewer that using logistic modeling would strengthen the work. To address this concern, we reanalyzed all of our data and switched to logistic modeling. This improved reproducibility for many figures. Changes that add or remove statistical significance to results include Fig 4A, loss of significance between Ca2+/DMSO and BAPTA/DMSO, Fig 6C, loss of significance for siRNA knockdown of A6 vs scrambled for ILY, and Fig 8A/B, gain of statistical significance for GFP-Dysf protecting SLO. We have updated our results accordingly.*

The SEMs of some data points (specific lysis LC50 scatterplots, for e.g. Fig. 2C, 4A, 4C, 8A and fMAX plots, for e.g. Fig. 3B) may not be apparently representative of the skew (e.g. and individual values (including outliers). A clarification of the statistical analysis behind the results may benefit in a clearer understanding of how the SEMs were calculated and presented in the main figures. Also, further elaboration on the meaning of the lettering in the scatterplots (denoted as a, b, c etc.) across the main figures may help improve the interpretation of the data. *

The SEMs were calculated by Graphpad and graphs also generated by Graphpad. To address the reviewer concern, we have switched all places where we plotted individual data points to median with no error bars. This will enable the reader to judge skew, outliers, etc without reliance on error bars.

We have now further elaborated on the lettering in the scatterplots. Samples sharing the same letter are NOT statistically significant. This is done to avoid a mess of stars and bars with multiple comparisons. This has now been explained in lines 981-985.*

Secondly, the authors present interesting results on the significance of Ca2+ on aerolysin's mechanism behind lytic activity and introduces dysfurlin-mediated patch repair as the primary cellular resistance mechanism against aerolysin mediated lysis. Results from Figure 2-4, indicate that extracellular Ca2+ plays a role in aerolysin's function and cell lysis (aerolysin triggers influx of extracellular Ca2+). However, the results presented in figure 8 suggest an impairment of dysferlin translocation from the cytosol to the plasma membrane upon removal of extracellular Ca2+. If this were the case, wouldn't dysferlin impairment sensitise cells to aerolysin? Thus, in these sets of experiments it seems that Ca2+ is a confounding factor.*

We agree that Ca2+ is a confounding factor, which is one reason we aimed to define better membrane repair mechanisms in response to different pore-forming toxins. Our interpretation is that Ca2+ triggers a death pathway that overcomes repair, and that aerolysin toxicity is due to the activation of this pathway. In this case, the impairment of Ca2+-dependent pathways does not reduce survival because the extent of damage is reduced/not present. Figuring out this death pathway is beyond the scope of the present manuscript, but a one future direction in which we are interested. This would also account for differences observed in different cell lines.*

• Can the authors further elaborate on how the function of dysferlin in protecting cells against aerolysin contrasts to how aerolysin kills cells? *

We have added the requested discussion to our manuscript, lines 519-525.

*Finally, it is also interesting to see that cells deploy different resistance mechanisms between different families of pores. In saying that, the usage of CDCs seems to be inconsistent between each set of results. For example, intermedilysin (ILY) was used in the siRNA knockdown experiments but not in others such as Ca2+ influx assays, while PFO was only used for the initial set of results. A comment on this would benefit in understanding the rationale for selecting certain CDCs for each set of experiments. *

We thank the reviewer for raising this point. We used SLO as the primary CDC in all the experiments because it is the CDC we have best characterized and have extensively published on. We included PFO in initial experiments to give readers a better idea of how multiple CDCs compare to aerolysin in target cells. However, since we’ve previously published on PFO, including it for later experiments would have increased cost and time of experiments without providing new knowledge.

We used ILY because it binds to the GPI-anchored protein human CD59, so its binding determinant is more similar to aerolysin, which binds GPI-anchored proteins. We included it where practical to determine the extent to which targeting may change repair responses. Since ILY does not bind to murine cells, it was omitted from experiments using murine cells.

We have added the rationale to the manuscript on lines 138-140.*

Minor comments Results (Nucleated cells are more sensitive to aerolysin and CDCs) - A statement of the EC50 values of aerolysin and CDCs from the haemolytic assays would be beneficial to compare activities between the two pores. *

The hemolytic activity is defined as the EC50 for the toxin in human red blood cells. The specific activity enables comparison of toxin activity, which is reported in Table 1. We have now added Supplementary Fig S10 which further plots the aerolysin and SLO specific activities against LC50 so that the reader can better assess batch-to-batch variability. In this study, we did not use enough batches of the other toxins to make this analysis useful for them.

* - Figure 1A: As stated in the introduction, pro-aerolysin exists as a precursor that is functionally inactive unless activated by trypsin, furin or potentially other proteases. It would benefit the reader if an explicit statement were made about this activity and how it may come about in HeLa and 3T3 cells. Why is pro-aerolysin not shown in the Casp 1/11-/- BMDM cells? *

The cell surface furin activity that activates aerolysin is not well-characterized across different cell types. We have revised the manuscript (line 76) to indicate these activities are present on the cell membrane.

We omitted pro-aerolysin from the Casp1/11-/- BMDM because we performed those experiments earlier in the study before we started including pro-aerolysin. Based on the other results, we judged that the time and resource costs of adding pro-aerolysin in this system outweighed the gain to the story.

* - Figure 1C: It was stated that "Casp 1/11 -/- Mo were ~100 fold more sensitive to pro-aerolysin and aerolysin compared to PFO and SLO" but did not show the activity for pro-aerolysin in these cells. *

We thank the reviewer for catching this typo, and have corrected this statement (line 172).

* - Supp fig 1E: Shouldn't 24 hr incubation of aerolysin to HeLa cells result in 100% specific lysis? *

We agree with the reviewer that these results were surprising. At 24 h, it is possible that cell growth interfered with the assay. The assay well has a finite surface area. If control cells are confluent near the start of the assay, but toxin-treated cells are not due to cell death by aerolysin, the growth rates between control and experimental wells may not be equal. Since our focus is the proximal membrane repair events, and not the late signaling events, pursuing this further is beyond the scope of the current manuscript.

* (Delayed calcium flux kills aerolysin-challenged cells) - What is the intracellular concentration of K+ normally in cells? Similarly, what is the intracellular concentration of Ca2+? *

Intracellular K+ is ~140 mM (see Ajiro et al 2008 PMID: 18294629), while cytosolic Ca2+ is ~100 nM at rest.

* - Figure 2C: Based on the description in the methods and results, both buffers are supplemented with 2 mM Ca2+ but one buffer (RPMI) shows more killing with SLO and ILY. Does this mean that both buffers contain 2 mM CaCl2? If so, what are the other potential reasons why one buffer enabled greater potency in CDCs? *

RPMI has 0.4 mM Ca2+ prior to Ca2+ supplementation. However, the 2.4 mM Ca2+ did not provide improved protection compared to RPMI alone (See Fig 2 in Ray et al 2018).

We suspect the various amino acids added to RPMI promote membrane integrity and account for the difference from Tyrode’s buffer. Glycine has previously been implicated in promoting membrane repair, but at higher concentrations than it is present in RPMI (0.133 mM in RPMI vs the mM concentrations used to protect cells). If other amino acids also protect, and/or why they protect is beyond the scope of the present work.

* - Figure 3H: The data for aerolysin (WT) would greatly benefit for comparison to the inactive mutant (and indicate the sustained Ca2+ increase). *

We have added this comparison, and updated the figure legend, line 1015.

* - Supplementary Video V1: The addition of Triton X-100 permeabilises cells; however, this wasn't evident in (A). - Video V2: Similar to previous comment on Supplementary Video V1 (for B). *

In V1A, the video was cut short to fit the play time with other videos. From addition, the triton takes a few minutes to diffuse to the cells and permeabilize them. In V2B, the cells do become permeabilized as shown by loss of the Ca dye. The cells are out of focus, which is why the nucleus TO-PRO is not detected.*

(Calcium influx does not activate MEK-dependent repair) - Figure 4A: Effective ionic concentration inside and outside cell is increased (if intracellular Ca2+ becomes chelated); therefore, Ca2+ may enter the cell by passive diffusion or transport by other intrinsic Ca2+ channels. *

There is already a very steep concentration gradient for Ca2+. The cytosolic Ca2+ is ~0.1 uM, compared with growth medium at 400 uM or assay buffer at 2400 uM. Chelation of the intracellular Ca2+ is not expected to increase Ca2+ import from outside the cell.*

(Caveolar endocytosis does not protect cells from aerolysin) - Figure 5C: What is the purpose of using HeLa cells as a control? *

We included HeLa cells to demonstrate the toxin was active and to rule out batch-to-batch variability as one interpretation of the reduced killing of differentiated 3T3-L1 cells.

* - "..with Alexa Fluor 647 conjugated pro-aerolysin K244C" - this should be introduced earlier as it was initially mentioned in Supp. Figure 3C. *

We have now introduced this earlier at line 190, instead of 300

* - Murine fibroblasts were used earlier (Figure 1). Following from this result (where the WT can be used as a positive control), can MEFs be used instead of adipocytes to see whether caveolar endocytosis plays any role in cellular resistance? *

The 3T3-L1 cells are murine fibroblasts prior to differentiation. Since they can also be differentiated into adipocytes, we used them instead of MEFs. The other reasons we used them include the availability of Cavin knockout cells, and the extensive caveolae present in adipocytes. We included analysis of 3T3-L1 prior to differentiation them in Fig 5B.

* - Further comment on the increased resistance of K5 knockout would benefit on the mechanism of aerolysin-mediated cytolysis. *

We agree further characterization of this line would be interesting in the future. At the present, however, any further comment would be speculative on our part. Since the resistance was not replicated in the second CRISPR line, we suspect it is either an unexpected mutation(s) in the cell line that arose during routine cell culture, or off-target effect(s) from the CRISPR used to generate the line.

* (Annexins minimally resist aerolysin) - Supplementary video V3 - it seems that annexin A6 is recruited to the membrane, to a greater extent (and also quicker) than SLO. This suggests that annexin recruitment is a cellular response against aerolysin challenge. *

We agree with the reviewer that annexins are recruited to the membrane during repair. However, individual knockdown did not enhance death. This is one reason we believe functional studies (i.e. cytotoxicity) are necessary when studying the cell biology of repair events. Recruitment of the protein, and it promoting repair may be two different things.

In V3, three of the SLO-challenged cells have translocated by the time focus is restored. In contrast, the first aerolysin cells translocate ~10 min. One complicating factor is that A6 cycles back off the membrane with the SLO challenge.

* o SLO also shows A6 recruitment (arrows pointed). However, supplementary figure 6B does not clearly illustrate this. *

Given the 45 min time scale, the rapid initial membrane enrichment is hard to see on the graph.

* - As annexin A1 is sensitive to calcium, further comment on the significance of intracellular/extracellular calcium in annexin A1 recruitment and aerolysin challenge would explain observations in Figure 4A. *

We have updated the manuscript, line 242 to include annexins and dysferlin as Ca2+-binding proteins in our discussion of intracellular calcium.*

(Patch repair protects cells from aerolysin) - Supplementary video V4 - the intensity decreases for the inactive mutant; is this due to lysis? *

We included TO-PRO in the experiment to rule out lysis. Since the cells remain in focus, we interpret the lack of TO-PRO to indicate no cellular lysis.

*- The next paragraph sounds like a contradiction: "GFP-dysferlin localized to the plasma membrane and vesicles independently of extracellular Ca2+ (Fig 8C D, Video V5) o Followed by "To study the Ca2+ dependency of dysferlin, we removed extracellular Ca2+ with 2 mM EGTA and challenged with sublytic toxin doses...found less depletion of dysferlin from cytosol". *

We thank the reviewer for pointing out our unclear language. In the second section, we intended to refer to dysferlin positive vesicles. We have rephrased the manuscript (lines 388-395) to clarify that we are focused on Ca2+-dependence of vesicle fusion, not steady-state.*

(Methods) - Table 1: The values presented in the methods section are, overall, confusing and require clarification. *

We have added Fig S10, and discussion of toxin activity and purity in the methods (lines 634-641) to provide further clarity on toxin activity.

* o 10-fold difference in SLO and PFO WT - do the authors think this might change the interpretation between different figures? *

We do not. The reason is that we changed the membrane affinity between SLO and PFO (Ray 2018), and this switches the properties of the respective toxins without changing their yields.

* o Understood how the haemolytic activity was calculated (referred to work in 2012), but how was the haemolytic unit originally derived? *

It was derived as a measure of activity for toxins by determining the EC50 in RBCs for a given toxin. Since species type of RBC and other factors can change the reported activity, we have normalized to using human red blood cells. This lets us assay human-specific toxins like ILY along with other toxins.

* o How were these values (from table 1) derived to toxin concentrations used for killing nucleated cells? *

Full discussion of our assay was recently published in Haram et al 2022 PMID: 36373947. For the cytotoxicity assays, we use the hemolytic activity. Suppose from Table 1, the toxin stock is 1.5 x10^5 HU/mL. Then to prepare a 2x working toxin stock, we dilute the toxin to 4 x10^3 HU/mL (this is a 1 in 37.5 dilution). To get the range of concentrations used in the dose response curve, we perform a 2-fold serial dilution. Finally we mix equal volumes of toxin and cells, giving us the final 1x toxin activity (2 x10^3 HU/mL for the highest concentration in this example).

* o Therefore, an EC50 haemolytic curve showing the activities for all toxins would greatly facilitate in understanding the derivation of values for table 1.*

The hemolytic unit already incorporates the EC50 hemolytic curve. 1 HU is the EC50 of the toxin in the human RBCs.

* - Flow cytometry assay: What is meant by gating out the debris? And would debris also contribute to the count in dead cells? *

We illustrate our gating strategy in Fig S1. The debris falls in the front left corner of the plot, and includes electronic noise, non-cellular debris and cellular fragments. Since one cell could give rise to multiple pieces of debris, we exclude the debris from analysis.

* o What was added as the high PI control? *

In Fig S1A, the high dose of toxin was used for maximal killing. In our cell populations, there is a low level (2-5%) of dead cells that serve as a control for PI staining. In the past, we’ve used 0.01% triton to validate permeabilization of the cells. We have also compared PI uptake with MTT assays (Keyel et al 2011, Ray et al 2018) to confirm that the PIhigh cells are dead.

*Elaborating reviewer #2's comment 7 regarding the addition of EDTA : with respect to measuring the binding if fluorescently labelled aerolysin, how can the authors differentiate between full functional pores versus prepores/incomplete pores? *

This requires electron microscopy, which is the beyond the scope of our current study. However, prior work and Fig 2D show that aerolysin forms pores without the need for Ca2+ (see next point).

How else can the authors validate whether aerolysin remains functional in the presence of EDTA?

Prior structural studies suggest that Ca2+ is not required for aerolysin pore formation. For example, Iacovache et al (2011) PMC3136475 induce oligomerization with low salt and pH 2+. Cryo-EM from the same group (Iacovache et al 2016 PMID: 27405240), showed pore formation under similar conditions.

In Fig S3, aerolysin kills in the presence of EGTA at higher concentrations, suggesting that it can form pores when EGTA is present. Also, in Fig 2D, we used Tyrode’s buffer, which was made without Ca2+ or EGTA. We added the indicated amounts of Ca2+ in, and observed a reduction in lysis at low [Ca2+]. This argues against EGTA interfering with toxin oligomerization/pore formation because EGTA was not present, and the toxin still failed to kill.

We have updated the manuscript (lines 203-205) to emphasize this point.

Significance

*While the work has investigated in-depth cellular resistance mechanisms, the significance and benefits of this study are unclear. For example, the authors have used different human cell lines to dissect how these cells are affected by different pores but have not stated the significance and potential benefit of studying these cell lines. Further elaboration in this aspect may increase the relevance of the study, to an audience who is interested in the field of infection and disease. *

We have updated our significance to better convey our advance, which is explained on lines 99-102, 128, 519-525. We also added benefits of testing the cell lines chosen on lines 167-168, and 277-278. We plan to add muscle cells to address the dysferlin points, which has relevance to necrotizing soft-tissue infections.

Description of analyses that authors prefer not to carry out

Not applicable

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Referee #3

Evidence, reproducibility and clarity

Summary

This body of work by Thapa & Keyel explores the differences in cellular resistance mechanisms between two different pore families (aerolysin versus CDCs). Herein, the authors were able to elucidate the toxin activities across a variety of different nucleated cells, using the haemolytic assay as a reference for normalising activity. Their findings revealed that, in general, aerolysins were relatively more potent than CDCs at damaging certain nucleated cell lines. Furthermore, the authors performed an exploration of different resistance mechanisms, including MEK-dependent repair, annexins, and patch repair by dysfurlin. The work provides some supporting evidence that patch repair is the main mechanism that cells deploy to prevent aerolysin-mediated cytotoxicity. Overall, the amount of work that was put in to craft the manuscript was impressive and the manuscript showed potential prospects in further investigating 1) mode of aerolysin killing in nucleated cells and 2) the role of patch repair and function of dysferlin in cellular resistance against aerolysin.

Major comments

In the first instance, the authors use a method of assaying the specific lytic activity of aerolysin in comparison to a number of different CDCs. Whilst it is acknowledged that these methods have been published in peer-review papers previously (e.g. Ray et al., Toxins, 2018), it would be great to have more information of how the specific activity is derived. Currently there is a convoluted method that makes a number of assumptions such as, but not limited to, 1) the number of dead cells measured in the FACS experiments is proportional to the activity of the different classes of PFPs however the authors do not show how they account for PFPs leading to loss of cells into debris which would involve a total cell count and 2) how the inflection or linear point is identified on individual experiments (e.g. Supp. Fig. 1B, 2A, 2B, 3A, 3B to name a few) and how reliable these points are (e.g showing the data points with model sigmoidal (?) curve and corresponding R values).

Furthermore, the batch-to-batch variability of protein samples presented in table 1 may be an issue where inactive but folded protein can affect the formation of homo-oligomer pores so more effort to reduce the effects of batch variation would be integral to the foundation of this paper. Given that aerolysin has a very different action on cells then this new characterisation should be provided regardless of what has been previously published by the authors on the activity of CDCs on the cells.

• Can the authors provide the raw data for the total FACS observations (scatterplot for all events) and show that there is no significant loss of cells? Or at least there is accountability of the cells?
• Can the authors provide more information about how the linear regression on Supp. Fig. 1B and other experiments showing the model sigmoidal curve performed such that this work is more reproducible?

The SEMs of some data points (specific lysis LC50 scatterplots, for e.g. Fig. 2C, 4A, 4C, 8A and fMAX plots, for e.g. Fig. 3B) may not be apparently representative of the skew (e.g. and individual values (including outliers). A clarification of the statistical analysis behind the results may benefit in a clearer understanding of how the SEMs were calculated and presented in the main figures. Also, further elaboration on the meaning of the lettering in the scatterplots (denoted as a, b, c etc.) across the main figures may help improve the interpretation of the data.

Secondly, the authors present interesting results on the significance of Ca2+ on aerolysin's mechanism behind lytic activity and introduces dysfurlin-mediated patch repair as the primary cellular resistance mechanism against aerolysin mediated lysis. Results from Figure 2-4, indicate that extracellular Ca2+ plays a role in aerolysin's function and cell lysis (aerolysin triggers influx of extracellular Ca2+). However, the results presented in figure 8 suggest an impairment of dysferlin translocation from the cytosol to the plasma membrane upon removal of extracellular Ca2+. If this were the case, wouldn't dysferlin impairment sensitise cells to aerolysin? Thus, in these sets of experiments it seems that Ca2+ is a confounding factor.

• Can the authors further elaborate on how the function of dysferlin in protecting cells against aerolysin contrasts to how aerolysin kills cells?

Finally, it is also interesting to see that cells deploy different resistance mechanisms between different families of pores. In saying that, the usage of CDCs seems to be inconsistent between each set of results. For example, intermedilysin (ILY) was used in the siRNA knockdown experiments but not in others such as Ca2+ influx assays, while PFO was only used for the initial set of results. A comment on this would benefit in understanding the rationale for selecting certain CDCs for each set of experiments.

Minor comments

Results

(Nucleated cells are more sensitive to aerolysin and CDCs)

• A statement of the EC50 values of aerolysin and CDCs from the haemolytic assays would be beneficial to compare activities between the two pores.
• Figure 1A: As stated in the introduction, pro-aerolysin exists as a precursor that is functionally inactive unless activated by trypsin, furin or potentially other proteases. It would benefit the reader if an explicit statement were made about this activity and how it may come about in HeLa and 3T3 cells. Why is pro-aerolysin not shown in the Casp 1/11-/- BMDM cells?
• Figure 1C: It was stated that "Casp 1/11 -/- Mo were ~100 fold more sensitive to pro-aerolysin and aerolysin compared to PFO and SLO" but did not show the activity for pro-aerolysin in these cells.
• Supp fig 1E: Shouldn't 24 hr incubation of aerolysin to HeLa cells result in 100% specific lysis?

(Delayed calcium flux kills aerolysin-challenged cells)

• What is the intracellular concentration of K+ normally in cells? Similarly, what is the intracellular concentration of Ca2+?
• Figure 2C: Based on the description in the methods and results, both buffers are supplemented with 2 mM Ca2+ but one buffer (RPMI) shows more killing with SLO and ILY. Does this mean that both buffers contain 2 mM CaCl2? If so, what are the other potential reasons why one buffer enabled greater potency in CDCs?
• Figure 3H: The data for aerolysin (WT) would greatly benefit for comparison to the inactive mutant (and indicate the sustained Ca2+ increase).
• Supplementary Video V1: The addition of Triton X-100 permeabilises cells; however, this wasn't evident in (A).
• Video V2: Similar to previous comment on Supplementary Video V1 (for B).

(Calcium influx does not activate MEK-dependent repair)

• Figure 4A: Effective ionic concentration inside and outside cell is increased (if intracellular Ca2+ becomes chelated); therefore, Ca2+ may enter the cell by passive diffusion or transport by other intrinsic Ca2+ channels.

(Caveolar endocytosis does not protect cells from aerolysin) - Figure 5C: What is the purpose of using HeLa cells as a control? - "..with Alexa Fluor 647 conjugated pro-aerolysin K244C" - this should be introduced earlier as it was initially mentioned in Supp. Figure 3C. - Murine fibroblasts were used earlier (Figure 1). Following from this result (where the WT can be used as a positive control), can MEFs be used instead of adipocytes to see whether caveolar endocytosis plays any role in cellular resistance? - Further comment on the increased resistance of K5 knockout would benefit on the mechanism of aerolysin-mediated cytolysis.

(Annexins minimally resist aerolysin)

• Supplementary video V3 - it seems that annexin A6 is recruited to the membrane, to a greater extent (and also quicker) than SLO. This suggests that annexin recruitment is a cellular response against aerolysin challenge. o SLO also shows A6 recruitment (arrows pointed). However, supplementary figure 6B does not clearly illustrate this.
• As annexin A1 is sensitive to calcium, further comment on the significance of intracellular/extracellular calcium in annexin A1 recruitment and aerolysin challenge would explain observations in Figure 4A.

(Patch repair protects cells from aerolysin)

• Supplementary video V4 - the intensity decreases for the inactive mutant; is this due to lysis?
• The next paragraph sounds like a contradiction: "GFP-dysferlin localized to the plasma membrane and vesicles independently of extracellular Ca2+ (Fig 8C D, Video V5) o Followed by "To study the Ca2+ dependency of dysferlin, we removed extracellular Ca2+ with 2 mM EGTA and challenged with sublytic toxin doses...found less depletion of dysferlin from cytosol".

(Methods)

• Table 1: The values presented in the methods section are, overall, confusing and require clarification.
  • 10-fold difference in SLO and PFO WT - do the authors think this might change the interpretation between different figures?
  • Understood how the haemolytic activity was calculated (referred to work in 2012), but how was the haemolytic unit originally derived?
  • How were these values (from table 1) derived to toxin concentrations used for killing nucleated cells?
  • Therefore, an EC50 haemolytic curve showing the activities for all toxins would greatly facilitate in understanding the derivation of values for table 1.
• Flow cytometry assay: What is meant by gating out the debris? And would debris also contribute to the count in dead cells?
  • What was added as the high PI control?

Referees cross-commenting

Elaborating reviewer #2's comment 7 regarding the addition of EDTA : with respect to measuring the binding if fluorescently labelled aerolysin, how can the authors differentiate between full functional pores versus prepores/incomplete pores? How else can the authors validate whether aerolysin remains functional in the presence of EDTA?

Significance

The work presents a foundation to further investigate into the mechanism of aerolysin function, following the discovery of the role of extracellular Ca2+ in its activity. As aforementioned, the role of dysferlin in resisting aerolysin also has potential, but the limitations of this work were discussed including the absence of performing a dysferlin knockout, although performing this experiment may help to strengthen the current finding.

While the work has investigated in-depth cellular resistance mechanisms, the significance and benefits of this study are unclear. For example, the authors have used different human cell lines to dissect how these cells are affected by different pores but have not stated the significance and potential benefit of studying these cell lines. Further elaboration in this aspect may increase the relevance of the study, to an audience who is interested in the field of infection and disease.

Section for special notes to the editor:

My major area of expertise and contribution to this paper is in the analysis and interpretation of activity (lytic) assays.

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Referee #2

Evidence, reproducibility and clarity

The current study explored an interesting question of aerolysin pore repair mechanism. An unusual feature of aerolysin is that unlike many other pore-forming proteins its pores remain "open" over a longer period of time, and this affects ion homeostasis influencing cell death and inflammatory response. Eventually, aerolysin pores are repaired, but what governs this process remains unknown.

In my opinion, the paper has several unresolved issues, some of which the authors mentioned in their discussion.

1. The effect of dysferlin overexpression does not indicate that patch repair is a protective mechanism or that dysferlin plays a significant role in aerolysin resistance. The authors should knock out dysferlin and assess cell resistance to lysis.
2. ESCRT complex was shown to play a role in plasma membrane repair following mechanical damage or perforin treatment of cells (Jimenez 2014, and Ritter, 2022). Whether ESCRT is important in aerolysin pore repair can be assessed by knocking out the Chmp4b gene or overexpressing dominant-negative mutant of VPS4a, E228Q.
3. I find the optimisation of lysin concentrations and data presentation quite confusing. I eventually understood, what was done, but I feel that the authors should be able to transform the data and plots so these are more accessible to a reader, eg a simple dose/time-response curves would be very helpful in that respect. For example, in Figure S1E, why does aerolysin appear to be less cytotoxic after 24 hrs than after 1 hr. In principle, I would expect to observe an additive effect, i.e. cell death at 1, 3, 6, 12, and 24 hrs should add to 100%; however, if 100% cells die at 500HU/ml, how can more cells die after 24hrs? Or am I missing something in the experimental design/data presentation? I also wonder whether using haemolytic units is appropriate (it may well be, if justified), given that the toxins used here have various membrane-binding properties. Wouldn't it make more sense to compare the cytotoxicity using nucleated cells?
4. The authors use "sublytic" concentrations of aerolysin (64HU) throughout most of the paper, but according to Figure S1C, 50% cells died at that concentration after 1hr, suggesting that when the cells were investigated over a shorter period of time, they were already dying - it's almost like the cells had life support turned off, but still being investigated as though they survived aerolysin treatment. This needs to be clarified or reassessed.
5. What effect does the addition of 150mM KCl have on the plasma membrane, trafficking/repair - wouldn't the plasma membrane be depolarised? There were a number of papers by John Cidlowski in mid 2000s, where his team explored the effect of potassium supplementation on apoptosis - this may be worth exploring.
6. Figure 3 and accompanied text: it would be more informative to show all the data rather than breaking it down to <5, 5-45 and >45 min. In my view, <5 min is an acute death due to lysis, where the toxins overcame all the protective mechanisms (membrane repair). If anything, I would dismiss that acute cell death altogether, and focused on the cells that survived the initial onslaught.
7. I am curious whether EGTA diffuses into the cytosol through aerolysin pores. If so, then unlike BAPTA-am it would affect Ca inside and outside the cell. Are the authors confident that in the absence of extracellular calcium (EGTA treatment), aerolysin formed the pores at all? Have they looked, for example, at intracellular Na/K, or have any other evidence of membrane disruption?
8. Figure 6 (and some other): I find the designation of statistical significance (a-f) quite confusing, as it is unclear which comparisons are statistically different. Looking at Figure S5, there was no difference between the effect of Annexin depletion on the toxicity of the three lysins.

Referees cross-commenting

I agree with the critique raised by the other two reviewers.

I am also happy to revise the time required to complete revisions to 3-6 months, but feel that even this may be optimistic considering substantial technical problems raised by Reviewer 1.

Significance

The paper attempts to address an interesting question of aerolysin pore repair, and it is interesting from the perspective of a potential difference between various pore-forming proteins.

The study will be potentially interesting to a broad audience of biochemists/cell biologists and microbiologists working in the field of pore-forming proteins/virulence factors.

My expertise is the biochemistry and cell biology of pore-forming proteins.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Thapa et al studied cellular mechanisms of membrane repair following pore forming toxin insult, namely aerolysin and CDCs. Out of four mechanisms tested, they show that under their conditions patch repair is the only mechanism able to counter aerolysin injury. The study is interesting but raises some concerns.

Major:

The authors conclusions contradict established results, which they cite. Yet experimental conditions are not similar in two ways: toxin concentration-wise and toxin treatment duration-wise. While we appreciate the efforts of the authors to standardize the concentration of toxins used based on hemolytic units, we note that the concentrations used are very much higher than in the other studies cited. Indeed, based on table 1, materials and methods, and the various experiments, aerolysin has a LC50 of approximately 200 HU/ml, which corresponds to about 2 ug/ml. This is approximately 200x more concentrated than for example in Gonzalez et al 2011 and Larpin et al. 2021. It makes the validity of direct comparison with those studies questionable. We noticed that the authors activate pro-aerolysin at high concentration (in the range of 1 to 5 mg/ml) and at room temperature. In our experience, under these concentration, activation leads to immediate oligomerization and massive precipitation. The final concentration of active toxin is thus unknown. The authors keep their cells in toxin-containing medium for the whole duration of the experiments, typically 45 minutes. This is in stark contrast with 45 seconds to 3 minutes transient exposure to toxin in Huffman et al 2004.

The authors do not report binding and oligomerization assays of the toxins. The only figure showing a western blot (fig. 7) is of low quality and shows unexpected observations. Aerolysin Y221G mutant is expected to bind and oligomerize. Yet, no band is present at about 250 kDa (expected oligomer) or at about 47 kDa (monomer). In addition, in aerolysin lanes (1 and 2) the oligomer is saturated, seems to be covering three lanes, indicating a possible spill-over.

Finally, while the patch repair hypothesis is interesting, it is unclear why the authors decided to overexpress dysferlin in cell lines that normally do not express it. Sure, there is a repair phenotype but this phenotype is artificially introduced. Dysferlin is not expressed at all in HeLa cells. Furthermore, dysferlin is not expressed in epithelial cells, which are the prime target of aerolysin. Why then focus on this protein? In order to show that patch repair is indeed protecting cells against aerolysin, the authors should disrupt patch repair of the cells under study and observe and increased toxicity.

Minor:

The graphic legends should be boxed out to be clearly separated from the data. In Figure 4A, it is mixed up with the data.

Some western blots are saturated, e.g. B-actin in figure 4B. Full blots should be provided.

In the methods, aerolysin sublytic dose for HeLa cells is specified at 62 HU/ml. In figure 5C and D, 31 HU/ml kills more than 50% of HeLa cells. This is not compatible.

Figure 2A and B have quite different LC50 for starting conditions ({plus minus} 200 HU/ml in A, 600-700 HU/ml in B). Why is it so different? Y-axis has a linear scale in A and a logarithmic scale in B. It would make comparison easier to have the same scale in both panels.

The letters detonating statistically significant groups are sometimes unclear. For example in Figure 1A and B, PFO belongs to group a and b simultaneously. What does this mean?

In Figure 8, aerolysin hat a LC50 in cells overexpressing GFP-Dysferin of approximately 1700 HU/ml in A and of approximately 400 HU/ml in B. Why is it so different?

In Figure S1, it is unclear what the plots « all events » vs « single cells » mean.

In the discussion, the authors write « First, survival did not correlate with overexpression, which would be expected if dysferlin acted as Ca2+ sink ». What is meant? GFP-dysferlin overexpression does correlate with survival in Figure 1A.

Referees cross-commenting

I notice quite a number of overlapping points between my comments and those of the other reviewers. In particular concerning the varying definition of sublytic concentrations and the need of a dysferlin-KO.

Significance

General assessment: The study strength lies in the several possible protection mechanisms that are tested. The weaknesses lie in the contradictions of the results reported here with established mechanisms, and in the statement that a cellular process that has been artificially introduced in the experimental system is the cellular protection mechanism against aerolysin attack. In order to prove that this process is a bona fide protection mechanism, the authors should show that it is present without the need of overexpressing a protein that is not expressed at all either in the used cell line (HeLa), or in the natural cellular target of aerolysin (epithelial cells). The significance of the proposed protection mechanism is therefore questionable.

Advance: The study contradicts previously established results but the experimental conditions used here are quite different to those used in the earlier studies, which makes the comparison quite difficult. As such it does not really fill a gap.

Audience: The study will be of interest of specialized audience.

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Reply to the reviewers

Manuscript number: RC-2022-01668

1. General Statements

We are grateful to reviewers for their thorough, insightful, and highly constructive feedback.

GENERAL REPLY #1.

We clarify a major misunderstanding. All instances of the phrase “failure of phagocytosis” or__ “phagocytic dysfunction” should be re-worded as “persistence of dead tissue”__ or simply “necro-slough.” The word “phagocytic” contains an implication of “cells” or micro-scale issues, which was not our thinking. We apologize for the ambiguity. Our claim is strictly about the millimeter-scale tissue outcome, not about cell activities to cause the tissue outcome. Please consider how strongly this miscommunication may have affected reviewers’ requests.

The relevant hypothesis statement now reads as follows: “Given that pressure ulcers often have slough or eschar, we hypothesize that mPI will have persistence of dead tissue in the wound bed, and that sterile mPI will have slough, despite the absence of bacterial biofilm.” This is a clinically-oriented claim about the relationship between bacterial infection and sloughing, not a cell biology claim about the relationship between macrophages and efferocytosis. We do not believe (and we do not wish to hypothesize) that mPI phagocytes are present and healthy while refusing to perform efferocytosis. To the contrary, such cells appear to be dead or absent at day 3 of mPI. Cells cannot clear debris when they are dead/absent. To satisfy the requests of peer review, we will perform some measurements looking for altered efferocytosis activity in monocytic cells, but we caution that the results might be negative.

GENERAL REPLY #2.

Reviewers asked us to characterize specific details of the immune system, especially for monocytes/macrophages. We did already measure many general immune-related factors at different timepoints, and found that surprisingly few of the general immunology analytes had any statistical significance (Supplementary Tables 3, 5 and 6), despite the large fold-changes seen in damage-related epitopes of oxidative stress.

We wish to avoid making any claims about the immune system in mPI, other than the absence of intact immune cells in untreated day-3 mPI wounds, and the DFO-induced increase in the presence/influx of immune cells at days 7 & 10. These serendipitous findings about immune cells are not required for any of the five chief hypotheses listed in our introduction. To characterize which cell types “should have been” present, and why they are absent, cannot possibly be established by the Reviewer’s request for greater rigor in the CTX-versus-mPI comparison, because CTX is the wrong comparison for that purpose.

To address reviewers’ requests, we provide multiple additional experiments characterizing limited aspects of the immune system. We are interested in how mPI wounds diverge from the dominant theory of what causes non-healing wounds. The dominant theory is that non-healing wounds are caused by excessive inflammation due to pre-existing morbidities (e.g., diabetes) and/or pro-inflammatory disruption (e.g., infection) that extend the duration of the inflammation phase. According to this theory, prolonged inflammation is what causes damage and blocks the granulation phase from progressing. Our mPI model violates expectations in two ways. Firstly, the dominant theory expects a non-healing wound to have elevated presence/infiltration of immune cells, but we found absence of intact immune cells at the earliest timepoint. The second is that mPI levels of oxidative damage were inversely correlated with immune cell abundance, suggesting that immune cells were not the largest source of oxidative damage. As expected, oxidative damage was highly correlated with poor healing (and was downstream of myoglobin iron). In summary, we will perform multiple additional studies toward “better describing the immune response ensuing the injury” which will promote the shared goal of understanding mPI and elucidating what the DFO drug is accomplishing.

We have one minor question about editorial policy for re-displaying the same control images for multiple experiments.

Reviewer Two initial comments (minor)

“- Figure 5 C, E, G: please provide illustrations for control treatment.

- Figure 6 K, L, M: please provide illustrations for control treatment."

The controls for Figure 5C, 5E, and 5G were already shown in Figure 2 (2I, 2L and 2B), and we hesitate to show them again without permission from the editor to duplicate figures. Similarly, controls for Figure 6K, 6L and 6M are already shown in Figure 2F-G.

2. Description of the planned revisions

__PLANNED ADDITION #1. __Measuring the iron system via immuno-staining.

*Reviewer Two initial comments: *

“2. Is myoglobin also released in the injured tissue after CTX and how does it compare to mPI (Mb+ surface area, quantity)?

• What about expression levels of proteins involved in heme/iron detoxifying proteins haptoglobin and hemopexin? Are they present in the injured tissue and are they differentially expressed between types of injury and mouse genotypes? Same goes for their receptors (CD163 and CD91, respectively): are they differentially expressed on macrophages found in the injured tissue?*

- Is hemoglobin found mPI and CTX wounds from WT or Mb-/- mice?”

Reviewer Two cross-comments:

“The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, __myoglobin deposition/accumulation in the wound, __better describing the immune response ensuing the injury. Hence my major comments 1 to 7.”

Authors' reply: We interpret this feedback to mean that measurements of myoglobin and iron detoxification factors would be help explain why mPI has higher iron and what consequences the iron may have. However, we believe the collapse or destruction of vessels in mPI (described below in the section for Completed Revision 2) might suffice to explain why iron-containing waste accumulates in mPI while the same waste gets removed in CTX. Furthermore, iron detoxification factors might be unable to enter the wound without the blood vessels.

For Planned Addition #1, we will perform the following five measurements, which should provide data for two core issues: globin protein presence in the wound, and iron detoxification factors in the wound. The methods we will perform are immunostaining for the following factors, comparing mPI against CTX at day 3 (each treated with saline and no other drugs, each with n=3 replicates).

1. Myoglobin
2. Hemoglobin
3. Hemopexin
4. Haptoglobin

5. Haptoglobin receptor CD163 Expected outcomes, regarding myoglobin abundance at day 3 post-injury in mPI vs CTX:

6. If Myoglobin is elevated in mPI vs CTX, then this finding would corroborate the increased ferric iron in mPI (measured by Prussian blue staining). It would also support the interpretation that myoglobin is the source of the excess iron that decreases upon myoglobin knockout.

7. If Myoglobin is not elevated in mPI vs CTX, then our myoglobin hypothesis could be in question and/or the myoglobin might have degraded to a state that remains redox active without binding the anti-myoglobin antibody (as occurs for hemoglobin [6]) and/or the day 3 timepoint could be too early/too late to observe the phenomenon. Expected outcomes, regarding iron detoxification factors (hemopexin, haptoglobin and CD163) at day 3 post-injury in mPI vs CTX:

8. If hemopexin and haptoglobin and/or haptoglobin receptor CD163 are elevated in mPI vs CTX, some readers will interpret this to mean that mPI has increased heme and/or increased scavenging of extracellular myoglobin/hemoglobin. Elevated hemopexin would corroborate our finding that Prussian blue staining is increased in mPI. One serious problem for interpretation of haptoglobin is that the haptoglobin-myoglobin complex has low affinity, while the haptoglobin-hemoglobin complex has high affinity, and some hemoglobin is probably present. Therefore, we also perform hemoglobin staining. Note also that CD163 is often used as a biomarker for “M2” macrophages, in addition to being the receptor for haptoglobin.

9. If hemopexin, haptoglobin, and/or haptoglobin receptor CD163 are not elevated in mPI vs CTX, some readers might interpret the measures to be irrelevant because the loss of blood vessels in mPI might prevent involvement of circulating factors. Other readers might interpret it to mean that mPI did not need, or did not utilize increased levels of the circulating factors. Other considerations are that the globin/heme source could degrade and the day 3 timepoint might be too early or too late to observe the phenomena of interest.
10. We cannot guarantee the primary antibodies will pass quality control and provide desired results. PLANNED ADDITION #2. Describing the immune response in vivo and in vitro.

Reviewer One initial comments:

“It would be interesting to see if myoglobin prevents monocyte or macrophage migration/chemotaxis. Another aspect is how cells reach the injured area… Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments…

Also the points mentioned above, about the role of myoglobin in immune cell infiltration and the role of myoglobin in the vessel properties should be at least discussed, if they are not experimentally addressed.”

*Reviewer Two initial comments: *

“8. The in vitro experiments with macrophages could be further supported by in vivo experiments, where types of injury (mPI vs. CTX) and mouse genotypes (Mb-/- vs. WT) could be evaluated for the ability of macrophages to perform efferocytosis: coupling apoptotic cell detection (Tunel staining) to macrophage immunostaining. Quantification of overlapping signal would give some information (albeit indirect) regarding the macrophages' ability to clear the tissue from dead cells. From my perspective, this would be the minimal set of data required to highlight a potential "efferocytic failure" in mPI.”

Reviewer Two cross-comments:

“The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, myoglobin deposition/accumulation in the wound, better describing the immune response ensuing the injury. Hence my major comments 1 to 7.

Beyond these points, the authors can then re-assess whether or not to include the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.”

Reviewer One cross-comments:

“Point 8 should be investigated if the authors wish to claim about efferocytosis.”

Authors' reply: We interpret this to mean that most readers will want to see more cell-specific and macrophage-specific data to complement the tissue experiments and molecular experiments. The specific choice of experiments is left up to us, but reviewers both agree something more is needed.

For Planned Addition #2, we will perform the following five measurements, which should provide quality data for at least three core issues: macrophages ex vivo, neutrophils ex vivo, and in vitro response of macrophages to myoglobin treatment. The methods we will perform are the following:

1. Perform Tunel staining alongside macrophage (F4/80) immuno-staining, to look for whether macrophages have engulfed apoptotic debris. We will compare mPI+Saline versus mPI+DFO at day 7, to see whether iron depletion affects the amount of engulfed debris inside macrophages.
2. Perform quadruple staining of pan-macrophage marker F4/80, pro-inflammatory macrophage marker iNOS, pro-regenerative macrophage marker Arginase-1, and DAPI nuclear stain.
3. Perform immuno-staining for Ly6G, a marker of neutrophils, and myeloperoxidase, a marker of neutrophil extracellular traps (NETs/NETosis).
4. Perform immuno-staining for CD38 and CD86. CD38 is a marker of CD4+, CD8+, B and Natural Killer cells. CD86 is a marker of dendritic cells, macrophages, B cells and other antigen-presenting cells. For greater information content, this staining might be multiplexed with the neutrophil staining, if antibody optimization is successful.
5. Measure the impact of Myoglobin on monocytic cell functions in vitro. We will test naïve and M1-differentiated RAW264.7 monocytes/macrophages, with or without treatment with myoglobin. The highest priority is to measure efferocytosis activity, but we will consider three functional assays: phagocytosis, efferocytosis, and transwell migration.

For the ex vivo studies (items 1-4 above), we will compare mPI+Saline versus mPI+DFO at day 7, using CTX+saline at day 3 as the positive control (n=3 per group). The in vitro treatment groups are naïve and M1-differentiated RAW264.7 monocytes/macrophages treated with or without myoglobin. The positive control is H2O2 treated RAW264.7 cells. The experiments will be carried out in quadruplicates.

Expected results for co-localization of Tunel+ F4/80 in mPI vs CTX.

• If Tunel+viable F4/80 co-localization is decreased in mPI vs CTX, then some readers might interpret a decrease in phagocytic activity by macrophages, which might help explain the persistence of dead tissue in mPI.
• If Tunel+viable F4/80 co-localization is not decreased in mPI vs CTX, then some readers may interpret that iron and its scavenger DFO cause no difference in the phagocytic function of macrophages, but some might question the timepoint or methods.

• Note that if we cannot see Tunel+F4/80 co-localization in day 3 samples of CTX injury (the positive control condition), then we consider that the assay has failed. Expected results for immuno-staining of Ly6G, myeloperoxidase, CD38 and CD86 in mPI vs CTX.

• If Ly6G, CD38, and CD86 are observed inside cells, they can indicate categories of immune cells present in the wounds. If observed extracellularly, they will be interpreted as debris from cells previously present.

• Myeloperoxidase is expected to be extracellular in the case of extracellular traps.

• Any observations will reflect only the timepoint measured, which may be before or after the peaok for that analyte. Note that we cannot guarantee that these antibodies will pass quality control and provide useful results, and we only have enough tissue samples to measure each analyte in triplicate. Expected results for cell-based assays of RAW264.7 cells when treated with myoglobin.

• If myoglobin-loaded macrophages exhibit decreased cell functions of efferocytosis, phagocytosis, and/or migration in vitro, then some readers might see this as the cellular mechanism for persistence of dead tissue and sloughing. However, such a finding would not rule out other causes of necro-slough. For example, the relative primacy of macrophages and fibroblasts in efferocytosis is subject to debate.

• If no change is detected, many readers will interpret this to mean mPI macrophages have no change in cell function after myoglobin loading.
• Our claims are unaffected either way, because what we see are dead immune cells and delayed presence/influx into the wound, and cells cannot clear debris when they are dead/absent.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Completed Revision #1. Major text amendments

Reviewer One initial comments:

“Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments. Rather, the reviewer suggests to reshape the text, remove unnecessary details to get straight to the points and to emphasize the important result. …Discussion sections about oxidative stress, endogenous iron, prevention studies, antiDAMPs strategies, slough and debridement are poorly informative and poorly referenced and should be either removed or shortened.”

Reviewer Two cross-comments:

“I also agree with Rev#1's assessment that some claims should be toned down… __Beyond these points, the authors can then re-assess whether or not to include __the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.”

Authors' reply: We have amended the main text as follows:

• Changed the title to omit the term “phagocytic dysfunction”.
• Changed the text to emphasize tissue physiology and not evoke concepts of cell biology. Changed terminology so that all “failure of phagocytosis” will be written as “persistence of dead tissue.”
• Shortened our discussion and conclusion by 33%, especially the sections entitled “the context of oxidative stress”, “anti-DAMP strategies”, and “debridement of slough.” Our abstract now says, “Unlike acute injuries (from cardiotoxin), mPI regenerated poorly with a lack of viable immune cells, persistence of dead tissue (necro-slough), and abnormal deposition of iron.” The old version had said, “mPI regenerated poorly with a lack of viable immune cells, failure of phagocytosis….”

Completed Revision #2. Surface area and vascular comparisons between CTX and mPI injuries.

*Reviewer Two initial comments: *

“I find the direct comparison made between the two types of injury, CTX and mPI, difficult to interpret. From my perspective, a more rigorous and systematic comparison between the two models of injury would be key to convincingly convey the findings of this work, especially regarding key features impacting repair.

1. • Time for tissue repair not only depends on the type of injury, but also on the extent of the injury. In other words__, how the mPI and CTX models compare in terms of surface of injured tissue__ (and resulting ischemia)?” Reviewer Two cross-comments:*

“The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area,….”

Authors' reply: We interpret the feedback to mean that the surface area of injured muscle should be comparable between CTX and mPI in order to claim that the tissue repair is different between the two injuries. We will provide the requested measurements showing similarity between the wounds, but we disagree with the premise that one should seek similarity. To that end, we will provide additional data (not requested), showing other forms of dissimilarity. Our scientific claims don’t rely on comparisons between CTX and mPI, and we urge readers to refrain from direct comparisons between dissimilar wounds.

We have revised the transferred manuscript as follows:

1. Added requested data (Suppl. Table 1) showing that both CTX and mPI have comparable size of dead muscle at the initial timepoint. To avoid making claims about CTX, we will delete the word “normal,” we will delete our phrase saying CTX lacks the dysfunctions seen in mPI, and we will explain that the purpose of CTX is to show a dissimilar example of muscle regeneration after an acute injury.
2. Added supplementary images (Suppl. Fig 2) showing dramatic differences in vasculature between CTX injury and mPI. Add accompanying text will explain that the goal of examining different types of wounds is model-description and hypothesis-generation, not hypothesis-testing Completed Revision #3: “Minor” edits suggested

Minor Edit #1

Suppl. Fig 4 is added to show intact immune cells at the wound margin and the absence of intact immune cells in the compressed region 3 days after mPI. This is as per Reviewer One’s suggestion to change Suppl. Fig 6 and call it in the results section that, “In the discussion section, there is reference to a SupplFig6, which seems to be not the good one in the document. In the FigS6 described in the text, it is mentioned that cells are kind of "stopped" at the boundaries of the damage… mPI Discussion end of page 10. Unfortunately, suplFig6 is missing (and is not called in the result section).”

Minor Edit #2

Main Fig 2L and Main Fig 5E have been changed to more representative images of HO-1 fluorescence in Day 3 saline- and DFO-treated mPI respectively, as per Reviewer Two’s minor comment, “Figure 2: HO-1 staining seem decreased in mPI compared to CTX and thus doesn't support the quantifications. Please 2x-check quantifications and images to provide consistent quantifications-illustrations pairing.”

Minor Edit #3

Suppl. Fig 5 (previously Suppl. Fig 3) has concentration and treatment times added to the figure caption as per Reviewer Two’s minor comment, “Provide concentrations and treatment times in figure legends (sup Fig3).”

Minor Edit #4

Suppl. Fig 6 (previously Suppl. Fig 4) has DNA gel electrophoresis results (Suppl. Fig 6D) added as requested by Reviewer Two in minor comment, “Show all the data mentioned in the manuscript (DNA gel electrophoresis supp fig 4)”

Minor Edit #5

Suppl. Fig 8, Suppl. Fig 10 and Suppl. Fig 12 have labels added to the image sets as per Reviewer Two’s minor comment, *“Missing information in supp Fig 5 A-D: which images from WT or Myb-KO?” *

Minor Edit #6

Suppl. Fig 11A-D have a different, more representative image set to show F4/80, CitH3 and DAPI triple-stain in saline-treated mPI (day 3). DAPI staining was not shown previously (only F4/80 and CitH3.)

Minor Edit #7

Suppl. Fig 12E has a blue dashed line added to the graph for the level of MerTK fluorescence in uninjured skinfold.

Minor Edit #8

Clarifying text and citation on the BODIPY 581/591 fluorescent probe that we used has been added in the Results section, as per Reviewer Two’s minor suggestion, “Bodipy is not a probe for lipid peroxidation. Due to its lipophilic nature, this dye can be used as a generic lipid satin to image intracellular lipid depots. Therefor the experiments using bodipy as a proxy for lipid peroxidation is incorrect and derived conclusions erroneous. Modulation in bodipy signals probably reflects modulation of intracellular lipid deposition.”

Minor Edit #9

The section, “Data availability”, which discloses the link to the Zenodo database containing the mice numbers and primary data has been moved from Suppl. Methods (Suppl. Text) to Methods in the main text.

Minor Edit #10

The acknowledgements section has been updated.

4. Description of analyses that authors prefer not to carry out

Reviewer Two initial comments:

“4. Does in situ Mb supplementation in Mb-/- mice worsens mPI repair to an extent that is comparable to WT mice?”

Authors' reply: Further study of knockout mice (Mb-/-) was mentioned by Reviewer Two, but the reviewer did not prioritize this experiment. We will not carry this out because we have already spent many years breeding descendants of our Mb-/- mice, trying to generate more Mb-/- pups, but the later years of breeding have had zero live births of homozygous knockouts. Because we have reached the ethical limit of wasted animals, any further study of myoglobin knockout would require an entirely new conditional knockout system, which is a long-term future investment.

Citations:

[1] Wang Y, Lu J, Liu Y. Skeletal Muscle Regeneration in Cardiotoxin-Induced Muscle Injury Models. Int J Mol Sci. 2022 Nov 2;23(21):13380.

[2] Averin AS, Utkin YN. Cardiovascular Effects of Snake Toxins: Cardiotoxicity and Cardioprotection. Acta Naturae. 2021 Jul-Sep;13(3):4-14.

[3] Naldaiz-Gastesi N, Goicoechea M, Alonso-Martín S, Aiastui A, López-Mayorga M, et al. Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells. Stem Cell Reports. 2016 Sep 13;7(3):411-424.

[4] Ahmed AK, Goodwin CR, Sarabia-Estrada R, Lay F, Ansari AM, et al. (2016). A non-invasive method to produce pressure ulcers of varying severity in a spinal cord-injured rat model. Spinal Cord, 54(12), 1096–1104.

[5] Turner CT, Pawluk M, Bolsoni J, Zeglinski MR, Shen Y, et al. (2022). Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion. Scientific Reports, 12(1), 12622.

[6] Bahl N, Du R, Winarsih I, Ho B, Tucker-Kellogg L, Tidor B, et al. (2011) Delineation of lipopolysaccharide (LPS)-binding sites on hemoglobin: from in silico predictions to biophysical characterization. J Biol Chem. 286(43), 37793-803.

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Referee #2

Evidence, reproducibility and clarity

Summary:

In this manuscript, Jannah and colleagues highlight a pathophysiological mechanism involving myoglobin for the poor repair capacity of ulcerative pressure wounds. The authors modeled muscle pressure injury (mPI) using a magnet and compared it to cardiotoxin (CTX)-induced muscle injury. They show a significant delay in repair kinetics for mPI compared to CTX, recapitulating the notoriously poor repair process associated with ulcerative pressure injuries. Using mice genetically invalidated for myoglobin (Mb), they show an improved mPI recovery, with improved tissue repair quality over a shorter period of time. Mechanistically, the authors link the poor repair of mPI to the oxidative and pro-inflammatory effect of Mb released from injured skeletal muscle fibers.

Major comments:

The authors hypothesis regarding the role of Mb in the pathophysiology of ulcerative pressure injuries is interesting. However, the work here seems quite preliminary with major points remaining to be clarified before considering reaching the author's conclusion. I find the direct comparison made between the two types of injury, CTX and mPI, difficult to interpret. From my perspective, a more rigorous and systematic comparison between the two models of injury would be key to convincingly convey the findings of this work, especially regarding key features impacting repair. My major comments are listed below.

1. Time for tissue repair not only depends on the type of injury, but also on the extent of the injury. In other words, how the mPI and CTX models compare in terms of surface of injured tissue (and resulting ischemia)?
2. Is myoglobin also released in the injured tissue after CTX and how does it compare to mPI (Mb+ surface area, quantity)?
3. Does Mb co-injection with CTX mimics mPI injury in terms of inflammation and repair kinetics?
4. Does in situ Mb supplementation in Mb-/- mice worsens mPI repair to an extent that is comparable to WT mice?
5. A better characterization of the inflammatory between types of injury and mice (Mb-/- vs. WT) before and after 3- and 10-days post-injury would be very informative. Comparing the relative proportions of leukocyte populations would provide valuable information regarding the kinetics of the repair process.
6. Macrophages in particular play a central role in the orchestration of tissue repair, through their immunomodulation abilities. On the same token, characterizing macrophage infiltrates (number per surface area of injured tissue) and phenotype would potentially provide valuable information to link observed differences between types of injuries to Mb. Ideally, assessment of leukocyte and macrophages infiltration and populations would be analyzed by flow cytometry after injured (vs. uninjured) tissue dissociation (enzymatic or mechanical). Otherwise, although less quantitative, this can also be done by cell infiltration using specific immunostaining and quantification (cell number/injured tissue surface area).
7. Macrophage phenotype (inflammatory vs. anti-inflammatory/reparative) can be achieved by RTqPCR, using well-define combination of mRNA encoding proteins associated with inflammatory (e.g. iNos, Cox-2, Cd86) or anti-inflammatory (Ym1, Arg-1, RELMa, Cd206).
8. The in vitro experiments with macrophages could be further supported by in vivo experiments, where types of injury (mPI vs. CTX) and mouse genotypes (Mb-/- vs. WT) could be evaluated for the ability of macrophages to perform efferocytosis: coupling apoptotic cell detection (Tunel staining) to macrophage immunostaining. Quantification of overlapping signal would give some information (albeit indirect) regarding the macrophages' ability to clear the tissue from dead cells. From my perspective, this would be the minimal set of data required to highlight a potential "efferocytic failure" in mPI.
9. What about expression levels of proteins involved in heme/iron detoxifying proteins haptoglobin and hemopexin? Are they present in the injured tissue and are they differentially expressed between types of injury and mouse genotypes? Same goes for their receptors (CD163 and CD91, respectively): are they differentially expressed on macrophages found in the injured tissue?

Minor comments:

• Figure 2: HO-1 staining seem decreased in mPI compared to CTX and thus doesn't support the quantifications. Please 2x-check quantifications and images to provide consistent quantifications-illustrations pairing.
• Figure 5 C, E, G: please provide illustrations for control treatment.
• Figure 5J: it would have been nice to add Mb-/- mice to the comparison.
• Figure 6 K, L, M: please provide illustrations for control treatment.
• Figure 8: please maintain consistency in the way you convey data between timepoints: area of regenerated (E, F) or unregenerated (G) tissue.
• Bodipy is not a probe for lipid peroxidation. Due to its lipophilic nature, this dye can be used as a generic lipid satin to image intracellular lipid depots. Therefor the experiments using bodipy as a proxy for lipid peroxidation is incorrect and derived conclusions erroneous. Modulation in bodipy signals probably reflects modulation of intracellular lipid deposition.
• Provide concentrations and treatment times in figure legends (sup Fig3).
• Show all the data mentioned in the manuscript (DNA gel electrophoresis supp fig 4)
• Indicate the number of experimental repeats and the statistical tests used in the figure legends.
• Missing information in supp Fig 5 A-D: which images from WT or Myb-KO?
• Is hemoglobin found mPI and CTX wounds from WT or Mb-/- mice?

Referees cross-commenting

I have read the report from Reviewer#1 and below are my cross-comments.

I agree with Rev#1's minor comments. I also agree with Rev#1's assessment that some claims should be toned down as data don't support them. The phagocytic dysfunction is certainly one of them, for the reasons mentioned, but not the only one. Indeed, I believe that virtually all the claims could use some dampening to various extent because the level of evidence is not very high throughout. A more rigorous description of the injury model would address this point in my opinion (narrower scope with a demonstration).

The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, myoglobin deposition/accumulation in the wound, better describing the immune response ensuing the injury. Hence my major comments 1 to 7.

Beyond these points, the authors can then re-assess whether or not to include the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.

Significance

The field of tissue repair and regeneration is an exciting field and improving our understanding of the molecular mechanisms involved in muscle tissue injury has clear and impactful clinical applications.

The pathophysiological mechanism involving Mb that the author address in this work has the potential to interest both basic science and clinical researchers and can potentially benefit not only the field of skeletal muscle regeneration but also the field of cardiac remodeling.

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Referee #1

Evidence, reproducibility and clarity

Summary: The study by Nasir et al. investigates the healing properties of skeletal muscle after a pressure injury and the impact of myoglobin in this process. First, they compared cardiotoxin versus pressure injuries and showed that the latter heals slowly and badly (this is shown through a large series of parameters). They then used myoglobin deficient mice or WT mice treated with the iron chelator DFO to show that in the absence of myoglobin, there is an improvement of the regeneration process.

Major comments:

The study is well written, clear, and the experiments are carefully presented and conducted. Although the text is usually very detailed and nicely referenced, some of the claims should be dampened. Notably the title since the phagocytic dysfunction is not evidenced by the results, not the wound enlargement (since DFO has no impact on it). <br /> Also the text is very long, notably the discussion, which contains 18 sections (!) and several sections are not very informative and poorly referenced, looking more as a thesis dissertation than as an article discussion.

Concerning immune cells, the conclusion cannot be that myoglobin impedes their phagocytic function. All the data concur that in the absence of myoglobin there are more immune cells in the regenerating muscle at day 3. Consequently, more macrophages will lead to a better cleansing of debris. Thus, the difference would not rely on phagocytic properties per se, but more on the number of macrophages that arrive at the site of injury. It would be interesting to see if myoglobin prevents monocyte or macrophage migration/chemotaxis. Another aspect is how cells reach the injured area. In the discussion section, there is reference to a SupplFig6, which seems to be not the good one in the document. In the FigS6 described in the text, it is mentioned that cells are kind of "stopped" at the boundaries of the damage. This is very interesting. If the vessels are physically flattened or squashed by the injury, extravasation can not occur properly, in comparison with cardiotoxin. Then the role of myoglobin in extravasation, or in the "reshaping" of the vessels after the removal of the magnet would be interesting to investigate. Of note, in the myoglobin deficient mice, the vascular network is increased, favoring immune cell infiltration.

Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments. Rather, the reviewer suggests to reshape the text, remove unnecessary details to get straight to the points and to emphasize the important results. Also the points mentioned above, about the role of myoglobin in immune cell infiltration and the role of myoglobin in the vessel properties should be at least discussed, if they are not experimentally addressed.

Minor comments:

• Page 5: "The panniculus layer of mPI was nearly devoid of intact immune cells." It is not clear here if the authors refer to the absence of immune cells or to damage of immune cells present in the injured area.
• Page 6: "In summary, measures of innate immune response became less abnormal after Mb knockout". Cardiotoxin injury is not the "normal" situation since this kind of injury is not physiological and is highly inflammatory as compared with others (Hardy et al., Plos One 2016).
• Discussion sections about oxidative stress, endogenous iron, prevention studies, antiDAMPs strategies, slough and debridement are poorly informative and poorly referenced and should be either removed or shortened.
• Discussion end of page 10. Unfortunately, suplFig6 is missing (and is not called in the result section).

Referees cross-commenting

I agree with the rev#2's review and later comments. His comments are quite complementary to those I raised. If the author have the capacity to make the experiments that are proposed by Rev#2, it would be super nice. Point 8 should be investigated if the authors wish to claim about efferocytosis.

Significance

This study is of interest because it provides insights on muscle regeneration after an injury that may occur in daily life just as contusion, or crush, to the contrary of the whole muscle necrosis induced by cardiotoxin (the main model used in the field). In that aspect, it is more physiological and of interest for the readers in the fields of in muscle regeneration and tissue trauma. The study is descriptive, but is very well conducted and well discussed. Additional experiments investigating the impact of myoglobin on vessel properties/extravasation of immune cells would raise the impact of the study but the study is publishable as it is (with editing as suggest above).

The field of expertise of the reviewer is muscle regeneration and inflammation.

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Reply to the reviewers

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Reply to the Reviewers

I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

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Referee #3

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). In this paper the authors develop an engineered uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo. This new model (3E-uterus) is used for mechanistic studies of embryo implantation. They hint that integrin-mediated adhesion of the embryo to the uterine wall is required for peri-implantation mouse development. The authors use this model also to study the role of tension for embryo development. They postulate that release of tension from the polar side of the embryo upon implantation allows for extra embryonic development. By using mathematical modeling of the implanting embryo as a wetting droplet, the authors link the embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Finally, the authors uncover the role of coordination between trophoblast motility and embryo growth, where trophoblast mobility displaces the Reichart's membrane giving the embryo space to grow. In summary, the authors technically advance the field of developmental biology by providing a model to study peri implantation morphogenesis of the mouse embryo.

Major comments:

• Are the key conclusions convincing?

The key conclusions the authors derive from their experiments are somewhat convincing. Suggested experiments below will strengthen their claims. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Claim 1: The 3E-uterus is representative of mouse embryo peri-implantation. To claim this a more extensive validation of the embryos cultured in their 3E uterus both via scRNA seq and IF for pluripotency, visceral and parietal endoderm markers is required. It is also interesting that embryos cultured in the 3E-uterus lose the correct timing of development. Could the authors please comment on this? scRNA seq of the embryos cultured in their system at different timepoint (i.e. Day 1-3) compared to control pre, peri and post implantation embryos could help answer this question.

Claim 2: The release of tension from the polar side upon implantation allows for extra embryonic development. To quantitatively measure the difference in tension before and after implantation is technically very challenging. However, this paper could benefit of further validations including IF stainings for markers such as E-cadherin, F-actin and Phospho myosin. In addition to this, treatments with Y27 and blebbistatin of the embryo would allow to further study the role of cell tension on embryo implantation. Finally, a laser ablation experiment at the cell junctions of the polar region before and after implantation would help to answer this question but this could be technically challenging due to the curvature nature of embryos.

Claim 3: Integrin-mediated adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. In Figure 2a the authors show that embryos cultured in 3E-uterus without RGD do not develop and hypothesize this is due to lack of integrin binding. A control experiment using a non-integrin binding peptide is beneficial here.

Claim 4: The spatial orientation of the embryo plays a key role in mouse peri implantation development. In Figure 5i-j, the authors place embryos in a downward (i) and upward (j) orientation. Could the authors also please comment on whether they believe the orientation, the way the embryo feels the gravity plays a role in implantation? Is the amount of space that the embryo has to grow in the limiting factor on development? Could the authors use 3E-uterus models with different lengths (by using molds with different spacing) to see the role of geometry and space that the embryo has for trophoblast mobility and embryo growth. What would happen if the embryo were very close to the bottom of the hydrogel?

Claim 5: A mathematical model based on the wetting droplet recapitulates the embryo in their system. Could the authors comment on whether their mathematical model considers proliferation, and would proliferation have an impact on the system's kinetics? What is the role of polar TE proliferation and how does that influence the trophectoderm morphology? If the embryo is geometrically confined, can the authors exclude that this confinement is influencing cell shape? - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

In this technical advance paper, the claims will become more robust with the suggested experiments above. Claims of implantation should be changed to accurately reflect that the 3E-uterus models peri implantation as there is no invasion in the 3D hydrogel matrix. In addition to this, the uterine cells are missing which are required to fully recapitulate the mechanisms of embryo peri-implantation. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

3- 6 months will allow the authors to address all questions above. Yet, the laser ablation experiment might be difficult to perform due to the curvature of the embryo. - Are the data and the methods presented in such a way that they can be reproduced?

We appreciate the details of the materials and methods section particularly of the imaging.<br /> - Are the experiments adequately replicated and statistical analysis adequate?

Yes

Minor comments:

• Specific experimental issues that are easily addressable.

• Laminin staining in Figure 2A is only done in in vitro embryos but not in in vivo embryos. Could the authors add the missing staining?

• It would be beneficial to have both active and normal integrin stainings in E4.5 embryos.
• Could the authors provide stainings for mesenchymal markers for E4.5 and D2 3E uterus?
• Can the authors comment on Figure Supp. 3D where the timing seems to be flipped?
• Why was 600 um chosen for the depth of the 3E-uterus?
• Are prior studies referenced appropriately?

How do the findings in this paper relate to the findings in Weberling et al (PMID 33472064), where they show that in vivo, the polar trophectoderm exerts physical force upon the epiblast, causing it to transform from an oval into a cup shape? - Are the text and figures clear and accurate?

Overall the text and figures are clear and accurate. In figure 2E and 2G, the outline covers the staining. Would it be possible to have it without the outline in the supplementary? - Do you have suggestions that would help the authors improve the presentation of their data and conclusions? It would be very informative to have for each panel in which a representative image is used for that image to be marked into the quantified data (graphs).

Overall, the manuscript is very well written and the conclusions are informative and clear.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The advance presented in this paper is technical. In this methodology paper, the authors use their novel model to investigate the mechanics of embryo peri-implantation and hint at new conceptual findings such as the role of wetting properties of the embryo onto the ECM of the uterus. - Place the work in the context of the existing literature (provide references, where appropriate).

Since embryos become hidden in the womb upon implantation, ex vivo cultures provide an experimental setting to monitor, measure and manipulate embryonic development. Ex vivo culture of peri-implantation (mouse) embryos so far relied on embryonic growth on 2D plastic surfaces (PMID: 4930085, 4562729 and 24529478) or 3D bioreactors (PMID: 33731940). Although important, these assays do not recapitulate the interaction with the uterine cells and ECM (the in vivo scenario). In this study initial steps are taken to recapitulate the interaction of the embryo with the uterine ECM during peri-implantation. Uterine cells are however missing from this new system, which is important for understanding the full mechanism of implantation. - State what audience might be interested in and influenced by the reported findings.

Developmental biologists - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Bioengineer, bioinformatician and developmental biologists working with embryo models and hydrogels. There is not sufficient expertise to evaluate the mathematical modeling.

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Referee #2

Evidence, reproducibility and clarity

The authors developed a new method of ex vivo culture of mammalian embryos. This engineered uterus recapitulates some features of peri-implantation development of the mouse embryo. The authors show that integrin adhesion to the uterine wall through integrin beta 1 is required for proper peri-implantation. They also demonstrate collective migration of the trophoblast on the synthetic hydrogel surface. The authors interpret their results through the physics of wetting, which allows them to conclude that a release of tension enables shape changes in the embryo. Finally, light sheet imaging allows the authors to visualize the interplay between growth and collective motion.

Major

1. The article will be of potential interest to a broader community than mammalian embryo peri-implantation researchers. This broader community will likely not be familiar with the structure and nomenclature of the embryo and surrounding tissues. The introduction of terms in the second paragraph of the introduction should be paralleled by a comprehensive image in Fig. 1. This image should clarify what is considered apical and basal in this context. Similarly, when the model is introduced a more comprehensive scheme should also be provided.
2. The failure of 2D hydrogels to support mouse blastocysts through peri-implantation (Supp Fig. 1) is insufficiently described. Some panels in this figure and not mentioned in the main text. This discussion should be expanded, especially considering that 2D approaches has been quite successful. A detailed discussion of the authors' cylinder approach compared to the best 2D systems published should be provided (Govindasamy et al, for example).
3. Is the hydrogel purely elastic or viscoelastic? The mechanical properties of the hydrogel (viscoelasticity and degradability) should be presented in the main text.
4. The authors call the finding that integrins are required for peri-implantation "striking", but a role for integrins in this process is are already known (see Sutherland et al, for example). The novelty of the authors findings in this regard should be better presented.
5. Figures 2ef (in utero) and 2gh (3E-uterus) show rather different results. In uterus, pERM and ZO1 look quite compartmentalized in the outer region. This is not the case in 3E-uterus shown in Figure 2gh. These data do not seem to support an agreement between in utero and 3E-uterus as mentioned in the text.
6. The authors claim that mTE cells lose cell polarity upon adhesion to the uterine matrix and acquire mesenchymal properties. This claim should be clarified. Cells protrude and become migratory but invasion seems to be collective, suggestive that epithelial features such as cadherin adhesion remain. Are these cells mesenchymal or are they simply epithelial cells with motile capacity (as in wound healing, for example)? How do mesenchymal vs epithelial features compare between in utero and 3E-uterus?
7. If I understand correctly, the model assumes that tension of the droplet-medium interface and is the same in the upper and lower sides of the embryo. However, the mechanical and geometrical properties of cells in both sides are quite different. Is the assumption of same tension justified? Can these tensions be measured or inferred to test this assumption?
8. Along similar lines, attributing a surface tension to a system that is thick (ie several cell layers) and that undergoes apical constriction (ie a bending modulus) is an oversimplification that should be justified. Cells in the pTE change (potentially) their apical, basal and lateral tension during apical constriction. How do these three components relate to what the model simply refers as tension? Additionally, how does the presence of a bending moment alter the wetting picture?
9. The physics of wetting were recently generalized to include additional terms attributed to active components (main associated with polarity, see works by Alert and Casademunt). These active components are not explicitly taken into account in the authors' model. Are they not needed? A brief discussion of this aspect should be provided.
10. In utero, the cavity in which the embryo is implanted is created during implantation. In this situation, the analogy with wetting seems harder to establish because the embryo spreads as it forms the cavity. How does this alter the authors interpretation?
11. The first paragraph of the supplementary note refers to Fig. 4D. This reference here does not seem correct.

Referees cross-commenting

The three of us coincide in appreciating the novelty and potential impact of the new method.

There is an agreement between all 3 referees to request additional evidence of how well the 3E-uterus captures the in vivo phenomenon. I believe the suggestions provided by my two colleagues in this regard are on point and seem feasible for the applicants within a 6 month period.

I also agree that tension measurements with laser ablation (or other inference techniques) would provide stronger support to the model.

Significance

This article provides an important technical advance to study peri-implantation of the mammalian embryo beyond current methods based on 2D substrates. This work will be of interest to the community of early mammalian embryogenesis but also to the broader field of engineering multicellular systems.

As list above, main limitations concern 1) The extent to which their method properly captures peri-implantation, 2) The novelty of some of the authors observations, 3) The soundness of the theoretical model.

My expertise is in experimental biophysics of multicellular systems.

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Referee #1

Evidence, reproducibility and clarity

Summary:

To recapitulate mouse peri-implantation development ex vivo, the authors engineered a uterus-like microenvironment by fabrication of topographically patterned hydrogels and identified the roles of the physical interaction between embryo and hydrogels for egg cylinder formation. Notably, integrin-mediated adhesion between trophoblast and matrix facilitates egg-cylinder shape. Moreover, Live-imaging with light-sheet microscopy led them to propose the hypothesis that the interaction between embryos and hydrogels appeared to be described by a droplet wetting process.

Major comments:

Although the authors claim that the interaction between the uterus and embryo is crucial for egg-cylinder formation, they did not utilize uterus-derived cells nor analyze these. They just observed how blastocysts grow autonomously into the egg-cylinder shape in the hydrogel which has solely physical properties of the uterus but not biochemical features except for the RGD peptide-mediated cell adhesion process. Thus, it is still uncertain if similar mechanisms contribute to egg-cylinder formation in utero. To fulfill the gap between ex vivo morphogenesis and in utero, the authors would be expected to analyze the interaction between trophoblast and uterus in utero if uterine mechanisms can follow the integrin-mediated adhesion and a droplet wetting process. For example, whether integrin can contribute to egg-cylinder formation in utero can be proved by analyzing knock-out phenotypes of integrin-related genes. It will take around six months to conduct such suggested experiments. Otherwise, the authors should modify their statement "the interaction between embryo and uterine" into "the interaction between embryos and uterine-like hydrogels" throughout the manuscript.

Specific point:

Supplemental figure 1h, page 4 lines 20-23: The authors claim that 1.5-2 % PEG generated the shear modulus at 100-300 Pa, which is in the stiffness range of the E5.5 mouse decidua (Govindasamy et al., 2021). In Govindasamy's paper, elasticity measurements were performed in Petri dishes using an MFP-3D Classic AFM (Asylum Research, Wiesbaden, Germany) and cantilevers with a force constant of 0.08 with spherical tips (2 mm; NanoWorld, Neuchatel, Switzerland). In the present paper, the shear modulus (G′) of hydrogels was determined by performing small-strain oscillatory shear measurements on a Bohlin CVO 120 rheometer with plate-plate geometry. Therefore, it is not appropriate that Govindasamy's modulus is compared to the authors' modulus directly. For a direct comparison of the two modulus values, the authors can measure the stiffness of PEG with AFM or the shear modulus of the E5.5 mouse decidua by their rheometer.

Significance

Strong points:

As described in summary, the authors have newly identified that integrin-mediated adhesion between trophoblast and matrix facilitates egg-cylinder shape and the interaction between embryos and hydrogels is followed by a droplet-wetting process. These findings are considered to be novel by their excellent ex vivo imaging method.

Limitations:

It remains unaddressed that ex vivo mechanisms they have identified contribute to the egg-cylinder formation in utero.

Audience:

The results demonstrated here by the authors are fascinating in terms of general interest after the above concerns are appropriately addressed.

Since I am an experimental biologist, I don't have sufficient expertise to assess if the physical droplet model can recapitulate the interaction of the embryo and pre-patterned hydrogel for egg cylinder formation in detail.

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Reply to the reviewers

We thank all the Reviewers for their highly constructive reviews. Below, I have pasted the Reviewer’s comments in black and my replies in red, for easy reading.

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

In their study, Zhu and colleagues study how the centrosome proteins Spd-2 and Cnn in Drosophila recruit gamma-tubulin complexes to centrosomes, which is an important step in mitotic spindle formation. The authors make use of mutant flies and RNAi and find that the two factors Spd-2 and Cnn together are responsible for mitotic centrosomal accumulation of gamma-tubulin. By inactivating Spd-2 or Cnn separately, the authors show that Cnn appears to recruit the large share of mitotic gamma-tubulin pool by its CM1-domain. Interestingly, this involves only gamma-TuSCs (subcomplexes of gamma-TuRC) and not gamma-TuRCs. A smaller pool is recruited by Spd-2, and this pool depends on gamma-tubulin complex proteins that are only present in pre-assembled, complete gamma-TuRCs. This suggests that Drosophila makes microtubule nucleation templates in two ways. First, as in yeast, by direct recruitment of gamma-TuSCs to mitotic centrosomes, where additionally oligomerization needs to happen. And second, by recruitment and activation of preassembled gamma-TuRCs. Inactivation of both Cnn- and Spd-2 pathways abolishes mitosis-specific gamma-tubulin recruitment, resulting in low, but not complete loss of gamma-tubulin at centrosomes. The authors show that these low-gamma-tubulin centrosomes are still able to organize microtubules, but these microtubules have different dynamicity. Inspired by existing literature in flies and other model organisms, the authors identify Msps/Xmap215 as an important nucleation factor in this scenario.

Major points:

1) The authors use fly embryos with mutant Grip71, Grip75 and Grip163 alleles, which are central to the study. Most conclusions are based on the assumption that some mutants contain only gamma-TuSC, whereas wildtype cells contain a mix of gamma-TuSC and gamma-TuRC. It would be important to show sucrose gradient analyses of extracts to confirm the expected presence/absence of gamma-TuSC/gamma-TuRC.

We agree that it would be nice to perform sucrose gradient analysis of γ-tubulin mutants in different mutant backgrounds, but unfortunately this is not as easy as the Reviewer may think. To clarify, we have used larval brain cells (not embryos) for the analysis of γ-tubulin recruitment to centrosomes. We cannot use embryos because most mutant combinations are lethal beyond larval stages, meaning that mutant adult females are not available for embryo collection (embryos use maternally loaded proteins and mRNA and so it is the genotype of the mother that is important). Performing sucrose gradients with larval brain extracts would be extremely challenging, if not impossible, because a relatively large amount of starting material is required for sucrose gradient centrifugation, and manually dissecting and preparing hundreds if not thousands of larval brains is unrealistic, especially as mutant larvae are rare.

Given that we are not able to carry out these experiments, we have modified the text to include the caveat that some higher-order complexes may partially form in certain mutants. For example, in relation to the ability of Grip71 to recruit γ-TuSCs in cnn,grip75,grip163 mutants, the text now reads: “Thus, Spd-2 appears to recruit a very small amount of γ-TuSCs (which may, or may not, be present as larger assemblies due to an association with Grip128-γ-tubulin) via Grip71 (i.e. the recruitment that occurs in cnn,grip75GCP4,grip163 GCP6 cells), but its recruitment of γ-tubulin complexes relies predominantly on the GCP4/5/4/6 core.”

Nevertheless, the most important conclusion is that Cnn can recruit γ-TuSCs independent of pre-formed cytosolic γ-TuRCs and this is based on the finding from one particular mutant – the spd-2,grip71,grip75,grip128,grip163 mutant – where γ-tubulin levels at mitotic centrosomes are only very slightly reduced compared to single spd-2 mutants (Figure 1B). This conclusion is based on three assumptions that we argue are all very reasonable:

Assumption 1: flies depleted of 2, if not all 3, GCP4/5/4/6 core components (grip75,grip128,grip163) do not have a functioning GCP4/5/4/6 core. The Grip75GCP4 allele is a null mutant and is combined with a deficiency chromosome that depletes the whole Grip75GCP4 gene, and the Grip163GCP6 allele is a very strong depletion allele and is also combined with a deficiency chromosome that depletes the whole Grip163GCP6 gene. Even if the efficiency of the RNAi against Grip128GCP5 were poor, it would be hard to form a GCP4/5/4/6 core without Grip75GCP4 and in the near absence of Grip163GCP6 (which together provide 3 of the 4 molecules of the complex, including the outermost ones).

Assumption 2: cells depleted of the GCP4/5/4/6 core cannot assemble cytosolic γ-TuRCs. This is reasonable given that even individual depletion of Grip75GCP4, Grip128GCP5 or Grip163GCP6 already strongly reduces the presence of cytosolic γ-TuRCs (Vogt et al., 2006; Vérollet et al., 2006). In spd-2,grip71,grip75,grip128,grip163 mutant brain cells, the only γ-TuRC protein not targeted, except for the γ-TuSC components, is Actin (Mozart 1 is expressed only in testes (Tovey et al., 2018) and Mzt2 does not exist in flies). In Xenopus and humans, Actin appears to facilitate γ-TuRC assembly via interactions with a GCP6-N-term-Mzt1 module, and so it would be unlikely to allow γ-TuSC assembly into higher-order complexes without GCP6 (i.e Grip163GCP6) and Mzt1.

Assumption 3: Were Cnn not able to recruit γ-TuSCs independently of pre-formed γ-TuRCs, we would expect a much stronger reduction in γ-tubulin recruitment to centrosomes in spd-2,grip71,grip75,grip128,grip163 mutant cells. It is reasonable to assume, even without sucrose gradients, that the assembly of γ-TuRCs is strongly impeded in spd-2,grip71,grip75,grip128,grip163 mutant cells. Nevertheless, γ-tubulin is still recruited to centrosomes at ~66% compared to ~77% in spd-2 single mutant cells. While statistically significant (as stated in the updated manuscript), this reduction would surely be much greater were Cnn not able to recruit γ-TuSCs.

In the absence of experimental data, we have therefore made these arguments in the main text by making some text modifications and adding a new paragraph, as follows:

*“….the centrosomes in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells had ~66% of the γ-tubulin levels found at wild-type centrosomes, only slightly lower than ~77% in spd-2 mutants alone (Figure 1A,B). Thus, the recruitment of γ-tubulin to mitotic centrosomes that occurs in the absence of Spd-2, i.e. that depends upon Cnn, does not appear to require Grip71 or the GCP4/5/4/6 core. *

While we cannot rule out that residual amounts of GCP4/5/4/6 core components in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells may support a certain level of γ-TuSC oligomerisation in the cytosol, we favour the conclusion that Cnn can recruit γ-TuSCs directly to centrosomes in the absence of the GCP4/5/4/6 core for several reasons: First, the alleles used for grip71 and grip75GCP4 are null mutants, and the allele for grip163GCP6 is a severe depletion allele (see Methods), and even individual mutations in, or RNAi-directed depletion of, Grip75GCP4, Grip128GCP5 or Grip163GCP6 are sufficient to strongly reduce the presence cytosolic γ-TuRCs (Vogt et al., 2006; Vérollet et al., 2006). Second, spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells are depleted for all structural γ-TuRC components except for γ-TuSCs and Actin (note that Mozart1 (Mzt1) is not expressed in larval brain cells (Tovey et al., 2018) and that Mzt2 does not exist in flies). In human and Xenopus γ-TuRCs, Actin supports γ-TuRC assembly via interactions with a GCP6-N-term-Mzt1 module (Liu et al., 2019; Wieczorek et al., 2019, 2020; Zimmermann et al., 2020; Consolati et al., 2020), and so Actin alone is unlikely to facilitate assembly of γ-TuSCs into higher order structures. Third, our data agree with the observation that near complete depletion of Grip71, Grip75GCP4, Grip128 GCP5, and Grip163GCP6 from S2 cells does not prevent γ-tubulin recruitment to centrosomes (Vérollet et al., 2006). Fourth, given the strength of mutant alleles used, one would have expected a much larger decrease in centrosomal γ-tubulin levels in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells were Cnn not able to directly recruit γ-TuSCs to centrosomes. Thus, our finding that Cnn can still robustly recruit γ-tubulin to centrosomes in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells strongly suggests that Cnn can recruit γ-TuSCs to centrosomes without a requirement for them to first assemble into higher-order complexes.”

2) Given the advantage of the CnnΔCM1 separation of function mutant, I do not understand why it is not used throughout the study. Instead, full Cnn loss is used, which results in strongly reduced Spd-2 levels (Figure 2C,D). Are the observed differences between wild-type and mutants in Figure 2-5 dependent on defective PCM or do they also occur in a CnnΔCM1 background?

This is a good point, and we agree that it would have been “cleaner” to use the CnnΔCM1 mutant in these experiments. The reason that the CnnΔCM1 mutant was not used is that this mutant allele was made only after we had already generated the multi-allele stocks and performed most of the other experiments in Figures 2-5. It would have taken a long time to go back and generate fly stocks containing the CnnΔCM1 allele instead of the cnn null mutant allele. As we have shown that the CnnΔCM1 mutant cannot recruit any γ-tubulin, we don’t believe that using this mutant would change the results regarding recruitment of γ-tubulin by the spd-2 pathway i.e. when we have examined γ-tubulin recruitment in the cnn mutant background (Figure 2). Nevertheless, in terms of the efficiency to which microtubules can be nucleated in the absence of γ-tubulin complexes, which was examined in a cnn,grip71,grip163 mutant background, it is likely that using a cnnΔCM1,grip71,grip163 mutant background would better maintain Spd-2 in the PCM and thus better allow Msps and Mei-38 to stimulate microtubule nucleation. We may therefore find that microtubules can be nucleated even more efficiently in the absence of γ-TuRCs. Note that we do state this caveat in the paper. That said, performing the experiments would not be essential to conclude that microtubules can be nucleated independent of γ-TuRCs, which is the main point of this part of the paper.

Should the Reviewer and Editor deem it necessary, we will generate CnnΔCM1,grip71,grip163 lines to test whether or not γ-tubulin can be recruited to mitotic centrosomes under these conditions, and, if no γ-tubulin is recruited, we will generate CnnΔCM1,grip71 ,grip163,Jup-mCherry lines to test the ability of these centrosomes to nucleate microtubules (using the CherryTemp). Please note, however, that this would be several months of difficult fly genetics and data collection and we would therefore appreciate it if you consider the cost/benefit ratio when making your decision on whether you expect this data or not.

3) Statistical tests should support the conclusions in the text. If the authors claim differences between different genetic backgrounds (e.g. that spd2-mutants only have ~77% of gamma-tubulin at mitotic centrosomes compared to wild-type), statistical tests must compare mutant mitosis vs. wild-type mitosis.

We agree. We have now carried out the appropriate statistical tests and included them in the new version of the paper. For more detail, see the response to Reviewer 2 point 2.

4) While Cnn, grip71, grip163 mutants do not accumulate gamma-tubulin at centrosomes in mitosis, they still have low levels of centrosomal gamma-tubulin. It is therefore misleading to refer to "gamma-tubulin negative centrosomes".

This is a fair point. While we suspect this small fraction of γ-tubulin is non-functional in regard to microtubule nucleation i.e. it is the interphase pool of γ-tubulin and interphase centrosomes do not organise microtubules, we agree that referring to them as "gamma-tubulin negative centrosomes" is misleading. We have now changed the text to refer to them simply as “cnn,grip71,grip163 mutant centrosomes” or “cnn,grip71,grip163 centrosomes”.

Minor points:

1) The abstract states that gamma-TuRC is a catalyst of microtubule nucleation. By definition, a catalyst takes part in a reaction but is not part of the final product. Although our knowledge of the nucleation mechanism is still incomplete, mechanistic studies suggest a non-catalytical mechanism since gamma-TuRC was found to stay attached to the microtubule end after nucleation (Consolati et al. 2020, Wieczorek et al. 2020).

We have now removed any reference to the γ-TuRC being a catalyst.

2) CnnΔCM1 flies: genotyping data should be provided besides describing gRNAs.

We are not entirely sure what the Reviewer means here. We had already stated in the main text and methods that the deletion region spanned from R98 to D167. For further clarity, we now included the word “inclusive” in both the main text and the methods: main text: “We therefore used CRISPR combined with homology-directed repair to delete the CM1 domain (amino acids 98-167, inclusive) from the endogenous cnn gene…”; Methods:“R98 to D167, inclusive”. Please do let us know if further information is required.

3) Is it important to combine spd-2 with all four mutants, grip75 grip128 grip163 and grip71? What about spd-2 grip71 cells and spd-2 grip75 grip128 grip163 cells? Should that not have the same effect?

This comment relates to Major point 1, as our main conclusion (that Cnn can recruit γ-TuSCs) is only possible when combining spd2 with all four mutants i.e. targetting all γ-TuRC specific proteins is the most likely way to deplete as many pre-formed γ-TuRCs as possible. Depleting only Spd-2 and Grip71 would leave fully assembled γ-TuRCs in the cytosol, as assembly does not require Grip71. Depleting Spd-2, Grip75, Grip128, and Grip163 would prevent cytosolic γ-TuRC assembly, but there is a possibility that Grip71 may still act as a link between γ-TuSCs and Cnn. It was therefore necessary to deplete Spd-2, Grip75, Grip128, and Grip163, and Grip71.

4) CM1-containing factors are the only known factors able to directly bind and activate gamma-TuRC. How do the authors envision activation of gamma-TuRC in the absence of Cnn?

This is a good question but remains unanswered. Phosphorylation of γ-TuRCs is the most obvious possibility. For example, Aurora A phosphorylates NEDD1 (homologue of Grip71) to promote microtubule nucleation (Pinyol et al., 2013). NME7 kinase has been shown to increase the activity of purified γ-TuRCs (Liu et al., 2014). Other γ-TuRC components are also phosphorylated, but the consequences on γ-TuRC activity are not known. Another possibility is that TOG proteins indirectly promote the closing of the γ-TuRCs while adding tubulin dimers onto γ-tubulin (Thawani et al., 2020).

5) Do the authors think that each identified pathway to microtubule nucleation (i.e. Spd-2/gamma-TuRC, Cnn/gamma-TuSC, Msps/mei38) as revealed by mutant genetic backgrounds contributes to a similar extent to overall nucleation capacity also in an unperturbed genetic background?

Another good question, but it is very difficult to answer. Our view is that when γ-TuRCs are present and active they will likely dominate microtubule nucleation, out-competing the ability of TOG domain proteins to stimulate microtubule nucleation independently of γ-TuRCs. Nevertheless, TOG proteins will likely help promote microtubule nucleation from γ-TuRCs when both are present, as has been previously shown in vitro (Thawani et al., 2018; King et al., 2020; Consolati et al., 2020) and in fission yeast (Flor-Parra et al., 2018). We also believe that both Spd-2 and Cnn γ-TuRC recruitment pathways will contribute simultaneously. Another question is whether Cnn recruits γ-TuRCs instead of γ-TuSCs when γ-TuRCs are present in the cytosol. We assume this will depend on Cnn’s affinity of γ-TuRCs versus γ-TuSCs and on the relative levels of γ-TuRCs and γ-TuSCs in the cytosol.

6) How does CM1 mediate binding to gamma-TuRC? Using recombinant Cnn fragments, the authors find that a Cnn triple mutant (R101Q, E102A and F115A) no longer binds gamma-tubulin, suggesting these residues together mediate binding to gamma-tubulin complexes. However, it is not tested to what extent R101, E102 and F115 individually contribute to gamma-tubulin binding. Does the binding mode in Drosophila resemble more the one in humans or in budding yeast? Also, was this done with extracts from Grip71, Grip75, Grip128RNAi, Grip163 embryos or normal embryos?

In future, we will test the relative contributions of R101, E102 and F115, but for this study we wanted only to show that the CM1 domain was necessary for Cnn binding (hence why we directly mutated all three residues). We apologise for not stating that the IPs were carried out using wild-type embryos extracts – we have now included this information in the main text and methods.

7) Figure 2C: Should the green channel not correspond to Spd-2?

Thank you for pointing out this mistake – now corrected.

8) I suggest to reconsider the color-coding of graphs. While the colored background of the dot plots in Figure 1 and 2 are a matter of taste, the coloring of graphs in Figure 4F-H is confusing. Here, genetic backgrounds of fly lines are colored in the same way as the microscopy channels in Figure 4A-E, but they do not belong together.

We have now modified the colour-coding of images/graphs in Figure 4A-E as suggested.

9) A tacc mutant allele is used in experiments, but is not further described. Please provide the necessary background information.

We thank the reviewer for pointing this out. We had also forgotten to include the msps alleles used. The information for msps and tacc are now included in the methods.

10) The authors assess spindle quality in Cnn, grip71, grip163 cells and show that spindle quality worsens with ectopic msps. For comparison it would be good to compare spindle quality side by side with a wild-type situation.

This data is now included in Figure S4A,B.

11) Introduction: "[...], however, as they depend upon each other for their proper localisation within the PCM and act redundantly." - Sentence is incomplete.

I think this was just to do with how we had phased the sentence (the position of “however” was confusing). We have now rephrased the sentence: “It is complicated, however, to interpret the individual role of these proteins in the recruitment of γ-tubulin complexes, as they depend upon each other for their proper localisation within the PCM and act redundantly”.

12) Introduction: "Cnn contains the highly conserved CM1 domain (Sawin et al., 2004), which binds directly to γ-tubulin complexes in yeast and humans (Brilot et al., 2021; Wieczorek et al., 2019)". - Choi et al 2010 should also be cited here.

This citation has been added.

13) Results: "Typically, interphase centrosomes have only ~5-20% of the γ-tubulin levels found at mitotic centrosomes, [...]". - Citation is needed

We now cite our Conduit et al., 2014 paper.

14) The authors should discuss that Msps was found to act non-redundantly with gamma-tubulin in interphase nucleation (Rogers, MBC, 2008), contrary to the conclusions in the current manuscript.

Thank you for pointing this out. We have now modified the relevant part of the discussion to read:

“TOG domain and TPX2 proteins have been shown to work together with γ-TuRCs (or microtubule seed templates) to promote microtubule nucleation (Thawani et al., 2018; Flor-Parra et al., 2018; Gunzelmann et al., 2018b; Consolati et al., 2020; King et al., 2020; Wieczorek et al., 2015). Consistent with this, co-depletion of γ-tubulin and the Drosophila TOG domain protein Msps did not delay non-centrosomal microtubule regrowth after cooling compared to single depletions in interphase S2 cells (Rogers et al., 2008). Nevertheless, several studies, mainly in vitro, have shown that TOG and TPX2 proteins can also function independently of γ-TuRCs to promote microtubule nucleation (Roostalu et al., 2015; Woodruff et al., 2017; Schatz et al., 2003; Slep and Vale, 2007; Ghosh et al., 2013; Thawani et al., 2018; King et al., 2020; Zheng et al., 2020; Tsuchiya and Goshima, 2021; Imasaki et al., 2022). Our data suggest that, unlike from non-centrosomal sites in interphase S2 cells, Msps can promote γ-TuRC-independent microtubule nucleation from centrosomes in mitotic larval brain cells. This difference may reflect the ability of centrosomes to concentrate Msps at a single location.”

**Referees cross-commenting**

This is a good paper in my opinion, they need to add some controls though, to determine the expected presence/absence of gTuSC/gTuRC in the different mutants. An important advance is the finding that gTuSC can function as nucleator in parallel to gTuRC, depending on the recruitment mechanism. Different recruitment mechanisms, nucleation templates, and regulatory strategies co-exist and provide complex regulation and robustness to nucleation/spindle assembly. We thank the Reviewer for their thorough and constructive review. We hope they will agree to allow publication without us having to perform the sucrose gradient experiments that, as discussed above, will be very difficult, if not impossible, to carry out.

Reviewer #1 (Significance (Required)):

This is a very well-executed study and the data is presented clearly. However, some findings would benefit from additional experiments to substantiate the main interpretations. If these points are addressed, the study would provide an important conceptual advance in the field, namely that animal cells may rely on two different gamma-tubulin complexes for nucleation at mitotic centrosomes, gamma-TuSC and gamma-TuRC, which differ not only in their composition of GCP proteins but also the mode of recruitment to the centrosome. The findings will be of interest to all cell biologists.

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

Summary This paper sets out to further our understanding of how two proteins, Cnn and Spd-2, independently recruit g-Tubulin ring complexes(g-TuRC) to mitotic centrosomes in Drosophila cells. It uses some robust classical genetics to generate mutants to reduce/remove GCP4/5/6, Dgrip71 and Cnn and Spd-2 from cells, monitoring the consequences using live imaging.

It begins by showing that Cnn can recruit g-Tubulin independently of the core g-TuRC components or Dgrip71, and that a mutant Cnn lacking the CM1 domain cannot, strongly suggesting that, similarly to other organisms, the CM1 domain is essential for this function.

It then demonstrates that Spd-2, in contrast, cannot localise g-Tubulin in the absence of the g-TuRC components or Dgrip71.

In the second half of the paper, then use this tool as a proxy for centrosomes that completely lack mitotic g-Tubulin recruitment, in order to explore spindle assembly in the absence of centrosomal g-Tubulin. The show that microtubules and spindle are still nucleated but do so with different dynamics. This section is particularly convincing, given the use of the live de/repolymerisation assays using the CherryTemp device.

Finally, the authors visualise spindle formation in the absence of centrosomal g-Tubulin, alongside a number of other MT associated proteins, including Msps.

Major Comments 1. The claims and conclusions relating to the first half of the paper are supported by the data, but they need to be caveated by a clear explanation of the alleles used. Some are well-characterised mutant lines but have they been previously shown to completely remove the associated protein products? For the RNAi lines, do the authors have evidence (via Western blots) that these remove the protein products? It is not necessary that they show Western blots for all the lines, and it does not invalidate the major conclusions that the fly line carrying mutations in cnn, grip71, grip163 completely fails to localise g-Tubulin to mitotic centrosomes. However, they need to help the reader understand much more clearly whether these lines are complete nulls and, consequently this may impact the strength of their interpretation of the relationship between Grip163 versus Grip75, discussed both at the end of the relevant section and in the Discussion.

We appreciate the reviewer’s concern and have now included a detailed description in the Methods section of the alleles we use and their known effect on protein levels (pasted below for convenience). We have also included western blots for cnn and spd2 mutants to show the absence of detectable protein in larval brains. Unfortunately, we could not provide western blots for the other mutants, as we don’t have working antibodies for these proteins (although for Grip71 we did make an antibody and did western blots that showed the absence of protein in grip71 mutants, but this antibody has now been commercialised and so the western blot is published on the CRB website: https://crbdiscovery.com/polyclonal-antibodies/anti-grip71-antibody/). Nevertheless, protein levels for the grip75, grip163, msps and tacc mutants have been shown previously (now cited in the new text). We have also modified the main text to allow the reader to better understand whether proteins are completely absent or strongly reduced. In response to the specific comment about interpreting the relationship between Grip163 and Grip75, as we mention in the new methods section, the Grip75 allele is a null mutant while the Grip163 mutant is a severe depletion; thus, the fact that the Grip163 mutant has a stronger effect on γ-tubulin recruitment is not due to a stronger depletion.

New text in methods: “For spd-2 mutants, we used the dspd-2Z35711 mutant allele, which carries an early stop codon resulting in a predicted 56aa protein. Homozygous dspd-2Z35711 mutant flies lack detectable Spd-2 protein on western blots and so the allele is therefore considered to be a null mutant (Giansanti et al., 2008). This allele no longer produces homozygous flies (which is common for mutant alleles kept as balanced stocks for many years), which combined dspd-2Z35711 with a deficiency that includes the entire spd-2 gene (dspd-2Df(3L)st-j7). On western blots, there was no detectable Spd-2 protein in extracts from dspd-2Z35711 / dspd-2Df(3L)st-j7 hemizygous mutant brains (Figure S4B). For cnn mutants, we combined the cnnf04547 and cnnHK21 mutant alleles. The cnnf04547 allele carries a piggyBac insertion in the middle of the cnn gene and is predicted to disrupt long Cnn isoforms, including the centrosomal isoform (Cnn-C or Cnn-PA) (Lucas and Raff, 2007). This mutation is considered to be a null mutant for the long Cnn isoforms (Lucas and Raff, 2007; Conduit et al., 2014). The cnnHK21 allele carries an early stop codon after Cnn-C’s Q78 (Vaizel-Ohayon and Schejter, 1999) and affects both long and short Cnn isoforms – it is considered to be a null mutant (Eisman et al., 2009; Chen et al., 2017a). On western blots, there was no detectable Cnn-C protein in cnnf04547 / cnnHK21 hemizygous mutant brains (Figure S4A). For Grip71, we used the grip71120 mutant allele, which is a result of an imprecise p-element excision event that led to the removal of the entire grip71 coding sequence except for the last 12bp; it is considered to be a null mutant (Reschen et al., 2012). We combined this with an allele carrying a deficiency that includes the entire grip71 gene (grip71Df(2L)Exel6041). On western blots, there is no detectable Grip71 protein in grip71120 / grip71df6041 hemizygous mutant brains (see blots on CRB website, which were performed by us). For Grip75GCP4, we used the grip75175 mutant allele, which carries an early stop codon after Q291. Homozygous grip75175 mutant flies lack detectable Grip75GCP4 protein on western blots and so the allele is therefore considered to be a null mutant (Schnorrer et al., 2002). We combined this with an allele carrying a deficiency that includes the entire grip75GCP4 gene (grip75Df(2L)Exel7048). In the absence of a working antibody, we have not confirmed the expected absence of Grip75GCP4 protein in grip75175 / grip75Df(2L)Exel7048 hemizygous mutant flies on western blots. For Grip128GCP5, we used the UAS-controlled grip128-RNAiV29074 RNAi line, which is part of the VDRC’s GD collection, and drove its expression using the Insc-Gal4 driver (BL8751), which is expressed in larval neuroblasts and their progeny. In the absence of a working antibody, we have not confirmed the absence or reduction of Grip128GCP5 protein on western blots. RNAi was used for grip128GCP5 as its position on the X chromosome made generating stocks with multiple alleles technically challenging. For Grip163GCP6, we used the grip163GE2708 mutant allele, which carries a p-element insertion between amino acids 822 and 823 (total protein length is 1351aa) and behaves as a null or strong hypomorph mutant (Vérollet et al., 2006). We combined this with an allele carrying a deficiency that includes the entire grip163GCP6 gene (grip163Df(3L)Exel6115). In the absence of a working antibody, we have not confirmed the absence or reduction of Grip163GCP6 protein in grip163GE2708 / grip163Df(3L)Exel6115 hemizygous mutant flies on western blots. For Msps, we used the mspsp and mspsMJ15 mutant alleles. The mspsp allele carries a p-element insertion within, or close to, the 5’ UTR of the msps gene and results in a strong reduction, but not elimination, of Msps protein on western blots (Cullen et al., 1999). The mspsMJ15 allele was generated by re-mobilisation of the p-element (the genetic consequence of which has not been defined) and also results in a strong reduction, but not elimination, of Msps protein on western blots (Cullen et al., 1999; Lee et al., 2001). For TACC, we used the taccstella allele which contain a p-element insertion of unknown localisation but that results in no detectable TACC protein on western blots (Barros et al., 2005). For Mei-38, we used the UAS-controlled mei-38-RNAiHMJ23752 RNAi line, which is part of the NIG’s TRiP Valium 20 collection, and drove its expression using the Insc-Gal4 driver (BL8751). In the absence of a working antibody, we have not confirmed the absence or reduction of Mei-38 protein on western blots. RNAi was used for mei-38 as its position on the X chromosome made generating stocks with multiple alleles technically challenging. Moreover, the only available mutant of mei-38 affects a neighbouring gene.”

I have an issue with the statistics in Figure 1 &2. I realise the t-tests in Figure 1 show the significant differences between g-Tubulin recruitment to centrosomes in interphase and mitosis, in order to demonstrate the difference between the Spd-2;Grip combination line in (B) and the Spd-2; CnnCM1 double mutant in (D). But in doing so, it draws attention to the fact that there is no similar t-test between mitotic g-Tubulin recruitment to centrosomes in WT, Spd-2 and the Spd-2;Grip combination lines. This lack of stats between conditions is further confused by the language used in the text: In the Figure legend, the authors claim mitotic centrosomal g-Tubulin levels between are WT, Spd-2 and the Spd-2;Grip combination lines "similar", and in the text they say: the spd-2 Grip combination line had g-Tubulin "similar to the levels found at spd-2 mutants alone". But then they give numbers - an average of 77% of wild type for spd2 and 66% of wild type for the spd-2 Grip combination. I'm sure if they did a t-test they would find a significant difference between these conditions. This doesn't invalidate the thrust of what they're claiming, but they do need to be consistent in language, analysis and interpretation.

We agree that we should have performed a statistical comparison between the γ-tubulin levels for “WT mitosis” vs “spd2 mitosis” and for “spd-2 mitosis” vs “spd2,grip71,grip75,grip128,grip163 mitosis” (Figure 1B). We have now done this and found statistically significant differences in both cases. We have included the new p-values in the figure and modified the main text to read: “In fact, the centrosomes in these spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant cells had ~66% of the γ-tubulin levels found at wild-type centrosomes, only slightly lower than the levels found at spd-2 mutants alone (Figure 1A,B).”; and we have modified the legend to read: “A one-way ANOVA with a Sidak’s multiple comparisons test was used to make the comparisons indicated by p values in the graph. Note that there is only a small reduction in mitotic centrosomal γ-tubulin levels in spd-2 mutants and in spd-2, grip71,grip75GCP4, grip128GCP5-RNAi,grip163GCP6 mutants, showing that Cnn can still efficiently recruit γ-tubulin complexes to mitotic centrosomes when only γ-TuSCs are present.” Note that due to performing comparisons multiple times with the same data sets, it was necessary to use a one-way ANOVA with a Sidak’s multiple comparisons test (rather than paired t-tests).

For Figure 1D, we did not compare WT mitosis vs cnn∆CM1,spd-2 mitosis, as the point here was to test whether there was an increase from interphase to mitosis in cnn∆CM1,spd-2 mutants and we wanted to maintain the statistical power of using a paired t-test (one is more likely to detect differences with a paired t-test than with a multiple comparisons ANOVA, making the conclusion that there is no difference between interphase and mitotic cnn∆CM1,spd-2 centrosomes even more solid).

Similarly, in Figure 2, it would be better to assess any statistically significant difference between mitotic accumulation of g-Tubulin between fly lines, rather than accumulation between interphase and mitosis (which is pretty clear cut). This would help to clarify whether differences between loss of grip subunits are merely additive or synergistic. Again, this doesn't invalidate the overall result that concomitant loss of cnn, grip71 and grip163 completely abolishes mitotic centrosomal accumulation of g-Tubulin, but it is a more complete analysis of the extant data.

As for Figure 2, we respectfully disagree that we should make comparisons between genotypes instead of, or in addition to, making comparisons between interphase and mitotic centrosomes within the same genotype. This is because we will lose statistical power by performing a multiple comparisons test. Indeed, if we were to compare both within and between selected genotypes (14 comparisons in total), then we lose the statistically significant differences between interphase and mitotic centrosomes in cnn,grip75,grip163 (p=0.04) and cnn,grip71,grip75 (p=0.08) genotypes, when there clearly appears to be a difference (as stated by the Reviewer). Given that the point of this experiment is to elucidate which proteins are required to allow maturation from interphase to mitosis, rather than which combination of mutations has the stronger effect, we feel that maintaining the paired t-test analysis is more appropriate.

One OPTIONAL experiment that would significantly improve the study would be similar CherryTemp live imaging of the cells lacking both centrosomal g-Tubulin and Msps. Currently the manuscript finishes with a fixed analysis of MT de/repolymerisation in these cells, which provides evidence that Msps has a role in MT nucleation in the absence of centrosomal g-Tubulin-nucleated MTs, but very little else can be concluded.

We would love to do this experiment but the genetics are complicated. We would have to generate stocks containing a cnn,grip71,GFP-PACT triple allele chromosome II and a grip163,msps,Jupiter-mCherry triple allele chromosome III. While live data would provide interesting insights into the dynamics of microtubules nucleated in the absence of γ-TuRCs and reduction of Msps, our fixed analysis is at least sufficient to implicate Msps in γ-TuRC-independent microtubule organisation.

1. There is, perhaps surprisingly, no mention of Augmin in the paper. Augmin has been shown to recruit g-TuRC to pre-existing MTs, through the grip71 subunit (Chen et al., 2017). So, presumably, in cnn, grip71, grip163, g-Tubulin cannot be recruited to pre-existing MTs either? This could add impact to the results - in that it implies the MT nucleation seen in the absence of cnn, grip71 and grip163 actually reflects, not just loss of centrosome function, but also loss of Augmin function. Mentioning this in the discussion could help increase the impact of the paper.

We apologise for this oversight. Indeed, it is perfectly possible that Grip71/Augmin-mediated amplification of microtubules during microtubule re-growth from centrosomes could influence the difference in recovery rates between control and mutant centrosomes. We have now modified the results section to read:

“Our data suggest that microtubules are more resistant to cold-induced depolymerisation when they have been nucleated independently of γ-TuRCs, but that microtubules are nucleated more efficiently when γ-TuRCs are present. However, it must be considered that, due to the loss of Cnn from centrosomes in the cnn,grip71,grip163 mutant cells, general PCM levels are reduced, likely reducing the levels of any protein involved in γ-TuRC-independent microtubule nucleation. Moreover, Grip71 is necessary for γ-TuRC recruitment to microtubules, most likely via the Augmin complex (Reschen et al., 2012; Chen et al., 2017b; Dobbelaere et al., 2008; Vérollet et al., 2006), enabling microtubules to be nucleated from the sides of pre-existing microtubules. Thus, the potential for Augmin-mediated amplification of centrosome-nucleated microtubules in control cells may also contribute to the increased microtubule recovery speed in control cells. Importantly, however, both of these caveats make it even clearer that microtubules can be nucleated independently of γ-TuRCs from mitotic centrosomes in Drosophila.”

Minor comments 1. The cnn, grip71, grip163 mutant image in Fig3 B after 40 min cooling appears to have 4 centrioles. Is this a cell that exited and re-entered mitosis?

Cnn mutant cells often have centrosome segregation problems, resulting in cells with variable numbers of centrioles (Conduit et al., 2010b, Current Biology). We have now mentioned this in the legends for Figure 3, Figure 4, and Figure S4.

Methods should contain more detail on the de/repolymerisation live imaging analysis (including the numbers of cells contributing to the analysis) and techniques such exponential curve fitting.

We have now included this information in the methods and updated this information in the figure legend (to include cell numbers, not just centrosome numbers, and to indicate that GraphPad Prism was used to generate the models.

P5 para 2 - "GPC4/5/4/6" should read "GCP4/5/6"

We actually use the GCP4/5/4/6 nomenclature throughout as it represents the 2 copies of GCP4 to one copy of GCP5 and GCP6 in the complex, as well as the order of these molecules.

Fig legend 1 - "error bar" should read "scale bar"

Thanks, now corrected

Reviewer #2 (Significance (Required)):

The experimental approach (genetics and cell biology) taken in this manuscript is very appropriate and the experiments are of high quality. It uses the strengths of Drosophila to cleverly engineer flies to pull apart the relationship between two different ways to recruit the main MT nucleator, g-Tubulin, to mitotic centrosomes. This is an important advance for the specific research field of centrosome biology.

By generating a fly that completely fails to localise g-Tubulin to mitotic centrosomes, the paper is able to explore whether MTs and the mitotic spindle can form in its absence. Again, there is very high quality imaging and image analysis, using a commercially available (but very cool) fast heating/cooling apparatus - the CherryTemp to explore the dynamics of MT generation. The limitation to this approach, though, is that g-Tubulin itself is still present and presumably able to nucleate MTs in the cytosol or elsewhere, albeit inefficiently. As such, it adds to a body of centrosomal and cell division research, rather than adding a highly significant conceptual advance.

Similarly, the finding that Msps is involved in nucleating MTs in the absence of centrosomal g-Tubulin, via fixed analysis, supports other work, rather than moving the field forwards.

Overall, assuming the caveats mentioned in the major comments are dealt with, I see this as a robust and very well carried out piece of research, that will be of interest to those investigating the broad field of cell division

My field of expertise is Drosophila cell division

We thank the Reviewer for their thorough and constructive review. We hope that the reviewer may agree with us and the other Reviewers that revealing the complexity of γ-TuRC recruitment and microtubule nucleation at centrosomes, particularly the finding that different types of γ-tubulin complexes are recruited to centrosomes by different tethering proteins, provides an important conceptual advance.

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

Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments below, particularly points 1 and 2, could be addressed.

1. The experiment in Fig. 2B examines what is required for Spd-2 to recruit g-tubulin to mitotic centrosomes that lack Cnn. This panel should include a cnn mutant-only control, for which the readers are currently referred to an older paper from 2014. Without repeating this control in parallel to one of the conditions in this panel, it is impossible to say whether the addition of the grip71 mutation has any effect on g-tubulin levels.

This is a good point. We will perform a cnn vs cnn,grip71 experiment and include this data in the new version of the paper. This will take a couple of months, as this will involve growing up fly lines, performing the necessary crosses, microscopy, data analysis, and manuscript updating.

1. The experiment in Fig. 2B is in the background of a Cnn loss-of-function mutation in which centrosomal Spd-2 is at just under 40% of its levels in brains with Cnn (according to Fig. 2D). So the Spd-2 doing the recruiting-is the non-Cnn-dependent population. The authors should also do one experiment in the background of their Cnn-CM1delete mutant or their Cnn CM1 g-tubulin recruitment mutant, because these backgrounds would be expected to have normal amounts of Cnn matrix and normal levels of Spd-2. Comparing the amount of g-tubulin recruitment in a cnn loss-of-function mutant to that in a cnn-CM1delete mutant would reveal whether the Cnn-bound Spd-2 can contribute to g-tubulin recruitment in the same way that the Cnn-independent Spd-2 can. These two populations could easily differ in their ability to recruit g-tubulin. Also, is it clear that these two pathways can act in parallel (i.e. that assembly of the Cnn matrix around the centriole does not mask the ability of Cnn-independent Spd-2 to recruit g-tubulin)? Thus, there are three possibilities- all interesting- for the outcome of this experiment. The Cnn-CM1delete mutant/Cnn-CM1 g-tubulin recruitment mutants could: (1) recruit less g-tubulin than the cnn loss-of function mutant (if Cnn matrix assembly inhibits the Cnn-independent Spd-2 pathway), (2) recruit the same amount of g-tubulin as the cnn loss-of-function mutant (if the Cnn matrix does not inhibit the Cnn-independent Spd-2 pathway but Cnn-dependent Spd-2 does not itself recruit g-tubulin), or (3) recruit more g-tubulin than the cnn loss-of-function mutant (if both the Cnn-dependent and Cnn-independent Spd-2 can recruit g-tubulin).

These are very interesting points that we have not considered before. As the reviewer suggests, we will perform an analysis of γ-tubulin levels at centrosomes in cnnnull vs cnn∆CM1 to test the ability of Cnn-dependent and Cnn-independent populations of Spd-2 to recruit γ-tubulin. This should take ~2 months.

1. The paper needs a summary model figure that the field can understand. The current model in Fig. 2E does not suffice in this regard. It would be nice to have this model appear at the end of the paper to outline the 3 pathways for centrosomal microtubule nucleation outlined by the work. Maybe have an arc for the centrosome at the bottom of the figure and show arrows from the gTuSC to the Cnn CM1 domain from the gTuRC to the Cnn CM1 domain and the gTuRC to Spd-2 or something like this. How you draw this could be impacted by the experiment outlined above in point 2. Also, there would be a g-tubulin-independent pathway in the figure. Not everyone reads papers carefully, and you want people to be able to get the takeaway message at a glance.

We have now completely modified the Figure and moved it to the end of the paper (new Figure 5). We thank the Reviewer for this suggestion as it really does provide a clearer message for the reader.

1. The authors show that this pathway is modulated by loss of Minispindles (Msps)-but as this is a critical microtubule assembly factor, it seems likely that Msps loss might modulate all of the pathways. From the data in Figure 4, my main takeaway would be that Msps is not the central player in the g-tubulin independent nucleation pathway. It might make the paper more impactful to end the story after Fig. 4, move the current Fig. 5 to the supplement and add a nice model figure at the end.

We agree that Msps may play a role beyond microtubule nucleation, including plus end growth, and that this may also influence the efficiency of spindle formation in cnn,grip71,grip163,msps mutants. Nevertheless, our microtubule regrowth data in original Figure 5A clearly show that Msps is a key player in the g-tubulin independent nucleation pathway at centrosomes. Perhaps the Reviewer missed this point as the data was in Figure 5 and not Figure 4. Moreover, the original Figure 5E shows that the effect of depleting Msps in addition to cnn, grip71 and grip163 is specific to cells containing centrosomes i.e. if Msps played a significant role in microtubule regulation beyond its role at centrosomes, then one would expect spindle formation to be worse when comparing mutant cells that lack centrosomes. Nevertheless, we now realise it would be better to include the microtubule regrowth from centrosomes data for cnn,grip71,grip163 vs cnn,grip71,grip163,msps in Figure 4, and move the spindle assembly data from original Figure 5C-E to a new supplementary Figure (Figure S4). We then end the paper on a model figure in new Figure 5.

Minor comments: 5. In Fig. 1E the sequence labels are confusing. Please label each sequence on the left with the residue numbers in the corresponding endogenous protein that are shown in the alignment.

You are absolutely right, I’m not sure why our labelling was like that. Now corrected.

In Fig. 1F, please label with location of molecular weight markers

Now added.

Reviewer #3 (Significance (Required)):

Repeating my text from above. Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments, particularly points 1 and 2, could be addressed.

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Referee #3

Evidence, reproducibility and clarity

Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments below, particularly points 1 and 2, could be addressed.

1. The experiment in Fig. 2B examines what is required for Spd-2 to recruit g-tubulin to mitotic centrosomes that lack Cnn. This panel should include a cnn mutant-only control, for which the readers are currently referred to an older paper from 2014. Without repeating this control in parallel to one of the conditions in this panel, it is impossible to say whether the addition of the grip71 mutation has any effect on g-tubulin levels.
2. The experiment in Fig. 2B is in the background of a Cnn loss-of-function mutation in which centrosomal Spd-2 is at just under 40% of its levels in brains with Cnn (according to Fig. 2D). So the Spd-2 doing the recruiting-is the non-Cnn-dependent population. The authors should also do one experiment in the background of their Cnn-CM1delete mutant or their Cnn CM1 g-tubulin recruitment mutant, because these backgrounds would be expected to have normal amounts of Cnn matrix and normal levels of Spd-2. Comparing the amount of g-tubulin recruitment in a cnn loss-of-function mutant to that in a cnn-CM1delete mutant would reveal whether the Cnn-bound Spd-2 can contribute to g-tubulin recruitment in the same way that the Cnn-independent Spd-2 can. These two populations could easily differ in their ability to recruit g-tubulin. Also, is it clear that these two pathways can act in parallel (i.e. that assembly of the Cnn matrix around the centriole does not mask the ability of Cnn-independent Spd-2 to recruit g-tubulin)? Thus, there are three possibilities- all interesting- for the outcome of this experiment. The Cnn-CM1delete mutant/Cnn-CM1 g-tubulin recruitment mutants could: (1) recruit less g-tubulin than the cnn loss-of function mutant (if Cnn matrix assembly inhibits the Cnn-independent Spd-2 pathway), (2) recruit the same amount of g-tubulin as the cnn loss-of-function mutant (if the Cnn matrix does not inhibit the Cnn-independent Spd-2 pathway but Cnn-dependent Spd-2 does not itself recruit g-tubulin), or (3) recruit more g-tubulin than the cnn loss-of-function mutant (if both the Cnn-dependent and Cnn-independent Spd-2 can recruit g-tubulin).
3. The paper needs a summary model figure that the field can understand. The current model in Fig. 2E does not suffice in this regard. It would be nice to have this model appear at the end of the paper to outline the 3 pathways for centrosomal microtubule nucleation outlined by the work. Maybe have an arc for the centrosome at the bottom of the figure and show arrows from the gTuSC to the Cnn CM1 domain from the gTuRC to the Cnn CM1 domain and the gTuRC to Spd-2 or something like this. How you draw this could be impacted by the experiment outlined above in point 2. Also, there would be a g-tubulin-independent pathway in the figure. Not everyone reads papers carefully, and you want people to be able to get the takeaway message at a glance.
4. The authors show that this pathway is modulated by loss of Minispindles (Msps)-but as this is a critical microtubule assembly factor, it seems likely that Msps loss might modulate all of the pathways. From the data in Figure 4, my main takeaway would be that Msps is not the central player in the g-tubulin independent nucleation pathway. It might make the paper more impactful to end the story after Fig. 4, move the current Fig. 5 to the supplement and add a nice model figure at the end.

Minor comments:

1. In Fig. 1E the sequence labels are confusing. Please label each sequence on the left with the residue numbers in the corresponding endogenous protein that are shown in the alignment.
2. In Fig. 1F, please label with location of molecular weight markers

Significance

Repeating my text from above. Centrosomes are complex and it has been appreciated for some time that they likely nucleate microtubules by more than one mechanism. However, what these mechanisms exactly are, and which are the most significant has not been clear. A major contributor to centrosomal microtubule nucleation the tubulin isoform gamma-tubulin (g-tubulin), which is present in two complexes, a smaller gTuSC that contains gamma tubulin along with GCP2 and 3 and a larger g-tubulin ring complex (gTuRC) whose assembly additionally requires GCP4/5/6. A second high-level question has been whether the centrosome has any g-tubulin-independent microtubule nucleation mechanisms. In this manuscript, the authors use a collection of mutants and RNAi conditions in the Drosophila brain to generate a picture of centrosomal microtubule nucleation pathways. They show that there are two g-tubulin-dependent and a third g-tubulin-independent microtubule nucleation pathways. They show that the first g-tubulin-dependent pathway depends on the CM1 domain of the centrosomal PCM matrix protein, Centrosomin (Cnn) and on the gTuSC components GCP2/3, but not on the components specifically required for gTuRC assembly. The second g-tubulin-dependent pathway depends on Spd-2 (and not Cnn) and requires the gTuRC-specific components and NEDD1/Grip71. By inhibiting both of these pathways, the authors also show that there is a robust g-tubulin-independent microtubule nucleation pathway. Overall, has the potential to be an impactful contribution from a conceptual point-of-view. I would be excited to recommend publication if the major comments, particularly points 1 and 2, could be addressed.

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Referee #2

Evidence, reproducibility and clarity

Summary

This paper sets out to further our understanding of how two proteins, Cnn and Spd-2, independently recruit g-Tubulin ring complexes(g-TuRC) to mitotic centrosomes in Drosophila cells. It uses some robust classical genetics to generate mutants to reduce/remove GCP4/5/6, Dgrip71 and Cnn and Spd-2 from cells, monitoring the consequences using live imaging.

It begins by showing that Cnn can recruit g-Tubulin independently of the core g-TuRC components or Dgrip71, and that a mutant Cnn lacking the CM1 domain cannot, strongly suggesting that, similarly to other organisms, the CM1 domain is essential for this function.

It then demonstrates that Spd-2, in contrast, cannot localise g-Tubulin in the absence of the g-TuRC components or Dgrip71.

In the second half of the paper, then use this tool as a proxy for centrosomes that completely lack mitotic g-Tubulin recruitment, in order to explore spindle assembly in the absence of centrosomal g-Tubulin. The show that microtubules and spindle are still nucleated but do so with different dynamics. This section is particularly convincing, given the use of the live de/repolymerisation assays using the CherryTemp device.

Finally, the authors visualise spindle formation in the absence of centrosomal g-Tubulin, alongside a number of other MT associated proteins, including Msps.

Major Comments

1. The claims and conclusions relating to the first half of the paper are supported by the data, but they need to be caveated by a clear explanation of the alleles used. Some are well-characterised mutant lines but have they been previously shown to completely remove the associated protein products? For the RNAi lines, do the authors have evidence (via Western blots) that these remove the protein products? It is not necessary that they show Western blots for all the lines, and it does not invalidate the major conclusions that the fly line carrying mutations in cnn, grip71, grip163 completely fails to localise g-Tubulin to mitotic centrosomes. However, they need to help the reader understand much more clearly whether these lines are complete nulls and, consequently this may impact the strength of their interpretation of the relationship between Grip163 versus Grip75, discussed both at the end of the relevant section and in the Discussion.
2. I have an issue with the statistics in Figure 1 &2. I realise the t-tests in Figure 1 show the significant differences between g-Tubulin recruitment to centrosomes in interphase and mitosis, in order to demonstrate the difference between the Spd-2;Grip combination line in (B) and the Spd-2; CnnCM1 double mutant in (D). But in doing so, it draws attention to the fact that there is no similar t-test between mitotic g-Tubulin recruitment to centrosomes in WT, Spd-2 and the Spd-2;Grip combination lines. This lack of stats between conditions is further confused by the language used in the text: In the Figure legend, the authors claim mitotic centrosomal g-Tubulin levels between are WT, Spd-2 and the Spd-2;Grip combination lines "similar", and in the text they say: the spd-2 Grip combination line had g-Tubulin "similar to the levels found at spd-2 mutants alone". But then they give numbers - an average of 77% of wild type for spd2 and 66% of wild type for the spd-2 Grip combination. I'm sure if they did a t-test they would find a significant difference between these conditions. This doesn't invalidate the thrust of what they're claiming, but they do need to be consistent in language, analysis and interpretation. Similarly, in Figure 2, it would be better to assess any statistically significant difference between mitotic accumulation of g-Tubulin between fly lines, rather than accumulation between interphase and mitosis (which is pretty clear cut). This would help to clarify whether differences between loss of grip subunits are merely additive or synergistic. Again, this doesn't invalidate the overall result that concomitant loss of cnn, grip71 and grip163 completely abolishes mitotic centrosomal accumulation of g-Tubulin, but it is a more complete analysis of the extant data.
3. One OPTIONAL experiment that would significantly improve the study would be similar CherryTemp live imaging of the cells lacking both centrosomal g-Tubulin and Msps. Currently the manuscript finishes with a fixed analysis of MT de/repolymerisation in these cells, which provides evidence that Msps has a role in MT nucleation in the absence of centrosomal g-Tubulin-nucleated MTs, but very little else can be concluded.
4. There is, perhaps surprisingly, no mention of Augmin in the paper. Augmin has been shown to recruit g-TuRC to pre-existing MTs, through the grip71 subunit (Chen et al., 2017). So, presumably, in cnn, grip71, grip163, g-Tubulin cannot be recruited to pre-existing MTs either? This could add impact to the results - in that it implies the MT nucleation seen in the absence of cnn, grip71 and grip163 actually reflects, not just loss of centrosome function, but also loss of Augmin function. Mentioning this in the discussion could help increase the impact of the paper.

Minor comments

1. The cnn, grip71, grip163 mutant image in Fig3 B after 40 min cooling appears to have 4 centrioles. Is this a cell that exited and re-entered mitosis?
2. Methods should contain more detail on the de/repolymerisation live imaging analysis (including the numbers of cells contributing to the analysis) and techniques such exponential curve fitting.
3. P5 para 2 - "GPC4/5/4/6" should read "GCP4/5/6"
4. Fig legend 1 - "error bar" should read "scale bar"

Significance

The experimental approach (genetics and cell biology) taken in this manuscript is very appropriate and the experiments are of high quality. It uses the strengths of Drosophila to cleverly engineer flies to pull apart the relationship between two different ways to recruit the main MT nucleator, g-Tubulin, to mitotic centrosomes. This is an important advance for the specific research field of centrosome biology.

By generating a fly that completely fails to localise g-Tubulin to mitotic centrosomes, the paper is able to explore whether MTs and the mitotic spindle can form in its absence. Again, there is very high quality imaging and image analysis, using a commercially available (but very cool) fast heating/cooling apparatus - the CherryTemp to explore the dynamics of MT generation. The limitation to this approach, though, is that g-Tubulin itself is still present and presumably able to nucleate MTs in the cytosol or elsewhere, albeit inefficiently. As such, it adds to a body of centrosomal and cell division research, rather than adding a highly significant conceptual advance.

Similarly, the finding that Msps is involved in nucleating MTs in the absence of centrosomal g-Tubulin, via fixed analysis, supports other work, rather than moving the field forwards.

Overall, assuming the caveats mentioned in the major comments are dealt with, I see this as a robust and very well carried out piece of research, that will be of interest to those investigating the broad field of cell division

My field of expertise is Drosophila cell division

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Referee #1

Evidence, reproducibility and clarity

In their study, Zhu and colleagues study how the centrosome proteins Spd-2 and Cnn in Drosophila recruit gamma-tubulin complexes to centrosomes, which is an important step in mitotic spindle formation. The authors make use of mutant flies and RNAi and find that the two factors Spd-2 and Cnn together are responsible for mitotic centrosomal accumulation of gamma-tubulin. By inactivating Spd-2 or Cnn separately, the authors show that Cnn appears to recruit the large share of mitotic gamma-tubulin pool by its CM1-domain. Interestingly, this involves only gamma-TuSCs (subcomplexes of gamma-TuRC) and not gamma-TuRCs. A smaller pool is recruited by Spd-2, and this pool depends on gamma-tubulin complex proteins that are only present in pre-assembled, complete gamma-TuRCs. This suggests that Drosophila makes microtubule nucleation templates in two ways. First, as in yeast, by direct recruitment of gamma-TuSCs to mitotic centrosomes, where additionally oligomerization needs to happen. And second, by recruitment and activation of preassembled gamma-TuRCs. Inactivation of both Cnn- and Spd-2 pathways abolishes mitosis-specific gamma-tubulin recruitment, resulting in low, but not complete loss of gamma-tubulin at centrosomes. The authors show that these low-gamma-tubulin centrosomes are still able to organize microtubules, but these microtubules have different dynamicity. Inspired by existing literature in flies and other model organisms, the authors identify Msps/Xmap215 as an important nucleation factor in this scenario.

Major points:

1. The authors use fly embryos with mutant Grip71, Grip75 and Grip163 alleles, which are central to the study. Most conclusions are based on the assumption that some mutants contain only gamma-TuSC, whereas wildtype cells contain a mix of gamma-TuSC and gamma-TuRC. It would be important to show sucrose gradient analyses of extracts to confirm the expected presence/absence of gamma-TuSC/gamma-TuRC.
2. Given the advantage of the CnnΔCM1 separation of function mutant, I do not understand why it is not used throughout the study. Instead, full Cnn loss is used, which results in strongly reduced Spd-2 levels (Figure 2C,D). Are the observed differences between wild-type and mutants in Figure 2-5 dependent on defective PCM or do they also occur in a CnnΔCM1 background?
3. Statistical tests should support the conclusions in the text. If the authors claim differences between different genetic backgrounds (e.g. that spd2-mutants only have ~77% of gamma-tubulin at mitotic centrosomes compared to wild-type), statistical tests must compare mutant mitosis vs. wild-type mitosis.
4. While Cnn, grip71, grip163 mutants do not accumulate gamma-tubulin at centrosomes in mitosis, they still have low levels of centrosomal gamma-tubulin. It is therefore misleading to refer to "gamma-tubulin negative centrosomes".

Minor points:

1. The abstract states that gamma-TuRC is a catalyst of microtubule nucleation. By definition, a catalyst takes part in a reaction but is not part of the final product. Although our knowledge of the nucleation mechanism is still incomplete, mechanistic studies suggest a non-catalytical mechanism since gamma-TuRC was found to stay attached to the microtubule end after nucleation (Consolati et al. 2020, Wieczorek et al. 2020).
2. CnnΔCM1 flies: genotyping data should be provided besides describing gRNAs.
3. Is it important to combine spd-2 with all four mutants, grip75 grip128 grip163 and grip71? What about spd-2 grip71 cells and spd-2 grip75 grip128 grip163 cells? Should that not have the same effect?
4. CM1-containing factors are the only known factors able to directly bind and activate gamma-TuRC. How do the authors envision activation of gamma-TuRC in the absence of Cnn?
5. Do the authors think that each identified pathway to microtubule nucleation (i.e. Spd-2/gamma-TuRC, Cnn/gamma-TuSC, Msps/mei38) as revealed by mutant genetic backgrounds contributes to a similar extent to overall nucleation capacity also in an unperturbed genetic background?
6. How does CM1 mediate binding to gamma-TuRC? Using recombinant Cnn fragments, the authors find that a Cnn triple mutant (R101Q, E102A and F115A) no longer binds gamma-tubulin, suggesting these residues together mediate binding to gamma-tubulin complexes. However, it is not tested to what extent R101, E102 and F115 individually contribute to gamma-tubulin binding. Does the binding mode in Drosophila resemble more the one in humans or in budding yeast? Also, was this done with extracts from Grip71, Grip75, Grip128RNAi, Grip163 embryos or normal embryos?
7. Figure 2C: Should the green channel not correspond to Spd-2?
8. I suggest to reconsider the color-coding of graphs. While the colored background of the dot plots in Figure 1 and 2 are a matter of taste, the coloring of graphs in Figure 4F-H is confusing. Here, genetic backgrounds of fly lines are colored in the same way as the microscopy channels in Figure 4A-E, but they do not belong together.
9. A tacc mutant allele is used in experiments, but is not further described. Please provide the necessary background information.
10. The authors assess spindle quality in Cnn, grip71, grip163 cells and show that spindle quality worsens with ectopic msps. For comparison it would be good to compare spindle quality side by side with a wild-type situation.
11. Introduction: "[...], however, as they depend upon each other for their proper localisation within the PCM and act redundantly." - Sentence is incomplete.
12. Introduction: "Cnn contains the highly conserved CM1 domain (Sawin et al., 2004), which binds directly to γ-tubulin complexes in yeast and humans (Brilot et al., 2021; Wieczorek et al., 2019)". - Choi et al 2010 should also be cited here.
13. Results: "Typically, interphase centrosomes have only ~5-20% of the γ-tubulin levels found at mitotic centrosomes, [...]". - Citation is needed
14. The authors should discuss that Msps was found to act non-redundantly with gamma-tubulin in interphase nucleation (Rogers, MBC, 2008), contrary to the conclusions in the current manuscript.

Referees cross-commenting

This is a good paper in my opinion, they need to add some controls though, to determine the expected presence/absence of gTuSC/gTuRC in the different mutants. An important advance is the finding that gTuSC can function as nucleator in parallel to gTuRC, depending on the recruitment mechanism. Different recruitment mechanisms, nucleation templates, and regulatory strategies co-exist and provide complex regulation and robustness to nucleation/spindle assembly.

Significance

This is a very well-executed study and the data is presented clearly. However, some findings would benefit from additional experiments to substantiate the main interpretations. If these points are addressed, the study would provide an important conceptual advance in the field, namely that animal cells may rely on two different gamma-tubulin complexes for nucleation at mitotic centrosomes, gamma-TuSC and gamma-TuRC, which differ not only in their composition of GCP proteins but also the mode of recruitment to the centrosome. The findings will be of interest to all cell biologists.

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Reply to the reviewers

Manuscript number: RC-2022-01573

Corresponding author(s): Helder Maiato and Niels Galjart

1. General Statements

The murine microtubule (MT) plus-end tracking protein CLASP1 has been extensively examined in cultured cells, revealing an important function for this protein in mitosis and the regulation of MT dynamics. Here we describe a major in vivo phenotype of Clasp1 knockout (KO) mice: we find that these mice die at birth due to respiratory problems. In the first version of our manuscript we tried to link this in vivo phenotype of the KO mice to CLASP1’s major roles in cultured cells, including mitosis, and we therefore included multiple results, obtained in cultured cells and in different organs.

We thank the reviewers for their thoughtful and constructive criticisms and for their judgment that our study is - in principle - worthy of publication. Based on suggestions by reviewers #2 and #3 we have decided to focus the revised manuscript on the lung phenotype of the Clasp1 KO mice, and on a possible cause for this phenotype. We believe that our new analysis, which was partly driven by the remarks of the reviewers, is revealing a mechanism for why the mice die at birth. This mechanism suggests a role for CLASP1 in controlling epithelial and endothelial cell differentiation in the neonatal lung, and in particular protein secretion in AT2 alveolar cells.

2. Description of the planned revisions

General remarks

We believe our new RNA-Seq analysis (explained in detail below, in point 3 “Description of incorporated revisions”) strongly suggests that four essential lung cell types (i.e. AT1 and AT2 cells, endothelial cells and immune cells) fail to properly differentiate in Clasp1 KO embryos. In particular AT2 cell differentiation and functioning are hampered in the KO mice.

Brief summary of planned experiments and table of old and new Figures

To support our new findings we will stain sections of wild type and KO lung with a selected set of antibodies and other reagents. To help the reviewers we have made a table with original Figures and Figures for the revision.

Figure Number

Original Figure

Fate of original

Revision Figure

1

Targeted inactivation of the Clasp1 allele

Remains

Targeted inactivation of the Clasp1 allele

2

Clasp1 KO mice show reduced rib-cage and delayed ossification

Minor revision

Clasp1 KO mice show reduced rib-cage and delayed ossification (Statistics will be added)

3

Innervation of the diaphragm is affected in Clasp1 KO mice from E14.5-E18.5

Moved to Supp

Newborn Clasp1 KO lungs show a drastic reduction in air inflation

4

Neurite outgrowth, branching capacity and microtubule dynamics are altered in Clasp1 KO neurons

Removed

Histological and immunological examination of the Clasp1 KO lungs demonstrating decreased air space

5

Histological and immunological examination of the Clasp1 KO lungs demonstrating decreased air space

Moved Up

(4)

Histo-morphological analysis of the developing lung throughout embryonic development (E14.5-PN1)

6

Transcriptome analysis of wild type and Clasp1 KO lungs

Major revision

Transcriptome analysis of wild type and Clasp1 knockout lungs reveals differentiation defects in four major lung cell types (New data added, old data moved to Supp)

7

Loss of Clasp1 alters the ratio of alveolar type I and type II cells in the lungs

Major revision

Cellular analysis of Clasp1 knockout lungs (New data will be added)

8

-

-

Role of Clasp1 in AT2 function (New data will be added)

S1

Incidental cell division defects in mouse embryonic fibroblasts derived from Clasp1 knockout mice

Removed

Innervation of the diaphragm is affected in Clasp1 knockout mice from E14.5-E18.5

S2

Ultra-structural analysis of diaphragms

Remains

Ultra-structural analysis of diaphragms

S3

Newborn Clasp1 knockout lungs show a drastic reduction in air inflation

Moved to Main (3)

Cellular analysis of late stage gestation mouse lungs

S4

Histo-morphological analysis of the developing lung throughout embryonic development (E14.5-PN1)

Moved to Main (5)

Exogenous administration of glucocorticoids promotes lung maturation and partially rescues postnatal lethality

S5

Cellular analysis of late stage gestation mouse lungs

Moved Up

(S3)

Analysis of signature genes and cell type signatures of the mouse and human lung

S6

Exogenous administration of glucocorticoids promotes lung maturation and partially rescues postnatal lethality

Moved Up (S4)

Transcriptome analysis of wild type, Clasp1, and Mll3 knockout E18.5 lungs

Below we react to specific comments of the reviewers, describing in more detail which experiments will be carried out and why we will do these experiments.

Specific remarks to the comments of the reviewers

Reviewer #1.

Comment:

p.17: Aqp5 expression was decreased in mutant lungs as shown by RNA-seq data and RT-qPCR. However, immunolabelling with T1a does not show a decrease in the number of Type I pneumocytes (Fig. 7D). According to the data presented, it is difficult to conclude that CLASP1 is involved in Type I pneumocyte differentiation.

A cell count should be done for Figure 7D. Immunolabeling with more markers for Type I pneumocytes, including AQP5 Ab, should be performed to determine if the decreased Aqp5 RNA expression correlates with less Type I cells. GSEA signature has to be confirmed by additional analyses.

Answer:

Given the flat appearance of the T1a-positive cells (see old Figure 7E) it is difficult to carry out a quantification for T1a (which is Pdpn). We will perform new IF experiments to examine AT1/2 cell numbers using additional markers (e.g. Hopx for AT1).

Comment:

p.17: The same comments can be made for Type II pneumocytes and SpC expression.

Answer:

We actually did do an Sftpc (Pro-SPC) count (see old Figure 7E), which reveals that the number of Sftpc-expressing cells is up in the Clasp1 KO. At first sight this seems surprising, given that Chil1 (a top AT2 signature gene at E18.5) is virtually absent from Clasp1 KO lungs. However, our new GSEA analysis (shown in the new Figure 6) shows that of all the E18.5 AT2 signature genes (403 genes in total) the majority is down-regulated, including Chil1 and 4 other top signature genes, but some genes are up, including Sftpc (see new Figure 6). Combined with the fact that we observe more Pro-SPC-expressing cells in the Clasp1 KO lung we hypothesise that AT2 cell numbers are up compared to wild type, giving rise to higher mRNA counts of some genes in the RNA-Seq. Differentiation of AT2 cells is significantly hampered, giving rise to lower expression of many AT2 signature genes in the RNA-Seq. By contrast, all AT1 signature genes are either down or not affected (see new Figure 6). We interpret this as evidence that AT1 cell numbers are down. The same goes for endothelial cells (EC, see new Figure 6). We will perform additional IF experiments to examine this hypothesis.

Reviewer #3.

Comment:

T1α-positive cells should be quantified (Figure 7D). From the images, the number of T1α+ cells in Clasp1 KO is not consistent with the qPCR result showing markedly reduced Aqp5 transcript levels in Clasp1 KO. It is unclear whether the reduction in Aqp5 is due to impaired water channel function as the authors suggest or instead due to reduced number of AT1 cells, further investigation should be conducted.

Answer:

Please see our answer to reviewer #1 above. To summarise, we now have evidence that AT1 cell numbers are down. We will perform additional IF experiments to examine this hypothesis.

Comment:

Additional AT1 markers (Hopx, Ager, Clic5 and Rage) should be assessed by qPCR and immunostaining to determine the effect of Clasp1 knockout on AT1 cells.

Answer:

Please see our answer to reviewer #1 above. To summarise, we will perform new IF experiments to examine AT1/2 cell numbers using additional markers (e.g. Hopx for AT1).

3. Description of the revisions that have already been incorporated in the transferred manuscript

General remarks

As explained in detail below, we believe that our new RNA-Seq analysis has uncovered a mechanism underlying the severe lung phenotype of Clasp1 KO mice, and that it has revealed the major cell types affected in embryonic Clasp1 KO lungs.

Brief summary of experiments

In the first version of the manuscript we used Gene Set Enrichment Analysis (GSEA, see https://www.gsea-msigdb.org/gsea/index.jsp) to compare our RNA-seq results to publicly available scRNA-Seq datasets of cell type signature gene sets, which contain cluster marker genes for cell types identified in single-cell sequencing studies of human tissue. As stated in our manuscript, this revealed “enrichment of alveolar epithelial type I cells and lung capillary intermediate cells in WT lungs ….”. However, the analysis was restricted to what is available in the Gene Set Enrichment Analysis database of the University of San Diego. Thus, we could only compare our embryonic mouse lung data to adult human lung scRNA-Seq data.

We recently discovered publicly available scRNA-Seq datasets of the mouse lung (see https://research.cchmc.org/pbge/lunggens/mainportal.html and https://lungcells.app.vumc.org). The data in these portals are not part of the common GSEA sets of the University of San Diego. In particular the LGEA web portal is very easy to use and data can be downloaded for individual applications. In the new version of our manuscript we compared our RNA-Seq data to scRNA-Seq data of the embryonic mouse lung, focussing on E18.5. We first overlaid differentially expressed genes in Clasp1 KO lungs with LGEA E18.5 scRNA-seq gene signatures for different cell types, and we subsequently compared all the genes in our dataset with the gene signature lists, using custom-built gene signature sets and the GSEA software. In addition, we interrogated LGEA to find out which signature genes are specifically turned on from E16.5-E18.5 in the different cell types in the developing mouse lung. We found, for example, that Chil1, which is the most severely down-regulated gene in our Clasp1 KO RNA-Seq, is a very prominent AT2 signature gene; Chil1 is hardly expressed at E16.5 and prominently comes up at E18.5.

Our combined analysis strongly suggests that four cell types (AT1, AT2, endothelial cells (EC), and immune cells (IC)) are affected in their differentiation in the Clasp1 KO lung, and that this defect occurs in the later stages of lung development (from E16.5 onward). As the top five differentially down-regulated genes in KO lungs (including Chil1) are all top signature genes of AT2 cells, these data strongly suggest that it is this cell type that is most affected in the KO. A Metascape analysis (which includes a GO enrichment analysis, see also our specific answer to comments of reviewer #3 below) is consistent with the scRNA-Seq comparison and suggests, among others, that the secretory pathway might be hampered in the Clasp1 KO. This analysis furthermore indicates that cholesterol metabolism might be affected in the Clasp1 KO, which bears relevance to our dexamethasone rescue experiments.

Specific remarks to the comments of the reviewers

Reviewer #1.

• *

Comment:

p.6: What is the justification to mention Nfib, Pdpn and Ndst1 mutant mice in the introduction? Do these genes have any cellular/molecular/functional relation with CLASP1?

Answer:

We initially wanted to provide examples of genes important for lung maturation, whose absence in knockout mice leads to lung collapse. Of the examples provided Pdpn (which is equal to the marker T1a) bears a relation with our data in that it is down-regulated in Clasp1 KO lungs (see Table S2, RNA-Seq); furthermore, we examined T1a localisation in IF stainings (see old Figure 7E). In the new version of the manuscript we modified this Introduction section, to better align with our recent results, and to introduce the papers mentioned by reviewer #3 (Nelson et al., 2017; doi:10.1242/dev.154823, Li, J. et al., Dev Cell, 44, 297-312 e5.), who points out that pressure plays an important role in lung development. In the Li et al manuscript Pdpn is mentioned as being expressed at E16.5 in so-called Id2+ cells, together with Sftpc. These cells are proposed to be the precursors of the AT1/2 epithelial cells that arise later.

Comment:

p.8: It is mentioned that CLASP1 is expressed in secretory cells of the lung. Which ones? Is CLASP1 expressed in nerves, muscle cells and/or fibroblasts of the diaphragm? These information are important according to the phenotypes described.

Co-immunolabelling experiments should be done.

Answer:

We apologize for our incorrect phrasing. With respect to the lung, we now state that “CLASP1 is expressed in the endothelium of blood vessels, as well as in all cells lining the airways of mouse lungs at E18.5 (Fig. 1A)”.

Comment:

p.11: To identify the cause of the respiratory failure, the authors looked at the innervation pattern of the phrenic nerve in the diaphragm. Mutants present decreased branching but larger nerve extensions covering a wider innervated area and less neuromuscular junctions. Despite the decreased innervation of the diaphragm, its morphology is normal as well as the ultra-structure of the sarcomeres suggesting a mild phenotype rather than the cause of death of the mutants as suggested by the authors (p.20).

Diaphragmatic muscle activity should be measured to establish if the contractile activity of the diaphragm is affected. This might support the statement of the authors.

Answer:

We thank the reviewer for these observations. We agree with the reviewer and have toned down our conclusions in this section. We now simply describe the innervation pattern because we believe it is interesting, and we tentatively conclude that it may contribute to the severe respiratory phenotype which is primarily due to impaired AT1/2, EC, and IC differentiation.

Comment:

p.13: The authors examined lung from mutants. Mutant lungs do not float and they are collapsed at birth. However, lung morphology appears normal and myofibroblasts, ciliated cells and Club cells are present as shown by IHC labeling. No difference in proliferation and apoptosis was reported.

It would have been more informative to do BrdU/EdU immunolabeling for proliferation in order to see if differences occur in specific cell types of the lung. It is not clear why the authors have limited their IHC analysis to these three specific cell types. A complete analysis should be done.

Answer:

As described above (general remarks), we compared our RNA-Seq data to publicly available scRNA-Seq data from the developing mouse lung (see new Figure 6). These comparisons reveal which cell types are affected in the Clasp1 KO lung (AT1/2, EC, IC), and which process might be hampered.

Comment:

p.14: The authors proposed a delay in lung development according to lung morphology that appears more collapsed starting at E15.5.

Measurement of branching would allow to quantify this delay. Since cell differentiation occurs ~E16.5, analysis of the onset of cell types can also support a delay in lung development.

Answer:

As described above (general remarks), we compared our RNA-Seq data to publicly available scRNA-Seq data from the developing mouse lung (see new Figure 6). This not only revealed which cell types are affected in the Clasp1 KO lung, but also suggest that a differentiation block occurs at E16.5 to E18.5. For example, Chil1, a top AT2 signature gene of E18.5, is hardly expressed at E16.5 and is strongly upregulated at E18.5. This gene fails to become up-regulated in the Clasp1 KO, indicating that epithelial precursor cells have problems differentiating to AT2 type cells. By contrast, Id2, a marker of precursor epithelial cells, is normally expressed in the Clasp1 KO, and two genes that are co-expressed with Id2 in these precursor cells (Pdpn and Sftpc) are slightly down and up, respectively, in the Clasp1 KO. Thus, while our lung morphology studies might suggest early defects, our RNA-Seq indicates that specific defects occur during the late terminal saccular stage, i.e. from E16.5 onward. We therefore agree with with Negretti et al (2021, doi: 10.1242/dev.199512, Discussion section) who state: the developmental stages of the lung are largely founded on histologically descriptive features. While this is important, such a categorization often results in debate regarding the function and identity of cell types within the boundaries of each stage. By contrast transcriptome analysis suggests that different cell types commit to change asynchronously during development, suggesting that the timing of the saccular-to-alveolar transition is fluid and highly cell-type specific.

As shown by Li et al (2018, doi.org/10.1016/j.devcel.2018.01.008) mechanical forces contribute to embryonic lung alveolar epithelial cell differentiation. Interestingly, RNA-Seq data from Nelson et al (2017; doi:10.1242/dev.154823) suggest that CLASP1 is a “pressure sensing gene” (see also below, our answer to comments of reviewer #3). Thus, Clasp1 KO lungs might fail to properly sense pressure, which could explain, at least in part, the observed failure in epithelial differentiation.

Comment:

p.15: Finally, the authors conclude this section by "these data support a direct role for CLASP1 in lung maturation".

Which direct role? How? This sentence appears premature according to the data presented. The authors should look at microtubule dynamics in lung cells from mutant embryos to see if a link exists between the proposed role of the protein and the lung phenotype observed.

Answer:

The reviewer is correct, knockout studies can not demonstrate a direct role of a protein in a perturbed process. We have therefore removed the word “direct” from this phrase.

Comment:

p.15: The authors attempted to rescue the defective lung maturation phenotype by treating pregnant females with dexamethasone at late gestational stages. Around 10% of mutants survive for more than 45 minutes to 2 hrs compared to 20-30 minutes for mutants obtained from untreated mothers (p.9). Even though it is an intriguing result, the very small numbers of "survivors" makes very difficult to reach a conclusion.

This section should be shortened.

Answer:

Our new Metascape analysis, which will be presented in the new Figure 8, suggests that cholesterol metabolism is affected in the Clasp1 KO mice. Cholesterol is an important component of mammalian cell membranes, of both alveolar and lamellar body surfactant, and it is a precursor of vitamin D and steroid hormones. A cholesterol defect would explain the partial rescue by dexamethasone in the Clasp1 KO, i.e. dexamethasone can rescue a steroid hormone defect but it cannot rescue other defects (e.g. surfactant production). Given these new results we decided to leave the section on glucocorticoids as it is and come back to it when we discuss the Metascape result in the revised manuscript.

Comment:

p.16: To determine which molecular mechanisms are responsible for the lung defect, the authors performed RNA-seq analysis on E18.5 lung specimens. The number of genes with significant differential expression was low and the highest scores were cathepsin E for the upregulated gene and chitinase-like 1 for the downregulated gene.

Are these two genes known for their role in lung development? Please describe.

Answer:

The Ctse gene, which encodes Cathepsin E, is indeed the most upregulated gene in the Clasp1 KO. Although it is up-regulated in all three KO mice, Ctse expression is quite low (normalised counts: ~2 in KO, up from ~0.2 in WT). Based on the comment of this reviewer we examined Ctse expression in the scRNA-Seq lung repositories, but we could not find any description, presumably because its expression is too low (scRNA-Seq has difficulty catching low abundance genes), consistent with our data. Furthermore, there is not much literature on the role of Cathepsin E in the lung. We therefore decided to remove any mention of Ctse in the manuscript. By contrast, the expression and function of Chil1 are described in detail.

Comment:

p.16: Except for the fact that Chil1 is also downregulated in mutant lungs for the H3K4 methyltransferase Mll3 gene, it is not clear why the authors compared these 2 sets of data.

Can CLASP1 and MLL3 interact together? How? Did the authors looked at the list of genes that are commonly differentially expressed? Does it provide some clues on the mechanisms? The RNA-seq data should be analyzed more deeply.

Answer:

The reviewer is correct, we compared the Mll3 (i.e. Kmt2c) RNA-Seq dataset because Chil1 is down-regulated in the Mll3 KO lung at E18.5, like in the Clasp1 KO. To examine a possible relation between Mll3 and Clasp1 in more detail, we overlaid the differentially expressed genes from the Mll3 dataset with the custom-built gene signature dataset of E18.5 lung (described above). The data suggest that Mll3 knockout affects AT1 differentiation (see new Supplementary Figure S6C). This mode of action is clearly different from that of CLASP1, and since Mll3 is nuclear and CLASP1 is cytoplasmic we do not believe these proteins interact. Given our new and exciting data on the Clasp1 KO lung phenotype, we moved the Mll3 data to the new Supplementary Figure 6, and only briefly we touch upon these data in the manuscript.

Comment:

p.16: There is also a Clasp2 gene with a more restricted expression pattern. Clasp2 mutant mice either die from hemorrhages or survive. It is not clear why the RNA-seq data of the lungs from Clasp2-/- mice are presented since no lung phenotype is mentioned for these mice. How the lack of change in Chil1 expression in Clasp2 mutant lungs is informative?

This should be clarified or the data should be removed.

Answer:

The reviewer is correct, i.e. in light of our new findings (Chil1 is a top signature gene of E18.5 AT2 cells) it makes little sense to include the Clasp2 KO RNA-Seq data, as these were generated in adult mouse lungs. We therefore removed these data from the manuscript.

Comment:

p.31: The authors mentioned a role for CLASP1 in the mesenchyme.

What are the experiments and data that support this sentence?

Answer:

We thank the reviewer for this remark, we have no evidence for a role of CLASP1 in the mesenchyme and have removed this phrase.

Comment:

How do the authors reconcile their observation of CLASP1 expression in lung secretory cells (p.8) with their conclusion of defective Type I cell differentiation (p.17)?

Answer:

We apologize for our incorrect phrasing. With respect to the lung, we now state that “CLASP1 is expressed in the endothelium of blood vessels, as well as in all cells lining the airways of mouse lungs at E18.5 (Fig. 1A)”.

Reviewer #2.

Comment:

Fig. 3. There is not a lot of detail how the analysis in B-E was done, and no primary data for the synaptic defects.

Answer:

We have removed these data from the manuscript.

Reviewer #3.

Comment:

1. The authors showed significant reduction in the rib cage size and abnormal diaphragm innervation in Clasp1 KO. Mechanical properties play a crucial role in regulating lung development and maturation. So changes in intrathoracic space and pressure are a major limiting factor that impairs lung development and maturation (Nelson et al., 2017; doi:10.1242/dev.154823, Li, J. et al., Dev Cell, 44, 297-312 e5.). Answer:

We thank the reviewer for these interesting papers and observations.

Nelson et al (2017; doi:10.1242/dev.154823) devised a method to culture lung-on-a-chip where they can induce pressure in culture. They apply this to examine lung development and they also do RNA-Seq. Interestingly, they find that Clasp1 is down-regulated at high pressure compared to low pressure (log2FC 0.5, Clasp1 goes down ~1.5 fold in high pressure). Thus Clasp1 appears to be a “pressure-responsive gene”. However, Nelson et al examine gene expression at much earlier time points than we do (E12-14 versus E18.5). In our view it therefore makes little sense to compare RNA-Seq data.

Li et al (2018 doi.org/10.1016/j.devcel.2018.01.008) show that mechanical forces help to control embryonic lung alveolar epithelial cell differentiation. More specifically, mechanical force from amniotic fluid inhalation ensures AT1 cell differentiation, whereas FGF10-mediated ERK1/2 signaling induces a protrusive structure in some cells that protects from mechanical force-caused flattening to specify AT2 fate. They conclude that future AT2 cells can “embed” into mesenchyme by exerting an acto-myosin based force and hence they can keep their cuboidal shape. The differentiation of the two cell types occurs at different time points, E16.5 for AT2, and E17.5 for AT1. In this manuscript they also mention that Id2+ tip cells express pro-SPC and Pdpn (which are up and down, respectively, in Clasp1 KO). These Id2+ cells would be the AT1/2 progenitors.

We believe that a smaller ribcage in the Clasp1 KO does not necessarily have to be a cause of increased pressure on the lung, if the lung is also smaller. Nonetheless, since CLASP1 is a “pressure-responsive gene”, Clasp1 KO lungs might experience aberrant pressure sensing (in addition to a possible pressure difference due to a smaller ribcage). This different sensing predicts altered differentiation pathways, which is exactly what we see. We have modified the revised version of the manuscript to reflect these thoughts and observations.

Comment:

Since CLASP1 was found to be highly expressed in the lung endothelium (Figure 1A), this suggests the importance of CLASP1 in the lung vasculature. GSEA analysis also showed significant downregulation of genes from the lung capillary intermediate 1 cell signature gene set in Clasp1 KO (Figure 7G). Extensive crosstalk between the lung endothelium and other lung cell types is critical for the regulation of lung development. However, no further investigation was carried out to elucidate this.

Answer:

We have performed a new comparison, which is extensively discussed above and shows that EC are affected in the Clasp1 KO lungs, as predicted by this reviewer. We will discuss crosstalk between cell types in the new version of the manuscript.

Comment:

Analysis of RNA-Seq data needs to be re-written. Pathway or GO enrichment was not performed. Although the authors have identified a number of key DEGs, only Chil1 was investigated. It is also unclear how it led the authors to identify Mll3 KO experiment on the Omnibus repository. A list of overlapped genes between Mll3 KO dataset and Clasp1 KO dataset were not provided. Aqp5 (AT1 marker gene) that authors claimed to be significantly reduced in Clasp1 KO is not on the DEGs list (Table S2).

Answer:

We initially focused on Chil1 because its expression is almost completely abrogated in all three Clasp1 KO lungs. The identification of the Mll3 dataset was coincidental; we mentioned it because Chil1 is also affected in these KO mice. A Venn diagram of overlapping significantly deregulated genes in both datasets is shown in the new Figure S6 of the revised manuscript. However, this analysis has been superseded by the new comparison with scRNA-Seq data from the LGEA web portal. As extensively explained above this new analysis provides a satisfying explanation for the lack of Chil1 in Clasp1 KO lungs. We also performed a Metascape analysis (which includes pathway and GO enrichment analyses), which will be included in the revised version of this manuscript. Finally, the reviewer is correct that Aqp5 is not in the DEGs list, this is because the adjusted p-value did not reach the required significance. We nevertheless showed its RNA-Seq values, first because the p-value is significant, second, because RT-PCR experiments confirm it to be down-regulated, and third, because Aqp1 (another AT1 marker) is also deregulated (with an adjusted p-value that is significant). In the revised manuscript we will examine Aqp5 levels by IF staining.

Comment:

There is a lack of cohesion between the experimental findings presented in the paper and the RNA Seq data analysis. Pathway or GO enrichment was not performed for the DEGs the authors identified. This would help identify the key functions of the deregulated genes in Clasp1 KOs and provide a fuller picture of what pathways/biological processes are dysregulated in the absence CLASP1. Instead, the authors have focused on one single gene, Chil1 in the subsequent analysis. The authors infer that overlapped DEGs between Mll3 KO and Clasp1 KO mean that same cell types or signalling pathways are affected in embryonic lungs of Mll3 and Clasp1 KO, this is an overinterpretation. A list showing the overlap in DEGs between Mll3 KO dataset and Clasp1 KO dataset should be provided.

Answer:

We have improved our RNA-Seq analysis and we have performed a Metascape analysis, which includes pathway and GO enrichment analyses. Results are shown in the new Figures 6 and 8. The Metascape analysis indicates which pathways/biological processes are deregulated in the absence CLASP1. We observe, for example, defects in endocytosis, and cholesterol metabolism. Given the new data, we decided to pay less attention to the Mll3-CLASP1 comparison.

Minor comments:

1. Figure 1A - please label the specific cell types to aid visualisation.
2. Figure 6B - present the Log2FC for KO vs WT instead of WT vs KO to facilitate data visualisation and interpretation
3. Figure 6E - provide the overlapping genes in a list and include it as a supplementary table
4. Figure 7D and 7F - Quantification is needed
5. The statistical tests used should be added to the figure legends.

6. There is some wording in the manuscript that is either unclear or inaccurate, please carefully check the manuscript. e.g. manuscript refers to alveolization- I would recommend changing this to the more widely used terms alveolarization or alveologenesis. The manuscript refers to 'catastrophe rate'- this term needs to be defined. Answers:

7. This has been done.

8. This has been done.
9. This panel has been moved to a Supplementary Figure, as the analysis is less relevant now we will not provide the list.
10. This will be done.
11. This has been/will be done.
12. This has been done. The term “catastrophe rate” has been removed.
13. *

4. Description of analyses that authors prefer not to carry out

General remarks

Based on the comments of reviewers #2 and #3 we have decided to fully focus our revised manuscript on the lung phenotype of the Clasp1 KO mice. We still do show the results on the ribcage (Figure 2) and diaphragm (Figure S2) because they might enhance the severity of the lung phenotype. We have decided not to carry out extra “non-lung” experiments.

Specific remarks to the comments of the reviewers

Reviewer #1.

Comment

p.10: Homozygous mutants are smaller. The authors reported minor skeletal phenotypes small rib cage and delayed ossification in sternum and occipital bone.

The number of specimens analyzed was not mentioned rendering difficult to establish if these observations are important or not. Stats should be included.

Answer:

Whereas the results of Figure 1H, I (growth deficits at E15.5 and PN1) are based on analysis of multiple animals, the embryonic skeleton data presented in Figure 2 are based on single mouse comparisons, i.e. one WT and one KO. Given the obvious growth deficit in the KO (Figure 1H, I) and the fact that gross morphological observation did not reveal a specific body part in the KO mice that is affected (Figure 1G), we were of the opinion that a representative comparison of the skeleton is allowed and we therefore kept Figure 2 intact. Since we focus in the revision on the lung phenotype, we have decided against examining the skeletons of more mice. We are willing to remove Figure 2, or make it Supplemental, if the reviewer feels that the skeletal phenotype is too prominently displayed.

Comment:

p.10: The authors established MEF used to study cell division. Multipolar spindles and additional centrosomes were detected in mutant cells.

No stats were provided to establish if the differences in numbers are significant. According to the authors, the cell division defects may explain the smaller size of mutants. The authors should check proliferation in MEF. The sentence of conclusion is not well supported according to the data presented.

Answer:

Based on the advice of reviewer #2, who states “I think it would be best to better focus the paper on the lung phenotype”, we have decided to remove the mitotic data on MEFs.

Comment:

p.12: The authors looked at the growth capacity of motor neurons and dorsal root ganglion neurons and showed a reduced growth in both cases.

How do the authors reconcile the observation made in the diaphragm in which nerve extensions are larger with the reduced growth capacity of neurons?

Answer:

We thank the reviewer for this remark, which is difficult to address, as CLASPs are expressed at different levels in neurons and as different isoforms, which may even have antagonistic functions. For example, in our recent publication (Sayas et al, 2019, DOI: 10.3389/fncel.2019.00005) we find through RNA-Seq that in cultured hippocampal neurons (3DIV) Clasp2β/γ levels are increased compared to Clasp2α-mRNA and that both in hippocampal and in DRG neurons Clasp2 mRNA levels are higher than Clasp1. As CLASP2b/g have a different function compared to CLASP2a, it is conceivable that absence of CLASP1 leads to different effects due to different CLASP2 activities. However, we recognize that these are speculations. Because of this and because reviewer #2 advices against inserting the neuronal data, we have decided to completely remove these results from the manuscript.

Comment:

p.12: The authors used cultured hippocampal neurons for imaging microtubule growth. According to the authors, the loss of CLASP1 deregulates microtubule dynamics.

No explanation was provided to justify the use of hippocampal neurons. What is a catastrophe rate? What is the justification to study this parameter? What does it tell us about microtubule dynamics?

Answer:

Although we have decided to remove the neuronal data from the revised manuscript, we would like to address this comment nonetheles. Hippocampal neurons are often used in the field, hence they represent a “golden standard”. Furthermore, the techniques to examine microtubule dynamics are well established in this system. Dynamic microtubule behaviour is described using five parameters: growth rate of microtubules, shrinkage rate of microtubules, catastrophe and rescue frequencies (the conversion of growth to shrinkage or from shrinkage to growth, respectively), and pauzing times. The marker used in our studies (EB3-GFP) accumulates at the ends of growing microtubules, allowing us to measure growth rate and the duration of a growth event. The latter is the inverse of the catastrophe frequency. Hence, using EB3-GFP we are able to examine two of the five parameters. Although this is not complete the parameters do allow us to draw (speculative) conclusions. For example, a higher growth rate indicates that free tubulin concentration is higher, as tubulin concentration is a main determinant of growth rate. This in turn means that there are less microtubules (tubulin must come from somewhere). If this correlates with the catastrophe frequency (which should be higher) than one can conclude that CLASP1 is a microtubule-stabilising protein.

Reviewer #2.

Comment:

Fig. S1. It would be good to indicate the number of cells / experiments analyzed. In panel D, there is only one multi-nucleated cell, which without further analysis does not mean much. The authors correlate this mitotic defect with smaller animal size although this connection is not at all conclusive. If both CLASPs are important for mitosis, do CLASP2 KOs have similar size defects? It is also mentioned above that CLASP1 KOs show microcephaly. Are there fewer neurons that might also be linked to a stem cell division defect? I understand that this is not the central point of the paper and important to include given previous work on CLASPs, but it would be good to discuss a little clearer. It seems the authors do not think this is the/a cause of the lung phenotype, but can that be completely excluded?

Answer:

Based upon suggestions of this reviewer (for example: “I think it would be best to better focus the paper on the lung phenotype”) we will not address this comment beyond a statement that Clasp2 knockout mice are indeed also smaller.

Fig. 4. Please indicate n of cells / experiments and statistics in the figure legend. In panel B and C, it would help to include the time on the figure itself and to scale the y-axis the same to better illustrate differences. It is very hard to see much in panel D. The quantifications in E and F do not make sense. How can the total neurite length (average of many neurons?) be larger than the longest neurite length?

The switch to MT dynamics in Fig. 4 is very abrupt and the relevance is unclear. Where were these kymographs located in the neuron (growth cones or neurites)? Primary data needs to shown here. The changes in catastrophe frequency are not that large and I doubt this can be accurately measured from kymographs as shown. Yes, MTs are important in neurite growth, but the potential link here is very vague. Are similar changes in MT dynamics also seen in the MEFs?

Minor:

Answer:

See above, we will not address these comments, since we will remove these data.

Reviewer #3.

Comment:

The lung morphological difference and disrupted lung cell differentiation in Clasp1 KO could be secondary to the biomechanical defects. This is crucially important but is not addressed in this study, ex vivo lung culture may help to answer this question.

Answer:

While the experiments suggested by this reviewer are interesting, we do not have sufficient expertise (nor the equipment) to carry out such specialised experiments.

Comment:

CLASPs are known to regulate directed cell migration (Myer and Myers 2017, doi: 10.1242/bio.028571) and this is a key process required for lung morphogenesis. Experiments to address whether directed cell migration is affected should be conducted in Clasp1 KO mice.

Answer:

We agree that migration assays would be interesting to perform. However, again, we do not have the expertise to do such assays in the developing lung. Experiments in MEFs are possible, and indeed, we previously showed a role for CLASP2 in directed cel migration in MEFs (DOI: 10.1016/j.cub.2006.09.065). However, lung epithelial cells are different from MEFs, and we have shown that CLASPs have cell type- (and isoform-)specific functions. Reviewer #2 actually advised us to focus on the lung phenotype.

Comment:

Higher magnification images of staining for microtubule associated proteins in neurons is required to show the details of the defects.

Answer:

Based on the reviewers’ advice we decided to take out the neuronal data and focus the manuscript on the lung phenotype.

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Referee #3

Evidence, reproducibility and clarity

In the manuscript, the authors used a Clasp1 KO mouse to investigate the roles of CLASP1, a microtubule-associated protein. The manuscript presents a number of phenotypes found in the homozygous knockouts including reduced intrauterine growth, altered respiratory muscle innervation, and perturbed lung maturation. The loss of CLASP1 leads to neonatal lethality due to breathing defects however, whilst the manuscript shows a number of different phenotypes in the mutants, The underlying mechanism of how disrupted CLASP1-mediated microtubule dynamics causes the phenotypes observed is not clear. The authors propose some mechanisms for the phenotypes but these are largely speculative and additional experiments are required to substantiate them.

Some major and minor comments are detailed below which we hope will be useful.

Major comments:

1. The authors showed significant reduction in the rib cage size and abnormal diaphragm innervation in Clasp1 KO. Mechanical properties play a crucial role in regulating lung development and maturation. So changes in intrathoracic space and pressure are a major limiting factor that impairs lung development and maturation (Nelson et al., 2017; doi:10.1242/dev.154823, Li, J. et al., Dev Cell, 44, 297-312 e5.).
2. The lung morphological difference and disrupted lung cell differentiation in Clasp1 KO could be secondary to the biomechanical defects. This is crucially important but is not addressed in this study, ex vivo lung culture may help to answer this question.
3. CLASPs are known to regulate directed cell migration (Myer and Myers 2017, doi: 10.1242/bio.028571) and this is a key process required for lung morphogenesis. Experiments to address whether directed cell migration is affected should be conducted in Clasp1 KO mice.
4. Since CLASP1 was found to be highly expressed in the lung endothelium (Figure 1A), this suggests the importance of CLASP1 in the lung vasculature. GSEA analysis also showed significant downregulation of genes from the lung capillary intermediate 1 cell signature gene set in Clasp1 KO (Figure 7G). Extensive crosstalk between the lung endothelium and other lung cell types is critical for the regulation of lung development. However, no further investigation was carried out to elucidate this.
5. Higher magnification images of staining for microtubule associated proteins in neurons is required to show the details of the defects.
6. Analysis of RNA-Seq data needs to be re-written. Pathway or GO enrichment was not performed. Although the authors have identified a number of key DEGs, only Chil1 was investigated. It is also unclear how it led the authors to identify Mll3 KO experiment on the Omnibus repository. A list of overlapped genes between Mll3 KO dataset and Clasp1 KO dataset were not provided. Aqp5 (AT1 marker gene) that authors claimed to be significantly reduced in Clasp1 KO is not on the DEGs list (Table S2).
7. T1α-positive cells should be quantified (Figure 7D). From the images, the number of T1α+ cells in Clasp1 KO is not consistent with the qPCR result showing markedly reduced Aqp5 transcript levels in Clasp1 KO. It is unclear whether the reduction in Aqp5 is due to impaired water channel function as the authors suggest or instead due to reduced number of AT1 cells, further investigation should be conducted.
8. Additional AT1 markers (Hopx, Ager, Clic5 and Rage) should be assessed by qPCR and immunostaining to determine the effect of Clasp1 knockout on AT1 cells.
9. There is a lack of cohesion between the experimental findings presented in the paper and the RNA Seq data analysis. Pathway or GO enrichment was not performed for the DEGs the authors identified. This would help identify the key functions of the deregulated genes in Clasp1 KOs and provide a fuller picture of what pathways/biological processes are dysregulated in the absence CLASP1. Instead, the authors have focused on one single gene, Chil1 in the subsequent analysis. The authors infer that overlapped DEGs between Mll3 KO and Clasp1 KO mean that same cell types or signalling pathways are affected in embryonic lungs of Mll3 and Clasp1 KO, this is an overinterpretation. A list showing the overlap in DEGs between Mll3 KO dataset and Clasp1 KO dataset should be provided.

Minor comments:

1. Figure 1A - please label the specific cell types to aid visualisation.
2. Figure 6B - present the Log2FC for KO vs WT instead of WT vs KO to facilitate data visualisation and interpretation
3. Figure 6E - provide the overlapping genes in a list and include it as a supplementary table
4. Figure 7D and 7F - Quantification is needed
5. The statistical tests used should be added to the figure legends.
6. There is some wording in the manuscript that is either unclear or inaccurate, please carefully check the manuscript. e.g. manuscript refers to alveolization- I would recommend changing this to the more widely used terms alveolarization or alveologenesis. The manuscript refers to 'catastrophe rate'- this term needs to be defined.

Significance

The authors have carefully documented a variety of phenotypes that occur in Clasp1 knockout mice, this is novel because this is the first report of genetic manipulation of Clasp1 in an animal model. It is clear that the homozygotes die because they cannot breathe properly once they transition to air breathing at birth. However, it is not clear in the current manuscript what the underlying reasons for the breathing defects are. The manuscript shows a number of respiration- related deficiencies including small rib cage, disrupted diaphragm innervation and lack of alveolar maturation but the manuscript documents a series of phenotypes rather than pulling together a hypothesis about the role of Clasp1 in the respiratory system.

Foetal breathing movements are essential for normal lung development and the maturation of cells into their differentiated phenotypes e.g. ATII to ATI cells in the alveoli. It could be that the reduced thoracic space, coupled with the diaphragm deficiencies are the underlying cause of the alveolar cell maturation defects and failure of normal breathing, due to impaired biomechanics. The authors could conduct further experiments to explore this avenue e.g. ex vivo lung culture to see if there are still developmental deficiencies in the absence of reduced intrathoracic space (small rib cage).

The manuscript details some interesting findings but in its current form, it lacks a coherent story. I am not convinced that all the details of the effects on DRG and motor neurons is required in the same manuscript as the analysis of lung biology. It may be clearer to split the findings into separate manuscripts.

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Referee #2

Evidence, reproducibility and clarity

The manuscript by Pereira et al. investigates the phenotype of loss of the MT-associated plus-end-bidding protein CLASP1 in mouse. They find that CLASP1 KO pups are not viable due to rapid respiratory failure and the paper presents an in-depth analysis of the lung phenotype that is quite striking. However, the mechanistic links to previously proposed cellular functions of CLASP1 in mitosis and MT dynamics are weak and confusing.

For example, the analysis of mitotic defects in MEFs or of MT dynamics in neurons is not convincing and does not really explain anything; if or if not these cellular phenotypes are related to the observed lung defect. (in fact, the discussion of the paper does not even mention again these functions of CLASP1). So, in the end, the reader is left with a menu of choice of whether CLASP1 is directly involved in lung development, required for innervation or for something else. Many other questions are left unanswered: Is innervation defective because lung development is abnormal, or does innervation control development (as it has been proposed in other organs)? How is CLASP1 controlling the lung transcriptome; does this have anything to do with its cellular functions or is this a completely indirect effect, again stemming from a deeper developmental defect? Overall, I think the lung phenotype is interesting and worth publishing. However, I do not exactly know how to resolve the mechanistic questions, but I think it would be best to better focus the paper on the lung phenotype and maybe rearrange the order data are presented (Fig. 4 seems oddly plopped in the middle of the lung analysis).

Specific comments:

Fig. S1. It would be good to indicate the number of cells / experiments analyzed. In panel D, there is only one multi-nucleated cell, which without further analysis does not mean much. The authors correlate this mitotic defect with smaller animal size although this connection is not at all conclusive. If both CLASPs are important for mitosis, do CLASP2 KOs have similar size defects? It is also mentioned above that CLASP1 KOs show microcephaly. Are there fewer neurons that might also be linked to a stem cell division defect? I understand that this is not the central point of the paper and important to include given previous work on CLASPs, but it would be good to discuss a little clearer. It seems the authors do not think this is the/a cause of the lung phenotype, but can that be completely excluded?

Fig. 3. There is not a lot of detail how the analysis in B-E was done, and no primary data for the synaptic defects.

Fig. 4. Please indicate n of cells / experiments and statistics in the figure legend. In panel B and C, it would help to include the time on the figure itself and to scale the y-axis the same to better illustrate differences. It is very hard to see much in panel D. The quantifications in E and F do not make sense. How can the total neurite length (average of many neurons?) be larger than the longest neurite length? The switch to MT dynamics in Fig. 4 is very abrupt and the relevance is unclear. Where were these kymographs located in the neuron (growth cones or neurites)? Primary data needs to shown here . The changes in catastrophe frequency are not that large and I doubt this can be accurately measured from kymographs as shown. Yes, MTs are important in neurite growth, but the potential link here is very vague. Are similar changes in MT dynamics also seen in the MEFs?

Minor:

Fig. 1A please indicate in legend what is CLASP staining (suppose the brown stuff).

Define HT in text.

Again, please include statistics in figure legends (and indicate n and p values)

Significance

Findings presented in regard to CLASP1 role in lung development are interesting and significant, also as a potential novel model system of newborn respiratory failure. The mechanistic link to known functions of CLASP1 however remains vague and would need substantial additional work to address properly.

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Referee #1

Evidence, reproducibility and clarity

The manuscript entitled "CLASP1 is essential for neonatal lung function and survival in mice" by Pereira et al. reports the characterization of the phenotype of a Clasp1 null mutant mouse line. CLASP1 is a microtubule plus-end tracking protein involved in the regulation of microtubule dynamics broadly expressed in the organism. All Clasp1-/- mice die at birth from respiratory failure.

General comment:

This is an interesting study about the characterization of a Clasp1 mutant mouse line. The manuscript is clear and well-written. The analysis is descriptive. Many aspects are studied but unfortunately they are covered superficially. However, they open the way to more deepened analyses.

Specific comments:

p.6: What is the justification to mention Nfib, Pdpn and Ndst1 mutant mice in the introduction? Do these genes have any cellular/molecular/functional relation with CLASP1?

p.8: It is mentioned that CLASP1 is expressed in secretory cells of the lung. Which ones? Is CLASP1 expressed in nerves, muscle cells and/or fibroblasts of the diaphragm? These information are important according to the phenotypes described. - Co-immunolabelling experiments should be done.

p.10: Homozygous mutants are smaller. The authors reported minor skeletal phenotypes small rib cage and delayed ossification in sternum and occipital bone. - The number of specimens analyzed was not mentioned rendering difficult to establish if these observations are important or not. Stats should be included.

p.10: The authors established MEF used to study cell division. Multipolar spindles and additional centrosomes were detected in mutant cells. - No stats were provided to establish if the differences in numbers are significant. According to the authors, the cell division defects may explain the smaller size of mutants. The authors should check proliferation in MEF. The sentence of conclusion is not well supported according to the data presented.

p.11: To identify the cause of the respiratory failure, the authors looked at the innervation pattern of the phrenic nerve in the diaphragm. Mutants present decreased branching but larger nerve extensions covering a wider innervated area and less neuromuscular junctions. Despite the decreased innervation of the diaphragm, its morphology is normal as well as the ultra-structure of the sarcomeres suggesting a mild phenotype rather than the cause of death of the mutants as suggested by the authors (p.20). - Diaphragmatic muscle activity should be measured to establish if the contractile activity of the diaphragm is affected. This might support the statement of the authors.

p.12: The authors looked at the growth capacity of motor neurons and dorsal root ganglion neurons and showed a reduced growth in both cases. - How do the authors reconcile the observation made in the diaphragm in which nerve extensions are larger with the reduced growth capacity of neurons?

p.12: The authors used cultured hippocampal neurons for imaging microtubule growth. According to the authors, the loss of CLASP1 deregulates microtubule dynamics. - No explanation was provided to justify the use of hippocampal neurons. What is a catastrophe rate? What is the justification to study this parameter? What does it tell us about microtubule dynamics?

p.13: The authors examined lung from mutants. Mutant lungs do not float and they are collapsed at birth. However, lung morphology appears normal and myofibroblasts, ciliated cells and Club cells are present as shown by IHC labeling. No difference in proliferation and apoptosis was reported. - It would have been more informative to do BrdU/EdU immunolabeling for proliferation in order to see if differences occur in specific cell types of the lung. It is not clear why the authors have limited their IHC analysis to these three specific cell types. A complete analysis should be done.

p.14: The authors proposed a delay in lung development according to lung morphology that appears more collapsed starting at E15.5. - Measurement of branching would allow to quantify this delay. Since cell differentiation occurs ~E16.5, analysis of the onset of cell types can also support a delay in lung development.

p.15: Finally, the authors conclude this section by "these data support a direct role for CLASP1 in lung maturation". - Which direct role? How? This sentence appears premature according to the data presented. The authors should look at microtubule dynamics in lung cells from mutant embryos to see if a link exists between the proposed role of the protein and the lung phenotype observed.

p.15: The authors attempted to rescue the defective lung maturation phenotype by treating pregnant females with dexamethasone at late gestational stages. Around 10% of mutants survive for more than 45 minutes to 2 hrs compared to 20-30 minutes for mutants obtained from untreated mothers (p.9). Even though it is an intriguing result, the very small numbers of "survivors" makes very difficult to reach a conclusion. - This section should be shortened.

p.16: To determine which molecular mechanisms are responsible for the lung defect, the authors performed RNA-seq analysis on E18.5 lung specimens. The number of genes with significant differential expression was low and the highest scores were cathepsin E for the upregulated gene and chitinase-like 1 for the downregulated gene. - Are these two genes known for their role in lung development? Please describe.

p.16: Except for the fact that Chil1 is also downregulated in mutant lungs for the H3K4 methyltransferase Mll3 gene, it is not clear why the authors compared these 2 sets of data. - Can CLASP1 and MLL3 interact together? How? Did the authors looked at the list of genes that are commonly differentially expressed? Does it provide some clues on the mechanisms? The RNA-seq data should be analyzed more deeply.

p.16: There is also a Clasp2 gene with a more restricted expression pattern. Clasp2 mutant mice either die from hemorrhages or survive. It is not clear why the RNA-seq data of the lungs from Clasp2-/- mice are presented since no lung phenotype is mentioned for these mice. How the lack of change in Chil1 expression in Clasp2 mutant lungs is informative? - This should be clarified or the data should be removed.

p.17: Aqp5 expression was decreased in mutant lungs as shown by RNA-seq data and RT-qPCR. However, immunolabelling with Ti does not show a decrease in the number of Type I pneumocytes (Fig. 7D). According to the data presented, it is difficult to conclude that CLASP1 is involved in Type I pneumocyte differentiation. - A cell count should be done for Figure 7D. Immunolabeling with more markers for Type I pneumocytes, including AQP5 Ab, should be performed to determine if the decreased Aqp5 RNA expression correlates with less Type I cells. GSEA signature has to be confirmed by additional analyses.

p.17: The same comments can be made for Type II pneumocytes and SpC expression.

p.31: The authors mentioned a role for CLASP1 in the mesenchyme. - What are the experiments and data that support this sentence?

• How do the authors reconcile their observation of CLASP1 expression in lung secretory cells (p.8) with their conclusion of defective Type I cell differentiation (p.17)?

Minor comment:

Legend of Figure S4 should be for Figure S5 and vice-versa.

Significance

In summary: a descriptive characterization of a Clasp1 mutant mouse line but no real clue on how this microtubule-associated protein acts to produce the phenotypes observed that likely cause the death of the mutant newborns.

This manuscript should interest researchers in lung developmental biology and cell biology,

My expertise: mouse models, lung development, gene regulation and networks

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Reply to the reviewers

The authors do not wish to provide a response at this time

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Referee #3

Evidence, reproducibility and clarity

Summary

In this manuscript, Rodríguez -Real and colleagues investigate how the centrosome may influence the repair of DNA double-stranded breaks (DSBs), building on the initial finding that relative HR frequencies (as measured using a standard split-GFP gene conversion reporter assay) are reduced in centrinone treated centrosome-depleted cells relative to mock treated controls cells. Such defects are found correlate to concordant reductions in immunofluorescence proxies for resection (RPA recruitment into foci) and upstream and downstream events in the HR cascade (BRCA1 and RAD51 recruitment, respectively), and a correlating increase in NHEJ repair of I-SceI induced repair in EJ5-like reporter assay. Taking a candidate approach to identifying which centrosome proteins link the centrosome to DSB repair regulation, the authors reveal cells depleted for subdistal appendage proteins show equivalent deviations in DSB repair reporter assays and show concordant defects in RPA recruitment, leading to the proposal that subdistal appendage proteins regulate DNA resection and thus optimal HR. Experiments are then used to show CEP170 (a subdistal appendage protein) may be phosphorylated by DDR kinases and some rescue experiments are used to support hypothesis that this phosphorylation may be involved in centrosome-DSB repair cross-talk signalling. Figure 3 experiments then show centrosome-depleted and heterozygous losses of CEP170 result in moderate sensitivities across a number of DSB-inducing treatments. Lastly meta-analyses of cancer datasets correlate low CEP170 expression to differences in cancer mutations signatures (Fig 4) and altered patient outcomes across a number of cancers (Fig 5), and propose that CEP170 - via a DSB repair repair function - may be causal in these alterations. Ultimately, the authors propose that the centrosome acts as a signalling node or 'centrosomal processing unit' (CPU) via distal appendage proteins to coordinate the signalling of DNA damage and its repair, and speculate this may link to the clinical phenotypic overlap between centrosome-related ciliopathies and DDR signalling disorders (e.g. ATR-Seckel).

Major comments

1. Concerning Figs 1-3, it is argued that the presented skews in pathway choice are not an indirect consequence of cell-cycle effects that accompany centrosome depletion (i.e. following centrinone treatments) or depleted centrosome factors. Indeed, S1B shows centrione depleted cell show reduced S-phase indices (where HR is most active) are concordant with increased G2(/M) cell indices, significant effects that may contribute (at least in part) to some of the reported. In the case of the reporter assays it will be difficult/impossible to normalise data vs cell cycle skew, however in the case of RAD51 IRIF frequencies and RPA recruitment, this can be done easily by monitoring the relative frequencies of these events specifically S-phase (BrDU/EdU positive) cells. This should be done if the case for indirect cell-cycle effects is to be dismissed.
2. Related to point (1): RPA/RAD51/BRCA1 measurements made quantitatively (i.e. by QIBC or equivalent) given % IRIF positive cells can be misleading given it is completely subjective to user defined thesholds.
3. Fig 3 - The fact that CEP170 KD decreases BRCA1 IRIF but does not increase RIF1 IRIF, is not indicative of a lack of NHEJ stimulation, nor does it infer the existence of a/some distinct mechanism stimulating NHEJ, or an 'undiscovered factor', as is stated. This is important as RIF1 IRIF are not an accepted, nor accurate surrogate marker of NHEJ pathway activity, only an indicator of RIF1 recruitment downstream of 53BP1, whose role in resection control is clear, yet whose contribution to NHEJ is highly context-specific.
4. Is CEP170 Ser-637 an evolutionarily conserved ATM/ATR site? - Conservation, at least in mammals/vertebrates would be expected if a regulatory event in DSB pathway choice. This should be commented on with supplementary alignment included to demonstrate whether this is likely to be a universally conserved mechanism of repair regulation.
5. Fig 3F-G: Important to show appendage localisation of wild-type and mutant CEP170 S637A/D proteins to inform whether these are functional, expressed at equivalent levels and support equal centrosome localisation intensities. Immunoblot data in support of CEP170 siRNA depletion and CEP170 transgene complementation efficiencies is missing, and needs to be included to reassure a reader the results are specific to defects in the phosphorylation (not stability/expression level/other).
6. Do the CEP170 P'n nmutations affect its physiological centrosome functions? If separation of function is not experimentally defined, it should be at least discussed.

Comments on interpretation and accuracy of stated conclusions:

1. P12. - The manuscript is lacks the necessary evidence to support the section title: "CEP170 Ser647 phosphorylation is critical for HR double strand break repair", and as such I find this and related textual conclusions in the manuscript body to be inaccurate and misleading. To make this claim would require generating a cell-line knockin of the S647A mutation, preferably at the endogenous CEP170 locus (or a robust complementation system), and its utilisation to establish that standard measures of HR e.g. RAD51 recruitment, PARPi sensitivity, and/or SCE frequencies are all affected as expected in cells bearing this mutation.
2. Abstract reads: "we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosome component involved in the regulation of recombination and resection... " - I disagree with this statement given that the study has not excluded other centrosome components/features of the centrosome in regulation of resection. Can the authors perform experiments to exclude a role for other centrosome components and substantiate the conclusion that this is a specific function of the subdistal appendages as is stated?
3. Based on the marginal sensitivity phenotypes shown in Fig 4 for heterozygous cell-lines, it seems unlikely that CEP170 is a central player in the DSB response.
4. The CPU model for DDR-centric role of the centrosome is premature based on the provided data, likewise the fact that a centrosome-regulated resection could explain the clinical overlap between seckel and and this model should be toned down. We probably don't need another acronym for the DDR.

Minor comments

• Abstract, lasts sentence needs correction: "suggesting this protein can act as a driver mutation but also..." - a protein cannot act as a driver mutation.
• Information regarding biological replicates, sample sizes, error bars should be made more clear throughout to better represent reproducibility; e.g. n=3 {plus minus} Dt. Dev, biological replicates consisting >500 cells/nuclei per condition

Significance

General assessment

In exploring for functional links between DSB repair and the centrosome, the results encompass a series of corelating results that collectively hint at a potential role for the centrosome in repair regulation. The indirect and perhaps boring explanation for the presented DSB repair imbalances is these are an indirect consequence of the inevitable cell cycle defects that accompany centrosome depletion. In S1 the authors make some effort towards dispelling this less interesting (indirect) explanation for the presented results, yet not really far enough to dismiss it as the unifying explanation. A major consequence of centrosome-loss is prolonged time spent in G2/M dues to sub-optimal spindle nucleation and assembly kinetics, and an extended transit through mitosis, defects that occur independently of the p53-dependent checkpoint to centrosome loss (in fact the defects have long been speculated precede and perhaps propagate p53 activation). Indeed, supplementary data indicates that in centrosome-depleted cells a reduction in S-phase index (when HR activity is highest) correlates to greater proportion of cells with DNA with G2(/M) content. While I agree that these cell-cycle skews are unlikely to be great enough fully account for the reductions in HR reporter and IF proxies, more targeted approaches to control for indirect cell cycle effects (one suggestion below) could strengthen the case for a direct role in repair regulation. The manuscript also falls short of a identifying a discrete mechanism that explains centrosome-repair crosstalk, and on this basis I feel some of the conclusions are too preliminary and speculative and thus the authors would benefit from being more nuanced in their conclusions. One clear example is the authors's oversimplistic attribution of DSB regulation to distal appendage components of the centrosome/cilia, yet doing so having only tested the appendage proteins on the basis of literature based exercise of protein segregation of DDR and centrosome proteins (S2A). I also find it premature to propose "CPU" models of DDR regulation, the results (while interesting) haven't gone far enough to rigorously challenge this hypothesis, and define its mechanistic basis. I also question the importance and relevance of the analyses in Figs 4-5: in the absence of scientific evidence to establish causation for low CEP170 expression in tumour mutation signature burden or patient prognosis, the presented remain correlates that might equally result from a number of phenomena unrelated to DSB repair. As such, I feel the manuscript does encompass results worthy of report that would be of interest to cell cycle and DNA repair biologists, it would be greatly improved by being more rigorous, objective and nuanced in its interpretation.

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Referee #2

Evidence, reproducibility and clarity

In this manuscript by Rodriguez-Real et al, the authors address the contribution of the centrosome to cellular process unrelated to organizing the microtubule cytoskeleton, namely DNA repair. As many proteins contributing to the DNA damage response physically associate with centrosomes, this appears a relevant question that has been neglected so far and led to a number of studies that appeared controversial. To do so, the authors exploit a variety of tissue culture models that are well established in the fields of centrosomes and DNA repair, including U2OS and RPE1 cells, exposed to perturbations promoting DNA damage (such as ionizing radiation or pharmacologic perturbation of DNA stability) in conjunction with siRNA mediated depletion of candidate centrosomal proteins., followed by the visualization of repair events either using fluorescent reporters, or visualizing endogenous repair foci by immunofluorescence. On this basis, the authors propose that a discrete centrosomal sub-structure, namely sub-distal appendages and the CEP170 protein therein concur to promote a particular nuclear DNA repair process, namely homologous recombination.

The manuscript suffers of two main limitation:

1. the authors provide no mechanistic understanding of how CEP170, a protein that resides at centriolar subdistal appendages and shows no nuclear translocation upon DNA damage, concurs to regulate processes in the nucleus. The fact that all reported phenomena appear to be independent of microtubules suggests that neither the LINC complex nor the precise position of the centrosome in the vicinity of nuclear pore complexes contribute to the reported phenomena.
2. some of the experimental perturbations performed in the manuscript might elicit the reported phenotypes due to spurious effects on cellular processes that have not been considered with sufficient caution.

Given that uncovering the mechanism underlying the contribution of CEP170 to homologous recombination might prove very demanding, my comments will focus primarily on the second point.

Major comments:

The centriolar depletion using centrinone is known to impinge on cell proliferation in p53 WT cells. Thus, I am not convinced that the data shown in Figure S1B and S1C will sufficiently document that the observed unbalance between HDR and NHEJ are not simply reflecting a different cell cycling speed/behavior. Moreover, it would be important to address whether centrinone or depletion of CEP170 (an essential gene, according to the authors!) will trigger DNA damage by themselves. In fact, even a small extent of chronic genotoxic stress caused by the perturbations used in the manuscript might explain the reported differential proficiency of HDR.

Minor comments:

It is a pity that CEP170 is not amenable to functional dissection using a complete knockout. The fact that in PMID: 27818179 a complete knockout of CEP128 has been achieved, suggests however that subdistal appendage mediated DNA repair is not the essential process in itself. As the authors employ other cell lines stemming from the same laboratory, they could consider acquiring CEP128 KO to complement their own experiments.

The proposal that CEP170 phosphorylation of by ATM/ATR upon DNA damage might require SDA localization of the protein is plausible, yet not circumstantiated by any experimental evidence. If the authors could monitor the phosphorylation of the endogenous CEP170 protein in WT vs CEP128 KO cells (phosphor-specific antibody, MS-based proteomics or simply "phos-tag" gels), this could provide a first spark towards a mechanistic understanding of the reported phenomenon.

The entire Figure 4 is based on quantifications of clonogenic potential.

1. it would be helpful if the data were accompanied by images displaying representative crystal violet stained dishes.
2. clonogenic potential potential is discussed as a mere readout of cell survival, yet a combination between survival and proliferation concur to the reported differential clonogenic potential

Odf2 contribution to both DAs and SDAs: while Odf2 has been initially proposed to be necessary for the assembly of both types of appendages, its contribution to distal appendages has been disputed by Tanos et al using siRNA (PMID: 23348840), also confirmed by our group using CRISPR (unpublished). Thus, the role of Odf2 in SDA assembly appears more crucial than for DA assembly.

CEP164 contribution to ATM/ATR activation: this has been disputed in this paper by the Morrison lab (PMID: 26966185). Thus, a cautionary note should be mentioned when referring to this concept.

Significance

Taken together, this manuscript addresses the contribution of the centrosome to DNA repair. This is in itself a very interesting topic with the potential to attract the interest of both cell/molecular biologists as well as cancer researchers. The major advance strength is represented by pinpointing a specific centriolar substructure, namely subdistal appendages, in the control of HDR. CEP170 had been previously shown to be target of phosphorylation by ATM/R and the present study highlights that the abovementioned phosphorylation is not a mere passenger event during DNA repair, but that potentially reflects a decisive event informing the repair pathway of choice. However, several experiments have alternative explanations/interpretations and no understanding of the underlying mechanism is provided.

The expertise of this reviewer is the study of cell cycle regulation and on the centrosome structure/function.

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Referee #1

Evidence, reproducibility and clarity

Summary

Rodríguez-Real, Huertas and colleagues here explore the roles of centrosomes in DNA damage responses, focussing on DNA repair activities. They show that centrosome depletion by PLK4 inhibition leads to reduced levels of homologous recombination and increased nonhomologous end-joining, along with altered level of nuclear focus formation by DNA repair proteins. Knockdown of genes that encode components of centriolar subdistal appendages (SDAs) cause reduced levels of RPA foci, with CRISPR-generated CEP170 heterozygotes also showing defects in focus formation. Knockdown of CEP170 impairs homologous recombination, although NHEJ activities are unaffected. Some increase in sensitivity to DNA damaging agents is seen in CEP170- or centriole-deficient cells, albeit with a modest effect size. CEP170 status is shown to affect mutational signatures and patient prognosis in different cancer samples.

While the experiments are generally well-presented and controlled, the effects seen are not large, so that the the conclusions that the authors draw are not entirely substantiated by the data presented, even without the suggestion of a mechanism. There are several additional experiments and clarifications that I consider necessary to provide appropriate support for the phenomenon.

Major points

1. The lack of cell cycle arrest or phenotype in the U2OS cells after a week's treatment with centrinone is somewhat surprising, given their p53 status. The initial description of centrinone showed a distinct impact on U2OS proliferation, albeit after 2 weeks' treatment (although the present paper shows robust impact on centriole numbers after only 1 week in centrinone). It would be useful to know the percentage of mitotic cells, or if there is any increased cell death observed at this stage of treatment.
2. In the I-SceI assays, were transduction efficiencies or apoptosis within the experiment impacted by centrinone treatment? If not, it would be useful to state that this was examined and that there were no confounding effects; having only normalised data does not allow the reader to exclude these potential confounding factors.
3. The authors present binary data for a given type of nuclear focus (positive or negative for RPA/ BRCA1/ RAD51), while the supporting images show altered numbers/ intensities. For example, the BRCA1 signals shown in Fig. 3D are less readily distinguished than they are in Fig. 1D. These data should be reconsidered: it is possible that these observations reflect different kinetics of focus formation, rather than a change in IRIF formation capacity. Numbers and a timecourse should be provided, with details of how these are quantitated provided in the Methods.
4. Are the BRCA1 and RAD51 results seen with centrinone treatment of U2OS cells recapitulated in the Saos-2 and RPE1 lines?
5. Some additional analysis is needed of the extent to which cells are sensitised to DNA damaging treatments by CEP170 deficiency or centrinone treatment. It should be confirmed that these experiments were performed in biological triplicate, rather than a technical triplicate (within a single experiment); if this is not the case, these experiments should be done in triplicate. Analysing p53-deficient hTERT-RPE1 clones, Kumar et al. (NAR Cancer 2020 PMID: 33385162) showed <10% survival with 100 ng/ml NCS. Hustedt et al. (Genes Dev 2019 PMID: 31467087) showed just over 50% survival with 10 nM CPT treatment, although their data for IR were comparable to the current study. Given the wide variation that these assays seem to incur, the extent to which a ≈20% difference in clonogenic survival is biologically significant may be limited. A rescue of the CEP170 siRNA, and/ or washout in the centrinone experiment would make these data more convincing. The knockdown of CEP170 in Figure 4 should be correctly labelled (not as CEP170+/-); given that the authors have generated CEP170 heterozygotes in Figure 2, this is potentially confusing.
6. Direct data for the (centrosomal) phosphorylation of CEP170 are limited; it has not been demonstrated that the S637A mutants are fully functional in terms of the centrosome functions of CEP170, so that the conclusion regarding a requirement for centrosomal CEP170 phosphorylation is not sufficiently supported by the available data. The CEP170-dependent changes in RPA focus positive cell percentages shown in Figure 3 are not very marked. The relevant sections should be revised, or the authors should include additional experiments showing directly a phosphorylation of CEP170.
7. It is difficult to interpret the mutational spectrum data and their significance. These should be compared with data for mutations in NDEL1 mutant cells, and/or other SDA components.
8. The Kaplan-Meier curves data are intriguing, but their interpretation is highly speculative, given that there are no data on treatment groups included in this study. It is unclear whether other genes that affect SDAs might also impact survival (in the same, or different cancers), so the presentation of those patient groups where CEP170 status impacted survival seems selective, given the ubiquity of HR and centrosomes. These data would be better included as Supplemental information.
9. The independence of p53 status/ responsiveness of the system is a crucial aspect of this study. Sir et al. (JCB 2013 PMID: 24297747) showed no DNA repair defect in centrosome-deficient chicken DT40 cells. This paper is very relevant to the current study and should be discussed. Similarly, the work by Lambrus et al (JCB 2015 PMID: 26150389) should also be considered.

Minor points

1. References for the RPE1 TP53/ SAS6 mutant cell lines should be provided (or controls for their generation presented).
2. Fig S1K should correct its x-axis to reflect the time intervals correctly.
3. Fig 2D should show blow-ups of the centrosomes.
4. To avoid any potential confusion, it would be helpful to indicate in the Figure proper which cells are used for the various analyses.
5. The 'basal side' of the centriole is not a standard term- this should be clarified. This may be confusing, given the role of centrioles in the basal body.
6. The consideration of Seckel syndrome seems somewhat speculative at this stage in the exploration of this phenomenon.

Referees cross commenting I think the comments from Reviewers #2 and #3 are reasonable and justified; there is good convergence between the comments that we all made and I have no issues to raise in this cross-commentary.

Significance

Strengths: Much previous work linking centrosomes and DNA damage responses has addressed cell cycle and checkpoint roles of the centrosome, so that a direct role in (nuclear) DNA repair is intriguing. Limitations:The present study shows a relatively moderate impact of centrosome defects on DNA repair, without a clear mechanism. There are some technical details that should be addressed. The relatively limited sensitization to DNA damaging treatments caused by centrosome deficiency questions the biological significance of the phenomenon.

Advance: The current study presents some new findings that potentially show DNA repair defects resulting from the loss of centrioles (or SDA proteins). This has not been demonstrated to date.

Audience: The idea of subdistal appendage components contributing to homologous recombinational repair of DNA damage is of potential interest to several fields, ranging from basic centrosome biology through translational to clinical cancer research.

Reviewer's expertise: basic/ cell biology.

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Reply to the reviewers

General Statements:

We were very pleased and appreciative of the reviewer’s comments, and constructive suggestions for improving the manuscript. In response to their suggestions, we have added new text to better emphasize the importance of the question, the novelty of our approach, the significance of the results, and the potential for future discovery,

To summarize our key findings, we have identified 3,500 instances where – despite their shared ancestry - only one of two paralogous proteins undergoes a specific post-translational modification. By comparing adjoining sequences across 1012 isolates of the same yeast species, we determined that sequence conservation near sites of modification is greater than at sites that are not modified. We postulate that these differences in sequence are partly responsible for the differences in post-translational modifications, and that differences in modification allow duplicated proteins to be differentially regulated. These differences may account for their retention after 100M years of evolution.

Our analysis is clearly distinct from earlier investigations. In particular, we use new and substantially larger proteomics datasets reporting multiple types of post-translational modifications, new tools to analyze protein structure (AlphaFold), as well as new and expanded protein interactome datasets. Perhaps most importantly, we rely entirely on in-species sequence conservation data, with particular emphasis on duplicated proteins. Finally, we developed a custom algorithm (CoSMoS.c.) and web site that quantifies sequence conservation, in an automated fashion, across all 1012 unique strain isolates.

We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins and/or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin. Comparison within a single species is powerful because it avoids non-biological sources of uncertainty, such as potential alignment errors and any accompanying structural differences. Thus, by comparing unique modifications in closely-related gene products and across closely-related strain isolates, investigators using CoSMoS.c. will be better able to predict new enzyme-substrate relationships, identify new motifs for post-translational modifications, and prioritize mechanistic investigations of those modifications.

All of the reviewers asked that we explain the motivation for the design choice, compare our design with those used in earlier studies, add new controls for the effects of protein abundance, and provide examples of how our novel approach may be useful to investigators who study post-translational modifications. We are pleased to report that we were able to address all of these issues with revised text, additional references, two new control experiments, and real-world examples of individual paralog-paralog comparisons that have been useful in the past.

Finally, we have changed the title to: Differential modification____ of protein ____paralogs reveals conserved sequence determinants of post-translational ____modification

And we have changed the running title to: In-species evolution of protein modification sites

Reply to the Reviewers:

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

Summary: This paper reports bioinformatics analysis of population variation in PTM sites in paralogs from the yeast whole-genome-duplication. If I understand it correctly, the main finding is that modified sites show less population variation than paralagous unmodified sites. The results are largely in line with what is expected based on previous studies, though the authors do not present their results in that context.

Major comments:

1. The study benefits from two clever design choices:

First, comparison of sites between paralogs is a very powerful test for an evolutionary hypothesis because paralogous sites are expected to have relatively similar structural context. Second, use of within species polymorphism data is much less susceptible to alignment errors that can be an issue for longer evolutionary comparisons.

However, these design choices are not discussed or motivated by the authors. Nor are they compared to the designs of previous studies. Examples of previous studies (PMID: 22588506, PMID: 21273632, PMID: 20594336,PMID: 20594336, PMID: 24465218, PMID: 22889910, PMID: 20368267, PMID: 28054638)** *

We were very pleased and appreciative of the reviewer’s comments, and constructive suggestions for improving the manuscript. We have added nearly all references suggested by the reviewer, as well as new text describing____ the central findings of these papers, as follows:

”Most importantly, and in contrast with previous studies, we restricted our analysis to modified and unmodified pairs of paralogous proteins. This represents a very powerful test for the hypothesis because paralogs have a shared evolutionary history and are expected to have similar secondary structures. Moreover, the use of within-species polymorphism data is much less susceptible to the alignment errors that often occur with longer evolutionary comparisons.”

and

“Our analysis is clearly distinct from - and complementary to - earlier investigations of post-translational modifications in yeasts. … Our analysis builds on these foundational studies, by considering new and substantially larger proteomics datasets, multiple additional types of post-translational modifications, new and sophisticated models of protein structure, large-scale kinase interactome data, and in-species sequence conservation data – with particular emphasis on duplicated proteins.

We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences.”

• 1. One essential control that needs to be added is how much of the effect the authors observe can be explained by protein abundance. In yeast, protein abundance is strongly negatively correlated with evolutionary rate, and is strongly positively correlated with identification of PTMs in MS and other assays (extensively discussed in some of the previous studies I listed above). The authors need to assess whether their findings are due to the slow evolution of highly expressed proteins, and the detection bias for these proteins in PTM identification experiments. As far as I could tell this was not discussed by the authors.*

This point was also raised by Reviewer #2. We have added additional text stating that detection of PTMs by mass spectrometry is correlated with protein abundance.____ In addition, and as suggested by the reviewers, we have now done a control experiment using cross-study conservation of PTMs and limiting our comparison to proteins of similar abundance. By both methods, and as detailed below, we were able to confirm our original findings:

“We then reanalyzed our data to account for possible effects of protein abundance, which in cross species comparisons was observed to negatively correlate with evolutionary rate and positively correlate with modification detection by mass spectrometry (39). Accordingly, we restricted our in-species analysis to a subset of 270 paralog pairs that have similar ( 100 instances each of phosphorylation, ubiquitylation and succinylation, where the target and paralog have the same amino acid, but only the target is modified. Even with this restricted dataset, we obtained similar results for all three types of analysis (Dataset S9). We also considered the potential effect of false positives and false negatives among the reported modification sites. False positives can result from ambiguous assignments, as might arise through misidentification of modified sites within peptides that contain multiple potential sites of modification. False negatives can result from difficulties in detecting modifications in poorly expressed proteins (39), or an overly strict reliance on high confidence sites. We then further restricted the data to only include modifications identified in multiple studies. After applying this additional filter, we were left with > 100 instances of phosphorylation. Once again, we obtained similar results for Symmetric Average Score and One-sided Average Score analysis, but not for Chemical Similarity Average Score, which is further restricted by splitting the data into five chemical categories (Dataset S10).”

• 3.A major weakness of the paper is its lack of focus. It includes a rambling historical introduction and discussion that omits discussion of the relevant recent research directly related to the questions at hand. For example, the paper describes historical work on phosphorylase, but gives not a single example of a paralog pair with a polymorphic PTM site identified in their study. The authors introduce gene duplication in a very general way, even though several papers have focused specifically on evolution of protein regulation in paralogs (e.g., PMID: 20080574, PMID: 27003913, PMID: 25474245) The paper of Nguyen Ba et al. 2014 (PMID: 25474245) seems especially relevant, as in addition to perfoming a genome-wide analysis, their abstract reads "We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation." It seems that the results of that study could be directly compared to the analysis performed here.*

This point was also raised by Reviewer 2. At the suggestion of the reviewers, we have moved or removed discussion of these foundational studies of PTM mapping and added discussion of well-characterized examples of paralog pairs with polymorphic PTM sites, based on the references provided, as follows:

“We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences. This is supported by a small number of prior studies, which compared four sets of paralogous proteins in yeast - Rck1 v. Rck2, Fkh1 v. Fkh2, Ace2 v. Swi5 (68), and Boi1 v. Boi2 (58), and concluded that divergence in short linear motifs is likely responsible for differences in phosphorylation. While paralogs are far less common in other organisms, a similar conclusion emerged from a comparison of predicted sites of phosphorylation in mammalian p53, p63 and p73 (69).

Our analysis of differentially-modified pairs of paralogous proteins revealed that the most common modifications – phosphorylation, ubiquitylation and acylation but not N-glycosylation – occur within regions of high sequence conservation. Further studies will benefit from the availability of our search algorithm CoSMoS.c.. For example, when studying a particular protein kinase, CoSMoS.c. can be used to identify specific motifs near potentially modified serines, threonines and tyrosines (Table 2). When studying a particular substrate of ubiquitylation, CoSMoS.c. can be used to prioritize conserved versus non-conserved sequences flanking potentially modified lysines. For rare modifications, CoSMoS.c. can also be used to locate highly conserved regions as the starting points for finding new sequence motifs. Thus, by comparing unique modifications in closely-related gene products and across closely-related strain isolates, we can prioritize mechanistic investigations of modifications that are likely to have functional importance, to identify recognition motifs for specific modifying enzymes, and to better predict new enzyme-substrate relationships.”

*Reviewer #1 (Significance (Required)):

The significance is hard to assess because the research is not given proper context and motivation.

I believe the study could be of interest to research studying cell signalling and its evolution, as well as those interested in gene family diversification. However, as written, no specific examples are given or clear hypotheses tested, making the paper seem largely descriptive.

My keywords: molecular evolution, signalling, intrinsically disordered regions, computational biology

*

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

Summary

The authors of this work study how S. cerevisiae paralogue pairs are differentially modified with respect to five major PTM classes: phosphorylation, ubiquitination, mono-acetylation, N-glycosylation, and succinylation. Emphasis is placed on paralogue pairs where a modification is found in only one of the two paralogues at homologous positions. A conservation analysis is then performed across 1011 S. cerevisiae isolates to check for differences in conservation between the modified target and its unmodified paralogue. The authors claim that, for most of the PTM classes, modified targets tend to be more conserved than their unmodified paralogues. Phosphorylation sites between paralogue pairs were also compared using AlphaFold2 and a database of kinase interactions (YeastKID), revealing differential interactions between paralogues but no significant structural differences. *

We were very pleased and appreciative of the reviewer’s comments, and constructive suggestions for improving the manuscript.____ * *

*Major:

1) A major issue with this work is that the problem of 'false negatives' for PTM detection is never adequately addressed or controlled for. As the authors allude to in the manuscript, the number of PTM sites detected is likely far below the number that exists and this is especially a problem for the less well characterised PTM classes. How then can the authors be confident that an 'unmodified' site is truly unmodified and not just undetected? The authors can refer to Freschi et al., 2011 (MSB) for a method that controls for the false negative (FN) PTM detection rate by comparing cross-study conservation with cross-study reproducibility. *

* 2) The second point follows closely from the first. The issue is that MS-based PTM detection is generally biased towards abundant proteins, and protein abundance also correlates strongly with evolutionary rate, with more abundant proteins tending to have higher conservation. Taken together, these two relationships could explain the observation that the modified paralogue tends to be more conserved than the 'unmodified' paralogue. The authors should try and control for the effect of protein abundance on the results observed; for example, by checking if the results/conclusions change when restricting the analysis to paralogue pairs with similar abundances. *

* 3) Alongside false negatives, there is the cognate issue of false positives and mislocalised PTM sites (see Lanz et al., 2021, EMBO Reports). If possible, the authors should check to see if their conclusions change when restricting the analysis to high-confidence PTM sites identified from multiple sources and/or validated by low throughput experimental assays.*

__This point was also raised by Reviewer #1. To address the concern, we have now done a new control analysis, one that uses only those modifications identified in multiple studies and comparing only proteins of similar (“We then reanalyzed our data to account for possible effects of protein abundance, which in cross species comparisons was observed to negatively correlate with evolutionary rate and positively correlate with modification detection by mass spectrometry (39). Accordingly, we restricted our in-species analysis to a subset of 270 paralog pairs that have similar ( 100 instances each of phosphorylation, ubiquitylation and succinylation, where the target and paralog have the same amino acid, but only the target is modified. Even with this restricted dataset, we obtained similar results for all three types of analysis (Dataset S9). We also considered the potential effect of false positives and false negatives among the reported modification sites. False positives can result from ambiguous assignments, as might arise through misidentification of modified sites within peptides that contain multiple potential sites of modification. False negatives can result from difficulties in detecting modifications in poorly expressed proteins (39), or an overly strict reliance on high confidence sites. We then further restricted the data to only include modifications identified in multiple studies. After applying this additional filter, we were left with > 100 instances of phosphorylation. Once again, we obtained similar results for Symmetric Average Score and One-sided Average Score analysis, but not for Chemical Similarity Average Score, which is further restricted by splitting the data into five chemical categories (Dataset S10).”

4) The authors define conservation here using 1011 wild and domesticated yeast isolates within one species (S. cerevisiae). While this is clearly valuable information, this reviewer wonders why orthologues from closely related species were not also leveraged to assess the evolutionary rate, as is traditionally done for studies on PTM evolution? Is there a strong rationale for this? Using more distantly-related genomes could give more statistical power for the detection of weak differences in selective constraint between paralogues.

We believe that a major strength of our study is the reliance on____ in-species sequence conservation data – with particular emphasis on duplicated proteins. To better emphasize this point, we have added new text as follows:

”Most importantly, and in contrast with previous studies, we restricted our analysis to modified and unmodified pairs of paralogous proteins. This represents a very powerful test for the hypothesis because paralogs have a shared evolutionary history and are expected to have similar secondary structures. Moreover, the use of in-species polymorphism data is much less susceptible to the alignment errors that often occur with longer evolutionary comparisons.”

and

“We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences.”

*Minor:

1) Both the Introduction and Discussion describe PTMs and the evolution of gene duplication in very general terms. However, literature concerning the evolution of PTMs and specifically the evolution of PTMs following gene duplication has been largely ignored. These studies give the most relevant context to this work and should be described and cited. Freschi et al., 2011 (Molecular Systems Biology) and Ba et al., 2014 (PloS Computational Biology) are particularly relevant. *

We have added references suggested by the reviewer, as well as new text describing the central findings of these papers, as follows:

“Our analysis is clearly distinct from - and complementary to - earlier investigations of post-translational modifications in yeasts. Previous analysis showed that duplicated proteins in Saccharomyces cerevisiae are more likely to be phosphorylated, and to have a greater number of phosphorylation sites, than non-duplicated proteins (58). The difference persisted when controlling for differences in protein abundance, coverage, essentiality, positioning within protein interaction networks and assembly into multi-protein complexes (58). When compared with a yeast species that diverged before the whole genome duplication event, it appears that the majority of phosphorylation sites in paralogs have either been lost or gained, with a strong bias toward losses (56). Subsequent cross-species comparisons noted a high degree of sequence conservation near sites of phosphorylation and other types of modification in yeasts (49, 59-65). The relationship was strongest for phosphosites with known function (49, 50, 61). A focused study of 249 unique high-confidence phosphorylation sites, targeted by 7 protein kinases in S. cerevisiae, confirmed that regions flanking sites of phosphorylation are significantly constrained, in comparison with other closely related yeast species (61). A similar relationship exists for sites phosphorylated by the cyclin-dependent protein kinase Cdk1 (66), and was the basis for predicting novel sites of phosphorylation by the cAMP-dependent protein kinase (67). Our analysis builds on these foundational studies, by considering new and substantially larger proteomics datasets, multiple additional types of post-translational modifications, new and sophisticated models of protein structure, large-scale kinase interactome data, and in-species sequence conservation data – with particular emphasis on duplicated proteins.

We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences. This is supported by a small number of prior studies, which compared four sets of paralogous proteins in yeast - Rck1 v. Rck2, Fkh1 v. Fkh2, Ace2 v. Swi5 (68), and Boi1 v. Boi2 (58), and concluded that divergence in short linear motifs is likely responsible for differences in phosphorylation. While paralogs are far less common in other organisms, a similar conclusion emerged from a comparison of predicted sites of phosphorylation in mammalian p53, p63 and p73 (69).”

*2) While I enjoyed to a limited extent the historical perspective on PTM discovery, there is far too much text given to this overall and the writing should be made more concise by removing excessive detail. This is especially the case for the Results section, where the emphasis should be on the analysis performed by the authors. *

This point was also raised by Reviewer 1. At the suggestion of the reviewers, we have moved or removed discussion of these foundational studies of PTM mapping and added discussion of well-characterized examples of paralog pairs with polymorphic PTM sites, based on the references provided, as detailed above.

*3) Description of the methodology should be reviewed for language and clarity. In particular, the authors should explain explicitly the meaning of new terms such as 'pairing structure' and how this may confer an 'advantage / disadvantage' to target proteins -- wording that this reviewer found especially confusing and unnecessary. The authors should also be explicit about how the distributions for each test are constructed; the current wording sometimes gives the impression that a distribution is derived from a single target or paralogue instead of being derived from a set of modified targets and the corresponding set of unmodified paralogues. Another confusion is that the Distribution Mean Test is contrasted with the Paralog Pairing Test in Fig S8 and yet on page 15 the Distribution Mean Test is described as 'paired' test on page 15 even though from the description the test seems unpaired? *

We are now more explicit about how the distributions for each test are constructed, and we have clarified the meaning of the terms 'pairing structure', 'advantage / disadvantage' and ‘Distribution Mean Test’, as follows:

“We then performed two statistical tests: the Distribution Mean Test, which determines whether the mean of the distribution of target protein conservation scores (that is, the mean conservation score for all modified target proteins) is significantly larger than that of the unmodified paralogs, and the Paralog Pairing Test, which tests whether the pairing structure confers an advantage for the target proteins. Figure 2 presents two possible pairing structures (panels A and C) and how these can advantage (panels A and B) or disadvantage (panels C and D) target proteins...”

“In this instance we applied a one-sided, paired Mann-Whitney-Wilcoxon Test (100), which determines whether the target protein conservation score distribution is significantly larger than the unmodified paralog conservation score distribution, without assuming that they follow a normal distribution. We used the paired test because the comparison is between the means of paired observations that have a relationship between the two groups (modified target and unmodified paralogs). Hereafter we refer to this as Distribution Mean Test.”

4) Following on from point 2) in the 'major' section above, the authors could consider normalising the conservation scores within a protein to control for the effect of protein abundance and other potential confounders acting at the protein level.

We have added additional text stating that detection of PTMs by mass spectrometry is correlated with protein abundance.____ In addition, and as suggested by the reviewers, we have now done a control experiment using cross-study conservation of PTMs and limiting our comparison to proteins of similar abundance. By both methods, and as detailed below, we were able to confirm our original findings:

“We then reanalyzed our data to account for possible effects of protein abundance, which in cross species comparisons was observed to negatively correlate with evolutionary rate and positively correlate with modification detection by mass spectrometry (39). Accordingly, we restricted our in-species analysis to a subset of 270 paralog pairs that have similar ( 100 instances each of phosphorylation, ubiquitylation and succinylation, where the target and paralog have the same amino acid, but only the target is modified. Even with this restricted dataset, we obtained similar results for all three types of analysis (Dataset S9). We also considered the potential effect of false positives and false negatives among the reported modification sites. False positives can result from ambiguous assignments, as might arise through misidentification of modified sites within peptides that contain multiple potential sites of modification. False negatives can result from difficulties in detecting modifications in poorly expressed proteins (39), or an overly strict reliance on high confidence sites. We then further restricted the data to only include modifications identified in multiple studies. After applying this additional filter, we were left with > 100 instances of phosphorylation. Once again, we obtained similar results for Symmetric Average Score and One-sided Average Score analysis, but not for Chemical Similarity Average Score, which is further restricted by splitting the data into five chemical categories (Dataset S10).”

*5) For the analysis of motifs, departure from the BLOSUM62 expectation may just reflect the fact that many of these PTMs fall in disordered regions - which have distinct amino acid propensities -- whereas matrices like BLOSUM62 were constructed mostly from ordered protein regions. *

We have modified the Materials and Methods section to reflect this alternative, as follows:

“If the observed changes differ substantially from expectation (BLOSUM62), this suggests the presence of selection pressure and functional importance. This might also arise from distinct amino acid propensities when comparing ordered protein regions, from which the BLOSUM62 matrices were constructed, and disordered regions, where most modifications are likely to occur. This is unlikely to impact our results, as we are comparing structurally similar paralogous proteins. In addition, we are using multiple score algorithms to support our conclusions.”

6) The analysis of sequence motifs could be extended by scoring phosphosites with yeast position weight matrices (PWMs) for protein kinases and comparing the results between modified targets and their unmodified paralogues. This can help distinguish true positive and false negative modification differences. See Freschi et al., 2011 (Molecular Systems Biology).

We have performed this analysis according to the reviewer’s suggestion and added new text to the Results, as follows:

“Finally, in an initial effort to match sites of phosphorylation with protein kinases, we used the position-weight matrices (PWMs) developed by Mok et al. (56, 57). That analysis determined phosphorylation site selectivity for 61 of the 122 kinases in Saccharomyces cerevisiae and proposed empirically-derived PWMs that enable the assignment of candidate protein kinases to known sites of phosphorylation (56, 57). We applied the PWMs to our dataset, which contains sites where one of the two proteins is known to be phosphorylated and the amino acid residue is the same in both. From this dataset, we kept 190 paralogous pairs where each protein contains at least one such phosphorylation site, so that both proteins would have kinase interactions to be compared. Using the PWMs from (57), we assigned the kinase that most likely corresponds to each phosphorylation site, as implemented in (56). Out of the 190 paralogous pairs, 130 interacted with different kinases. Together, these results indicate that most kinases regulate one or the other of the protein paralogs. They suggest further that differential modifications reported here may be the result of differential interactions with modifying enzymes.”

*Reviewer #2 (Significance (Required)):

This work is potentially of specialist interest to researchers studying the evolution of PTMs. While the evolution of phosphorylation following gene duplication has been studied previously (Freschi et al 2011, MSB), this work considers other PTM classes and takes advantage of a much larger data set. Potentially, clear examples of paralogue PTM divergence could be used as a basis for follow-up experiments. However, the web-server as it is now is designed to facilitate the easy analysis of a single protein at a time and not comparisons across paralogue pairs.

*

We have added new text to better emphasize the importance of the question, the novelty of our approach, the significance of the results, and the potential for future discovery, as follows:

“Post-translational modifications are critical functional elements within proteins, and are therefore expected to be conserved in evolution. Here, we have identified several thousand instances where, despite a shared ancestry, only one of two paralogous proteins undergoes a specific post-translational modification. We also developed a custom algorithm that quantifies sequence conservation, in an automated fashion, across 1012 unique strain isolates. By comparing adjoining sequences in multiple isolates of the same species, we determined that sequence conservation near sites of modification is greater than at sites that are not modified. In addition, many of the modifications were associated with characteristic sequence elements nearby. We postulate that these differences in sequence conservation are partly responsible for differences in post-translational modifications, that differences in post-translational modifications allow duplicated proteins to be differentially regulated, and these differences may account for their retention after 100M years of evolution.

Our analysis is clearly distinct from - and complementary to - earlier investigations of post-translational modifications in yeasts. Previous analysis showed that duplicated proteins in Saccharomyces cerevisiae are more likely to be phosphorylated, and to have a greater number of phosphorylation sites, than non-duplicated proteins (58). The difference persisted when controlling for differences in protein abundance, coverage, essentiality, positioning within protein interaction networks and assembly into multi-protein complexes (58). When compared with a yeast species that diverged before the whole genome duplication event, it appears that the majority of phosphorylation sites in paralogs have either been lost or gained, with a strong bias toward losses (56). Subsequent cross-species comparisons noted a high degree of sequence conservation near sites of phosphorylation and other types of modification in yeasts (49, 59-65). The relationship was strongest for phosphosites with known function (49, 50, 61). A focused study of 249 unique high-confidence phosphorylation sites, targeted by 7 protein kinases in S. cerevisiae, confirmed that regions flanking sites of phosphorylation are significantly constrained, in comparison with other closely related yeast species (61). A similar relationship exists for sites phosphorylated by the cyclin-dependent protein kinase Cdk1 (66), and was the basis for predicting novel sites of phosphorylation by the cAMP-dependent protein kinase (67). Our analysis builds on these foundational studies, by considering new and substantially larger proteomics datasets, multiple additional types of post-translational modifications, new and sophisticated models of protein structure, large-scale kinase interactome data, and in-species sequence conservation data – with particular emphasis on duplicated proteins.

We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences. This is supported by a small number of prior studies, which compared four sets of paralogous proteins in yeast - Rck1 v. Rck2, Fkh1 v. Fkh2, Ace2 v. Swi5 (68), and Boi1 v. Boi2 (58), and concluded that divergence in short linear motifs is likely responsible for differences in phosphorylation. While paralogs are far less common in other organisms, a similar conclusion emerged from a comparison of predicted sites of phosphorylation in mammalian p53, p63 and p73 (69).

Our analysis of differentially-modified pairs of paralogous proteins revealed that the most common modifications – phosphorylation, ubiquitylation and acylation but not N-glycosylation – occur within regions of high sequence conservation. Further studies will benefit from the availability of our search algorithm CoSMoS.c.. For example, when studying a particular protein kinase, CoSMoS.c. can be used to identify specific motifs near potentially modified serines, threonines and tyrosines (Table 2). When studying a particular substrate of ubiquitylation, CoSMoS.c. can be used to prioritize conserved versus non-conserved sequences flanking potentially modified lysines. For rare modifications, CoSMoS.c. can also be used to locate highly conserved regions as the starting points for finding new sequence motifs. Thus, by comparing unique modifications in closely-related gene products and across closely-related strain isolates, we can prioritize mechanistic investigations of modifications that are likely to have functional importance, to identify recognition motifs for specific modifying enzymes, and to better predict new enzyme-substrate relationships.”

__In addition, and in response to the reviewer’s suggestion, we are currently expanding the web site to facilitate comparisons across paralogue pairs. ____

__

Currently, the major problems stated above 1) correction for the problem of false negatives, and 2) correction for the confounding effects of protein abundance need to be addressed before the results can be fully interpreted and evaluated.

As detailed above under Points 1-3,____ we have now done a control experiment using cross-study conservation of PTMs and limiting our comparison to proteins of similar abundance. By both methods, we were able to confirm our original findings, as detailed above.

*Reviewer field of expertise: phosphosite evolution, PTM evolution, protein evolution.

*

* *

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

This manuscript describes the evolutionary conservation of yeast post-translationally modified residues and sequence motifs surrounding them.

Reviewer #3 (Significance (Required)):

Although this is not new, (Beltrao, Cell 2012, Minguez, MSB 2012, Hendriksen 2012) all show that sites of acetylation, phosphorylation and other modifications are more conserved in yeast than would be expected. Beltrao and Minguez also provide webservers http://ptmfunc.com/ http://ptmcode.embl.de where the link of conserved modified sites is made to protein structures and protein-protein interactions.

The novelty of this study is in studying the duplicated proteins after whole genome duplication as well as providing an online interactive server where the conservation can be retrieved in detail, different scoring functions are provided. In addition, the conservation is calculated in closely related species rather than long evolutionary distances as previous studies have done.

I am missing a concrete example of how a researcher would use the resource that the authors introduce here, and how it is an advance to previously proposed methods. For example, are there sites found conserved in this set of more closely related organisms, that are not conserved in yeast versus metazoa? Is the more fine-grained methodology useful to detect motif sequences that can otherwise not be detected? Can the authors provide proof that indeed the conserved sites are more functional than non-conserved?

*

*At the moment the manuscript describes very little results, and only a possible advance compared to previous methods, no proof is given that an actual advance is made. *

The authors should compare their work to previous work in this field.

We were very pleased and appreciative of the reviewer’s comments, and constructive suggestions for improving the manuscript.____ We have added new text to better emphasize the importance of the question, the novelty of our approach, the significance of the results, and the potential for future discovery, as follows:

“Post-translational modifications are critical functional elements within proteins, and are therefore expected to be conserved in evolution. Here, we have identified several thousand instances where, despite a shared ancestry, only one of two paralogous proteins undergoes a specific post-translational modification. We also developed a custom algorithm that quantifies sequence conservation, in an automated fashion, across 1012 unique strain isolates. By comparing adjoining sequences in multiple isolates of the same species, we determined that sequence conservation near sites of modification is greater than at sites that are not modified. In addition, many of the modifications were associated with characteristic sequence elements nearby. We postulate that these differences in sequence conservation are partly responsible for differences in post-translational modifications, that differences in post-translational modifications allow duplicated proteins to be differentially regulated, and these differences may account for their retention after 100M years of evolution.

Our analysis is clearly distinct from - and complementary to - earlier investigations of post-translational modifications in yeasts. Previous analysis showed that duplicated proteins in Saccharomyces cerevisiae are more likely to be phosphorylated, and to have a greater number of phosphorylation sites, than non-duplicated proteins (58). The difference persisted when controlling for differences in protein abundance, coverage, essentiality, positioning within protein interaction networks and assembly into multi-protein complexes (58). When compared with a yeast species that diverged before the whole genome duplication event, it appears that the majority of phosphorylation sites in paralogs have either been lost or gained, with a strong bias toward losses (56). Subsequent cross-species comparisons noted a high degree of sequence conservation near sites of phosphorylation and other types of modification in yeasts (49, 59-65). The relationship was strongest for phosphosites with known function (49, 50, 61). A focused study of 249 unique high-confidence phosphorylation sites, targeted by 7 protein kinases in S. cerevisiae, confirmed that regions flanking sites of phosphorylation are significantly constrained, in comparison with other closely related yeast species (61). A similar relationship exists for sites phosphorylated by the cyclin-dependent protein kinase Cdk1 (66), and was the basis for predicting novel sites of phosphorylation by the cAMP-dependent protein kinase (67). Our analysis builds on these foundational studies, by considering new and substantially larger proteomics datasets, multiple additional types of post-translational modifications, new and sophisticated models of protein structure, large-scale kinase interactome data, and in-species sequence conservation data – with particular emphasis on duplicated proteins.

We propose that in-species comparisons of paralogs will prove to be more reliable than cross-species comparisons of orthologous proteins, or in-species comparisons of non-homologous proteins. Comparison of paralogs is powerful because they are likely to have similar structures and functions, due to their shared evolutionary origin (56, 58, 68). Comparison within a single species is powerful because it allows us to avoid important non-biological sources of uncertainty, such as potential alignment errors and unknown structural or functional differences. This is supported by a small number of prior studies, which compared four sets of paralogous proteins in yeast - Rck1 v. Rck2, Fkh1 v. Fkh2, Ace2 v. Swi5 (68), and Boi1 v. Boi2 (58), and concluded that divergence in short linear motifs is likely responsible for differences in phosphorylation. While paralogs are far less common in other organisms, a similar conclusion emerged from a comparison of predicted sites of phosphorylation in mammalian p53, p63 and p73 (69).

Our analysis of differentially-modified pairs of paralogous proteins revealed that the most common modifications – phosphorylation, ubiquitylation and acylation but not N-glycosylation – occur within regions of high sequence conservation. Further studies will benefit from the availability of our search algorithm CoSMoS.c.. For example, when studying a particular protein kinase, CoSMoS.c. can be used to identify specific motifs near potentially modified serines, threonines and tyrosines (Table 2). When studying a particular substrate of ubiquitylation, CoSMoS.c. can be used to prioritize conserved versus non-conserved sequences flanking potentially modified lysines. For rare modifications, CoSMoS.c. can also be used to locate highly conserved regions as the starting points for finding new sequence motifs. Thus, by comparing unique modifications in closely-related gene products and across closely-related strain isolates, we can prioritize mechanistic investigations of modifications that are likely to have functional importance, to identify recognition motifs for specific modifying enzymes, and to better predict new enzyme-substrate relationships.”

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Referee #3

Evidence, reproducibility and clarity

This manuscript describes the evolutionary conservation of yeast post-translationally modified residues and sequence motifs surrounding them.

Significance

Although this is not new, (Beltrao, Cell 2012, Minguez, MSB 2012, Hendriksen 2012) all show that sites of acetylation, phosphorylation and other modifications are more conserved in yeast than would be expected. Beltrao and Minguez also provide webservers http://ptmfunc.com/ http://ptmcode.embl.de where the link of conserved modified sites is made to protein structures and protein-protein interactions.

The novelty of this study is in studying the duplicated proteins after whole genome duplication as well as providing an online interactive server where the conservation can be retrieved in detail, different scoring functions are provided. In addition, the conservation is calculated in closely related species rather than long evolutionary distances as previous studies have done.

I am missing a concrete example of how a researcher would use the resource that the authors introduce here, and how it is an advance to previously proposed methods. For example, are there sites found conserved in this set of more closely related organisms, that are not conserved in yeast versus metazoa? Is the more fine-grained methodology useful to detect motif sequences that can otherwise not be detected? Can the authors provide proof that indeed the conserved sites are more functional than non-conserved?

At the moment the manuscript describes very little results, and only a possible advance compared to previous methods, no proof is given that an actual advance is made.

The authors should compare their work to previous work in this field.

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Referee #2

Evidence, reproducibility and clarity

Summary

The authors of this work study how S. cerevisiae paralogue pairs are differentially modified with respect to five major PTM classes: phosphorylation, ubiquitination, mono-acetylation, N-glycosylation, and succinylation. Emphasis is placed on paralogue pairs where a modification is found in only one of the two paralogues at homologous positions. A conservation analysis is then performed across 1011 S. cerevisiae isolates to check for differences in conservation between the modified target and its unmodified paralogue. The authors claim that, for most of the PTM classes, modified targets tend to be more conserved than their unmodified paralogues. Phosphorylation sites between paralogue pairs were also compared using AlphaFold2 and a database of kinase interactions (YeastKID), revealing differential interactions between paralogues but no significant structural differences.

Major:

1. A major issue with this work is that the problem of 'false negatives' for PTM detection is never adequately addressed or controlled for. As the authors allude to in the manuscript, the number of PTM sites detected is likely far below the number that exists and this is especially a problem for the less well characterised PTM classes. How then can the authors be confident that an 'unmodified' site is truly unmodified and not just undetected? The authors can refer to Freschi et al., 2011 (MSB) for a method that controls for the false negative (FN) PTM detection rate by comparing cross-study conservation with cross-study reproducibility.
2. The second point follows closely from the first. The issue is that MS-based PTM detection is generally biased towards abundant proteins, and protein abundance also correlates strongly with evolutionary rate, with more abundant proteins tending to have higher conservation. Taken together, these two relationships could explain the observation that the modified paralogue tends to be more conserved than the 'unmodified' paralogue. The authors should try and control for the effect of protein abundance on the results observed; for example, by checking if the results/conclusions change when restricting the analysis to paralogue pairs with similar abundances.
3. Alongside false negatives, there is the cognate issue of false positives and mislocalised PTM sites (see Lanz et al., 2021, EMBO Reports). If possible, the authors should check to see if their conclusions change when restricting the analysis to high-confidence PTM sites identified from multiple sources and/or validated by low throughput experimental assays.
4. The authors define conservation here using 1011 wild and domesticated yeast isolates within one species (S. cerevisiae). While this is clearly valuable information, this reviewer wonders why orthologues from closely related species were not also leveraged to assess the evolutionary rate, as is traditionally done for studies on PTM evolution? Is there a strong rationale for this? Using more distantly-related genomes could give more statistical power for the detection of weak differences in selective constraint between paralogues.

Minor:

1. Both the Introduction and Discussion describe PTMs and the evolution of gene duplication in very general terms. However, literature concerning the evolution of PTMs and specifically the evolution of PTMs following gene duplication has been largely ignored. These studies give the most relevant context to this work and should be described and cited. Freschi et al., 2011 (Molecular Systems Biology) and Ba et al., 2014 (PloS Computational Biology) are particularly relevant.
2. While I enjoyed to a limited extent the historical perspective on PTM discovery, there is far too much text given to this overall and the writing should be made more concise by removing excessive detail. This is especially the case for the Results section, where the emphasis should be on the analysis performed by the authors.
3. Description of the methodology should be reviewed for language and clarity. In particular, the authors should explain explicitly the meaning of new terms such as 'pairing structure' and how this may confer an 'advantage / disadvantage' to target proteins -- wording that this reviewer found especially confusing and unnecessary. The authors should also be explicit about how the distributions for each test are constructed; the current wording sometimes gives the impression that a distribution is derived from a single target or paralogue instead of being derived from a set of modified targets and the corresponding set of unmodified paralogues. Another confusion is that the Distribution Mean Test is contrasted with the Paralog Pairing Test in Fig S8 and yet on page 15 the Distribution Mean Test is described as 'paired' test on page 15 even though from the description the test seems unpaired?
4. Following on from point 2) in the 'major' section above, the authors could consider normalising the conservation scores within a protein to control for the effect of protein abundance and other potential confounders acting at the protein level.
5. For the analysis of motifs, departure from the BLOSUM62 expectation may just reflect the fact that many of these PTMs fall in disordered regions - which have distinct amino acid propensities -- whereas matrices like BLOSUM62 were constructed mostly from ordered protein regions.
6. The analysis of sequence motifs could be extended by scoring phosphosites with yeast position weight matrices (PWMs) for protein kinases and comparing the results between modified targets and their unmodified paralogues. This can help distinguish true positive and false negative modification differences. See Freschi et al., 2011 (Molecular Systems Biology).

Significance

This work is potentially of specialist interest to researchers studying the evolution of PTMs. While the evolution of phosphorylation following gene duplication has been studied previously (Freschi et al 2011, MSB), this work considers other PTM classes and takes advantage of a much larger data set. Potentially, clear examples of paralogue PTM divergence could be used as a basis for follow-up experiments. However, the web-server as it is now is designed to facilitate the easy analysis of a single protein at a time and not comparisons across paralogue pairs.

Currently, the major problems stated above 1) correction for the problem of false negatives, and 2) correction for the confounding effects of protein abundance need to be addressed before the results can be fully interpreted and evaluated.

Reviewer field of expertise: phosphosite evolution, PTM evolution, protein evolution.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This paper reports bioinformatics analysis of population variation in PTM sites in paralogs from the yeast whole-genome-duplication. If I understand it correctly, the main finding is that modified sites show less population variation than paralagous unmodified sites. The results are largely in line with what is expected based on previous studies, though the authors do not present their results in that context.

Major comments:

1. The study benefits from two clever design choices:

First, comparison of sites between paralogs is a very powerful test for an evolutionary hypothesis because paralogous sites are expected to have relatively similar structural context. Second, use of within species polymorphism data is much less susceptible to alignment errors that can be an issue for longer evolutionary comparisons.

However, these design choices are not discussed or motivated by the authors. Nor are they compared to the designs of previous studies. Examples of previous studies (PMID: 22588506, PMID: 21273632, PMID: 20594336,PMID: 20594336, PMID: 24465218, PMID: 22889910, PMID: 20368267, PMID: 28054638) 2. One essential control that needs to be added is how much of the effect the authors observe can be explained by protein abundance. In yeast, protein abundance is strongly negatively correlated with evolutionary rate, and is strongly positively correlated with identification of PTMs in MS and other assays (extensively discussed in some of the previous studies I listed above). The authors need to assess whether their findings are due to the slow evolution of highly expressed proteins, and the detection bias for these proteins in PTM identification experiments. As far as I could tell this was not discussed by the authors. 3. A major weakness of the paper is its lack of focus. It includes a rambling historical introduction and discussion that omits discussion of the relevant recent research directly related to the questions at hand. For example, the paper describes historical work on phosphorylase, but gives not a single example of a paralog pair with a polymorphic PTM site identified in their study. The authors introduce gene duplication in a very general way, even though several papers have focused specifically on evolution of protein regulation in paralogs (e.g., PMID: 20080574, PMID: 27003913, PMID: 25474245) The paper of Nguyen Ba et al. 2014 (PMID: 25474245) seems especially relevant, as in addition to perfoming a genome-wide analysis, their abstract reads "We examine changes in constraints on known regulatory sequences and show that for the Rck1/Rck2, Fkh1/Fkh2, Ace2/Swi5 paralogs, they are associated with previously characterized differences in posttranslational regulation." It seems that the results of that study could be directly compared to the analysis performed here.

Significance

The significance is hard to assess because the research is not given proper context and motivation.

I believe the study could be of interest to research studying cell signalling and its evolution, as well as those interested in gene family diversification. However, as written, no specific examples are given or clear hypotheses tested, making the paper seem largely descriptive.

My keywords: molecular evolution, signalling, intrinsically disordered regions, computational biology

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Reply to the reviewers

Manuscript number: RC-2022-01707

Corresponding author(s): Sarah Butcher, Richard Lundmark

1. General Statements [optional]

We thank the reviewers for their insightful comments. The inclusion of the points raised by the referees have strengthened the manuscript. However, some of the reviewer suggestions are beyond the scope of the work (see below), but will doubtlessly be touched upon in future studies by the authors. In addition to incorporating changes relevant to answering the reviewers’ comments, we have edited the manuscript for increased clarity and precision.

2. Description of the planned revisions

1. Liposome flotation assay Reviewer #1 suggested that we should perform a liposome floatation assay to separate possible C protein aggregation from membrane binding: "I would strongly recommend supplementing the current liposome sedimentation assay by liposome flotation assay. In contrast to liposome co-sedimentation, the flotation assay can discriminate protein aggregates from proteins bound to liposomes. Although the SDS PAGE shown in Fig. 1A looks pretty convincing, a faint protein band in the „P" lane of the middle panel for the (-) sample is evident. Therefore, C protein aggregation cannot be ruled out and it would be indistinguishable from liposome binding examined by mere co-sedimentation assay”

Response: We agree that this is a necessary control experiment to add, and we will perform it with liposomes containing 40 % POPS. As we detected complete C protein co-sedimentation with this lipid composition, performing the flotation experiment with the same composition will prove that the earlier result indicates lipid binding and not protein aggregation.

3. Description of the revisions that have already been incorporated in the transferred manuscript

1. Reviewer #1
2. “In addition, it needs to be clarified which TBEV C protein construct, whether full-length or truncated, was used for co-sedimentation fragmentation.”

Response: We have clarified in this section of the manuscript that the full-length C protein construct was used for the liposome co-sedimentation assays by adding “full-length” prior to instances of “C protein” e.g. in the paragraph starting line 118.

1. “How to understand the finding that „the C protein forms a very rigid layer when adsorbed to the membrane". Can the aggregation of C-protein be ruled-out? Following the 1M NaCl wash of C-protein-bound to SLB, the authors stated: „This shows that initial membrane recruitment of C protein is strongly dependent on its interactions with the negatively-charged lipid headgroups. However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization": does it mean that there are several layers of C protein, the first held by electrostatic interactions, overlayed by non-electrostatically bound C protein? If yes, the illustration of single-layered C-protein adsorbed onto SLB in Fig. 2A, B is not correct. ”

Response: We understand the confusion regarding the term “rigid” which was used as a way to describe how we interpret the relatively minor change in the dissipation upon membrane binding. What we intended to describe was that this indicates that the protein is attached in a stable way that does not add viscoelastic properties to the system. These data indicate that the protein does not form large aggregates that non-specifically attach to the membrane in different protrusive orientations. We have clarified this in the manuscript and specified that the as there is no dissipation change, there is no aggregation. We added the following to line 168 “This, in turn, indicates that the C protein does not bind as non-specific aggregates as these would have changed the viscoelastic properties of the system.”

We do not mean that there are several layers of C protein. We consider, due to the highly charged nature of C, that the most likely explanation is that there are multiple modes of C binding but the result is only one layer, with multiple C-proteins interacting with each other within that layer. We have modified the text at line 184 to: “However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization within the bound layer.”

1. “The sentence: “To confirm that the C protein is biologically active, we investigated its ability to bind RNA" seems to be a little odd because it suggests the model membrane binding assays do not require biological active proteins. However, considering that the interactions leading to binding either negatively-charged lipid or negatively-charged RNA are electrostatic - this sentence must be rewritten.” Response: We thank the reviewer and have now rephrased this sentence to the following at line 249 “Since RNA binding is crucial for the NC assembly, we investigated the C protein’s ability to perform this function.”

2. “The authors´ statement in the Abstract: „....we investigate nucleocapsid assembly..." is too speculative because the assembly was not studied in their work. It needs to be reformulated.” Response: We agree, and the statement has been removed from the abstract.

3. “Despite this clear and valuable methodological contribution, the authors' contribution to our knowledge of the coordination of the nucleocapsid components to the sites of assembly and budding is not so obvious. Contrary to the earlier idea that the flavivirus is asymmetrically charged (that is, hydrophobic on one side (α2) and positively charged on the other side (α4), recent studies show that the entire surface of the protein is highly electropositive (Mebus-Antunnes et al., 2022). Therefore, a well-ordered neutralization of the flaviviral C proteins' highly positive surface seems critical for the proper organization and assembly of nucleocapsid. I am afraid that the authors do not shed much light on this issue.” Response: The recent structure of the TBEV C protein, published after we submitted the manuscript, shows that indeed the C protein is highly positively charged on all surfaces (updated Supplementary Figure 1 and Selinger et al., 2022). The recruitment of C protein to the membrane, that we demonstrate is dependent on negatively-charged head groups, provides a biologically relevant mechanism for charge neutralization on the C protein surface that interacts with the lipids. The remaining surface charge can be then neutralized by RNA recruitment. Mebus-Antunnes et al. made their observations with just RNA and C protein from Dengue virus in the context of artificial surfaces e.g. mica. However, our experiments utilize the TBEV C protein and specifically include a membrane, the third critical component of NC assembly. Thus, we build upon the work of Mebus-Antunnes et al. by adding a second biologically relevant charge-neutralising component and comparing with a distantly-related virus. We have changed the discussion section of the manuscript to reflect this new structure and to emphasize the advance here. Starting from line 371 we changed the text to: “Recently, it has been shown that the neutralization of the C protein surface positive charge is important for RNA binding in the distantly-related Dengue virus (DENV) (Mebus-Antunes et al, 2022). The recruitment of C protein to the membrane, that we demonstrate is dependent on negatively-charged head groups, provides a biologically relevant mechanism for charge neutralization on the C protein surface that interacts with the lipids. The remaining surface charge can be then neutralized by RNA recruitment.”

Reviewer #2 1. “What results demonstrate C protein inserts into membrane? The current results support the C protein interacts with membranes with positive charge, but do not seem to demonstrate membrane insertion. If the C protein inserts into the membrane, which regions (helices) play this role?”

Response: The Langmuir-Blodgett trough tensiometry experiments with monolayers directly measure the insertion of a protein into the monolayer. By determining the maximum insertion pressure of the C protein constructs, we also show that the membrane insertion can occur in bilayers. We show that the N-terminus is not inserting into the membrane, further work, beyond the scope of this manuscript, is needed to pinpoint the residues responsible for insertion, for instance by hydrogen-deuterium exchange or FRET measurements that would not affect folding. To clarify the use of the LB trough, we added the following at line 216: “To investigate if the C protein membrane binding includes insertion into the membrane after the initial electrostatic binding, we used Langmuir-Blodgett trough monolayer experiments. In this approach, the insertion of a protein into a lipid monolayer can be detected by following the pressure (π) of the monolayer after protein injection into the aqueous subphase, with increases in π corresponding to protein injection (Brockman, 1999; Liu et al, 2022).“

1. “The authors should discuss several previous papers reporting the effect of partial deletions of the C gene on the replication of TBEV, West Nile virus, and other flaviviruses.” Response: We agree that this is a necessary addition, and have now added a paragraph in the discussion section starting at line 333: “N-terminally truncated flaviviral C proteins have been shown to be assembly competent and in vitro, able to bind RNA, which is consistent with our results with N-terminally truncated TBEV C protein (Khromykh & Westaway, 1996; Kofler et al, 2002; Patkar et al, 2007; Schlick et al, 2009). One role of C is in the modulation of host responses to infection and the N-terminus maybe involved in that (Yang et al, 2002; Limjindaporn et al, 2007; Colpitts et al, 2011; Bhuvanakantham & Ng, 2013; Katoh et al, 2013; Urbanowski & Hobman, 2013; Samuel et al, 2016; Slomnicki et al, 2017; Fontaine et al, 2018). The membrane insertion directly detected in our experiments is central to C protein function. Other studies have found that deletions in the hydrophobic region of the α2 helix significantly impair particle assembly (Kofler et al, 2002; Patkar et al, 2007; Schlick et al, 2009). In the light of this evidence, we consider that the α2 helix could be responsible for membrane insertion (Markoff et al, 1997; Kofler et al, 2002; Nemésio et al, 2011, 2013).”

Reviewer #3 1. “In Figure 4, the band (256:1) that are supposedly in the wells (red arrow) is not clear- it is only slightly darker than the other wells.”

Response: This confusion was the result of unclear wording. We have now revised the figure legend at line 278 to : “The black arrow indicates the bands of freely-migrating RNA, and the red arrow the wells. On lanes 624:1 and 256:1, RNA has been immobilized in the wells.”

1. “Figure S1A, the N-terminal end (which is truncated in the mutant) should be colored on the cyan molecule.” Response: We have coloured the truncated part of the cyan molecule in the figure (now S1B) according to the reviewer’s comment.

Other 1. As the nuclear magnetic resonance structure of the truncated TBEV C protein has recently been released (Selinger et al, 2022), we have updated the manuscript and Figure S1 to include the information from this structure. We have also generated a new homology model of the full-length TBEV C protein using this structure as a template and included that in Figure S1.

4. Description of analyses that authors prefer not to carry out

1. Reviewer #1
2. “However, we do not know whether in the infected cells, the C protein is pre-bound to ER membrane or to viral RNA. Having such a unique assay in their hands, I wonder whether the authors could use the pre-bound C protein with genomic RNA (i.e. the experiment shown in Fig. 4A) ribonucleoprotein complex in the SLB binding assay. If doable, this experiment would be exciting and could bring some important information about NC assembly.”

Response: We agree that it would be very interesting to decipher if the C-protein first binds to RNA or to membranes using the QCM-D methodology. Yet, our data on pre-incubated C-protein and RNA suggests that large aggregates are formed which would hamper the interpretation of the QCM-D data. Furthermore, based on the suggested experiment, we will not be able to firmly conclude whether or not the C-protein first binds to RNA or to membranes since the time of the experiment will allow rearrangement of preformed complexes between C-protein and RNA. Additionally, the QCM-D measurement cannot differentiate if the preformed complexes bind on their own, or if excess unbound C protein binds the membrane and then recruits the complex. Therefore, addressing this question would require major adjustments to the RNA model system and methodology that we feel are beyond the scope of this study.

Reviewer 2 1. “The authors should use the lipids detected in the virions to confirm C protein binding experiments.”

Response: In the mass spectrometry characterization of the TBEV virions, we detected lipids from 9 classes (Car, PE, PS, PI, PG, PC, Cer, HexCer & TG). We have tested four of them (PE, PS, PI, PC) in the liposome sedimentation assay. Additionally, we tested GalCer, which, like HexCer, are cerebrosides. Our liposome binding experiments clearly demonstrate that the C protein does not bind to a specific lipid class, but instead to lipids with negatively-charged headgroups. Therefore, we would argue that doing additional sedimentation experiments with Car, PG, Cer, and TG would not add extra insight to the manuscript.

Additionally, while the population of lipid species in the TBEV envelope is diverse, the diversity mostly comes from differences in the lipid tails, which do not generally affect the head group-mediated binding of proteins. Therefore, performing additional lipid binding experiments with varying tail lengths would not likely lead to new observations.

Finally, to perform the authentic experiment of testing C protein binding to liposomes formed from lipids extracted from purified virions would require orders of magnitude more virus sample than our research laboratory is capable of producing. Therefore, we argue that this experiment is beyond the scope of this study.

1. “The study may be strengthened by performing virus mutagenesis experiments.” Response: While we agree that, ultimately, experiments on virus and cells would help to understand the role of the C protein in the biological context, we think these experiments are beyond the scope of this study. For virus mutagenesis, candidate residues should be first identified with biochemical and biophysical studies, which is already beyond the scope of this work. Additionally, the C protein has multiple functions in the host cell in addition to NC assembly, and interpreting the effect on the mutations on e.g. virus titer is difficult.

Reviewer #3 1. “In all figure legends, authors should write a conclusion line after the description of the experiments - what conclusion is drawn from each experiment.”

Response: While we agree that adding such a conclusion line would make it easier for the reader to understand each figure, the format of the figure legends is highly subject to journal policy. Therefore, we think that the addition of such lines will be an editorial decision and will depend on the journal. We have, however strived to make the figure titles as informative as possible in lieu of such concluding lines.

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Referee #3

Evidence, reproducibility and clarity

Pulkkinen and co-authors, title: Simultaneous membrane and RNA binding by TBE virus capsid protein.

This paper characterizes the ability of purified TBE capsid proteins to bind to different composition of lipids by biophysical methods and found that it prefers to bind to negatively charge lipids. The capsid then partially inserts into the membrane. Using mass spectrometry, they analyze the lipid composition of the purified TBE virus and showed they composed of negatively charge lipids thereby further supporting that the virus is likely first assembled where the negatively charge lipids are located in the endoplasmic reticulum. They also characterize the membrane bound capsid protein's ability to bind RNA and show they are able to bind. For all these experiments, they also included a capsid mutant with its N-terminal end deleted and show the mutant capsid protein activity doesn't not differ much from the whole capsid protein- thus showing the N-terminal end is likely not important for these processes. The experiments are well conducted and the manuscript is very clearly written.

Comments:

1. In Figure 4, the band (256:1) that are supposedly in the wells (red arrow) is not clear- it is only slightly darker than the other wells.

Minor comments:

1. In all figure legends, authors should write a conclusion line after the description of the experiments - what conclusion is drawn from each experiment.
2. Figure S1A, the N-terminal end (which is truncated in the mutant) should be colored on the cyan molecule.

Referees cross-commenting

I agree with comments by Reviewers #1 and #2

Significance

The study here although done in an in vitro system illuminates the virus assembly process - the positively charged capsid protein binds to the negatively charge area of the endoplasmic reticulum membrane, the capsid then partially insert into the membrane, then capsid interacts with viral RNA genome to facilitate virus assembly process. This is a very detailed study of the initial steps of the virus assembly process.

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Referee #2

Evidence, reproducibility and clarity

Pulkkinen et al. performed biochemical and biophysical experiments to suggest (1) negatively charged lipids are required for TBEV C protein interacting with membrane, (2) the membrane-associated C protein could simultaneously bind viral RNA, (3) the first 17 amino acids are not required for (1) and (2), and (4) TBEV virions contain negatively charged lipids. The study is important and provides molecular insights in flavivirus assembly. The following points can substantiate the manuscript.

Major points

1. The authors should use the lipids detected in the virions to confirm C protein binding experiments.
2. What results demonstrate C protein inserts into membrane? The current results support the C protein interacts with membranes with positive charge, but do not seem to demonstrate membrane insertion. If the C protein inserts into the membrane, which regions (helices) play this role?
3. The study may be strengthened by performing virus mutagenesis experiments.
4. The authors should discuss several previous papers reporting the effect of partial deletions of the C gene on the replication of TBEV, West Nile virus, and other flaviviruses.

Significance

This is an important study as indicated in the comments to authors.

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Referee #1

Evidence, reproducibility and clarity

The authors characterized the interactions of recombinant, bacterially expressed full-length and N-terminally truncated C proteins of tick borne encephalitis virus with model membrane systems. They used a unique combination of biophysical methods, including protein liposome co-sedimentation, QCM-D measurement and Langmuir-Blodgett trough monolayer experiments. Their experiments showed that the binding of TBEV C to both liposomes and supported lipid bilayer (SLB) is strongly dependent on the presence of negatively charged lipids. They also showed that following the initial electrostatic binding to the model lipid membrane, both C protein variants absorb to the SLB and form rigid layers which are stabilized by non-electrostatic interactions. By Langmuir-Blodgett trough monolayer experiments they demonstrated that negatively charged lipids are needed for C protein membrane insertion. The SLB bound C proteins, either full-length or N-terminally truncated, were shown to bind in vitro transcribed TBEV genomic RNA. Finally, to prove their major finding that negatively charged lipid head groups are crucial for C protein interaction with the lipid membrane, the authors analyzed the lipid content of the purified virions.

This work deals with the central role of the C protein, namely with its binding to the lipid membrane and genomic RNA. In the infected cells, this process leads to nucleocapsid assembly, a step which is poorly understood. The authors demonstrate that the membrane affinity of the C protein is conditioned by the presence of negatively charged polar heads. The text and figures are clear and accurate. The results obtained from three independent methodological approaches are solid and confirm the importance of electrostatic interactions for a contact of C protein with the membrane. As highly interesting, I considered the observation that the C protein, while bound to the model membrane (SLB), still retains its ability to bind RNA. Although their data did not show anything about the orientation of the C protein in SLB, this methodology opens the way to how, using suitable mutants of TBEV C, this can be found. I am sure that the authors are aware of the possibilities of studying a series of the TBEV C mutants with impaired membrane or RNA binding. Therefore, I assume that the authors' primary focus here is to show new methodological approaches to the simultaneous measurement of C protein interactions with model membranes and RNA, and some data obtained on the abovementioned mutants will be published afterwards.

Major comments:

1. One of the fundamental challenges of the work with flaviviral capsid proteins is that they tend to form amorphous aggregates to neutralize their highly positive surface charge. As the authors state themselves, „ We cannot rule out that the C protein preparation is heterogeneous..." I would strongly recommend supplementing the current liposome sedimentation assay by liposome flotation assay. In contrast to liposome co-sedimentation, the flotation assay can discriminate protein aggregates from proteins bound to liposomes. Although the SDS PAGE shown in Fig. 1A looks pretty convincing, a faint protein band in the „P" lane of the middle panel for the (-) sample is evident. Therefore, C protein aggregation cannot be ruled out and it would be indistinguishable from liposome binding examined by mere co-sedimentation assay. In addition, it needs to be clarified which TBEV C protein construct, whether full-length or truncated, was used for co-sedimentation fragmentation.
2. In section: Initial C protein recruitment to the membrane is of an electrostatic nature How to understand the finding that „the C protein forms a very rigid layer when adsorbed to the membrane". Can the aggregation of C-protein be ruled-out?

Following the 1M NaCl wash of C-protein-bound to SLB, the authors stated: „This shows that initial membrane recruitment of C protein is strongly dependent on its interactions with the negatively-charged lipid headgroups. However, once bound, the C protein-membrane interaction is complemented with non-electrostatic interactions such as membrane insertion or protein oligomerization": does it mean that there are several layers of C protein, the first held by electrostatic interactions, overlayed by non-electrostatically bound C protein? If yes, the illustration of single-layered C-protein adsorbed onto SLB in Fig. 2A, B is not correct. 3. C protein inserts into membranes It is beyond the frame of this work; however, it would be nice to show whether mutations of amino acid residues within the hydrophobic segment of TBEV C, which are in other flaviviral C proteins considered responsible for hydrophobic interaction, can abolish the membrane interaction. 4. Membrane-bound C protein can recruit TBEV genomic RNA. The sentence „ To confirm that the C protein is biologically active, we investigated its ability to bind RNA" seems to be a little odd because it suggests the model membrane binding assays do not require biological active proteins. However, considering that the interactions leading to binding either negatively-charged lipid or negatively-charged RNA are electrostatic - this sentence must be rewritten. 5. The authors state, "These data show that membrane-bound C protein is capable of recruiting TBEV genomic RNA at the membrane, suggesting that this also happens in the context of NC assembly". However, we do not know whether in the infected cells, the C protein is pre-bound to ER membrane or to viral RNA. Having such a unique assay in their hands, I wonder whether the authors could use the pre-bound C protein with genomic RNA (i.e. the experiment shown in Fig. 4A) ribonucleoprotein complex in the SLB binding assay. If doable, this experiment would be exciting and could bring some important information about NC assembly.

Minor comments:

The authors´ statement in the Abstract: „....we investigate nucleocapsid assembly..." is too speculative because the assembly was not studied in their work. It needs to be reformulated.

Referees cross-commenting

I agree with the Reviews by reviewers #2 and #3

Significance

This manuscript's major novelty and originality are in using a unique combination of biophysical methods, including quartz crystal microbalance with dissipation monitoring and Langmuir-Blodgett trough. Using quartz crystal microbalance with dissipation, the authors confirmed the necessity of negatively charged lipid components of the model lipid membrane for C-protein binding. Furthermore, this method also allows them to measure the formation of a rigid layer of C protein stabilized by non-electrostatic interactions. By Langmuir-Blodgett trough monolayer experiments, they demonstrated the insertion of TBEV C protein into the model membrane. However, I do not have sufficient expertise to evaluate the correctness of the experiments done by these two methodologies.

Despite this clear and valuable methodological contribution, the authors' contribution to our knowledge of the coordination of the nucleocapsid components to the sites of assembly and budding is not so obvious. Contrary to the earlier idea that the flavivirus is asymmetrically charged (that is, hydrophobic on one side (α2) and positively charged on the other side (α4), recent studies show that the entire surface of the protein is highly electropositive (Mebus-Antunnes et al., 2022). Therefore, a well-ordered neutralization of the flaviviral C proteins' highly positive surface seems critical for the proper organization and assembly of nucleocapsid. I am afraid that the authors do not shed much light on this issue.

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Reply to the reviewers

The authors do not wish to provide a response at this time since they are submitting a Revision plan and not a Full revision.

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Referee #3

Evidence, reproducibility and clarity

The objective of this manuscript was to determine the role of TZPs in mouse oocyte quality. The experimental plan was to compare the phenotypes of global Myo10-/-, oocyte Myo10-/-, and Myo10+/+ follicles. The results indicate that global loss of Myo10 did not prevent oocyte growth, but resulted in lower density of TZPS. Whole ovary image analysis revealed that Myo10-/- follicles actually contained more TZPs than wt, despite the fact that TZP density was decreased in Myo10-/- follicles. In mature knockout females, oocyte growth proceeded, but with impaired oocyte-zona integrity and alterations in gene expression including upregulation of numerous protein encoding genes. Oocytes from Myo10-/- knockout females produced a normal-appearing spindle but exhibited reduced capacity to mature beyond MI. Analysis of ovulated oocytes from mated females revealed an increase in the number of unfertilized and dead oocytes, many of which exhibited gaps between the zona pellucida and the oocyte plasma membrane. Those oocytes that were successfully fertilized exhibited a higher than normal of developmental arrest by the blastocyst stage. Lastly, mating trials revealed that Myo10-/- females were sub-fertile.

The results are clearly described with high quality imaging to demonstrate phenotypes. The data appear reproducible based on sample size and the number of repetitions. In most cases, statistical analysis demonstrates significance of observed differences.

Minor comments:

1. Fig. 2B does not provide statistical evidence that the two data sets differ.
2. Fig. 6A Was the zona pellucida functional in unfertilized oocytes from Myo10-/- females? That is, were sperm bound to the zona or within the perivitelline space?
3. The observation that oocytes from Myo10-/- females have more TZPs but lower TZP density raises questions as to how more TZPs (even if less densely spaced) could fail to support oocyte development. Dye diffusion assays comparing the rate of injected dye from Myo10+/+ and Myo10-/- (GV stage) or (maturing) stage oocytes into their attached granulosa cells might reveal an explanation.

Significance

The manuscript addresses an important aspect of follicle development using state of the art methodology to test the requirement of Myo10 for successful TZP-oocyte interaction during follicle development. The authors demonstrate significant findings as to the mechanism by which TZPs enable granulosa cell-oocyte contact which is required for transfer of critical components from granulosa cell to oocyte. The requirement of Myo10 in this process in oocyte competence is demonstrated clearly, however the mechanism by which Myo10 ablation causes defective fertilization and development remains unclear. In any case, the results demonstrate new and interesting findings that will be of great interest to basic scientists including oocyte biologists working on diverse animal species. The results could lead to further understanding of TZP-oocyte interaction and reveal ways to improve or restore communication between these two cells.

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Referee #2

Evidence, reproducibility and clarity

Summary:

The authors have investigated the effect of knocking out the Myosin-X gene (Myo10) on oocytes in mice. The major finding was that transzona processes (TZPs), which are filipodia-like structures that cross the oocyte's extracellular matrix shell (zona pellucida, ZP), were greatly reduced when the gene was globally knocked out. In comparison, an oocyte-specific knockout had no effect on TZPs. Using a machine learning algorithm developed by one of the authors, it was found that characteristics of the ZP were changed, and the oocyte shape was altered in the knockouts. RNAseq showed that many genes were upregulated in oocytes from knockout females. Oocytes from knockouts also failed to complete meiotic maturation at a higher rate and produced embryos that were fertilized less frequently and whose embryos were impaired in reaching the blastocyst stage. Finally, litters per female and pups per litter were lower in knockouts, indicating lower female fertility.

Major comments:

Overall, this is a very well done and comprehensive study that indicates a major role for MYO10 in oogenesis and oocyte developmental competence. There are some relatively major issues that should be resolved, however:

1. An experiment was done to assess the number of follicles per ovary, which is shown in Fig. S3. No significant difference in follicle number (per unit area) was detected. However, there are two problems here. One is that only four repeats were done, and the lack of significance would appear to be driven by only one of the knockout repeats which had a high number of oocytes compared the others. It is possible that there is really not a biologically significant difference between the controls and somatic knockouts, but there are an insufficient number of repeats to determine this (technically, P>0.95 would mean they are the same). Second, it is unclear that the number of follicles per unit area is the relevant parameter for fertility rather than the absolute number of follicles. Both measures should be reported and tested statistically.
2. A main function of TZPs is to transfer metabolites and other small molecules into the oocyte via Cx37-containing gap junctions. As the authors note, the phenotype here is different from the Cx37 knockout, where oocytes failed to develop. This implies some connectivity remains in Myo10 knockouts, but how much has not been determined. The amount of connectivity should be measured. The techniques are fairly straightforward and involve only microinjection of a fluorophore into the oocyte and measuring the spread into the surrounding somatic cells. This also has implications for the lack of effect on GVBD and resumption of meiosis, since Laurinda Jaffe's group has shown that diffusion of cGMP out though the gap junctions is important in this process.
3. The TZP-like structures that remain are intriguing, but this was not followed up. They apparently are visible optically but contain neither actin nor membrane. Is it possible that these are tracks left from degenerated TZPs? Electron microscopy might resolve this question and should be considered. In any case, a more extensive discussion is warranted since the data are contradictory, with fluorescence-based methods indicating a decrease in TZPs but optical methods indicating an apparent increase.
4. The apparent delay in formation of a perivitelline space is interesting. The perivitelline space forms gradually as the ZP detaches from the oocyte independent of meiotic maturation (see, e.g., Richard et al., 2017, J Cell Physiol 232:2436-46). Could this not be a delay in detachment and therefore transient (and dependent on when the assay was performed relative to oocyte isolation)?
5. While GO analysis was done and shown in Table 1, this is not treated in any depth in the paper. There should be more description of the GO pathways that were upregulated and the implications.

Minor comments:

1. The comparisons that were done for whole-body knockout vs. oocyte-specific knockouts were only done by comparing each to its control. There is no direct comparison showing whether the two knockouts differ significantly from each other. The comparisons should be done using ANOVA with appropriate post-hoc tests to test all four groups against each other.
2. The experiment in which 5-ethynyl uridine incorporation was used to show that global transcription was not increased may not actually be conclusive, since a large amount of RNA synthesized is not mRNA. A global increase in mRNA synthesis could still be occurring but the signal swamped by RNAs such as rRNA and other non-coding RNAs.

Referees cross-commenting

It looks like the reviewers basically agree that this is interesting but there are questions remaining about whether cumulus-oocyte coupling is affected (and could explain the phenotype) and why there is an apparent discrepancy between the results for detecting the numbers and densities of TZPs. These should be addressed.

Significance

This work has fundamental implications for understanding oocyte development and the role of the surrounding somatic cells in oogenesis and oocyte developmental competence. It also has direct implications for human and animal fertility and assisted reproduction.

This is a fundamental new set of results that establishes a role for Myo10 and adds to the knowledge about the role of transzonal processes. It is a substantial advance over previously published research.

The audience will primarily be basic biomedical researchers in the general field of reproductive biology as well as those investigating filipodia and should extend to those interested in translational research in infertility.

I have direct and extensive expertise in the field of oogenesis in mice.

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Referee #1

Evidence, reproducibility and clarity

In the manuscript by Crozet et al. the authors investigated the contribution of transzonal projections (TZPs) to the oocyte development and acquisition of competence. The results were obtained using two Myo10 knockout mice models: a full knockout for Myo10 (Myo10-/- full) and an oocyte-conditioned knockout (Myo10-/- oo). The major findings due to the global depletion of Myo10 include the decrease in TZP density, discrete morphological alterations in the oocytes, alterations in oocyte gene expression, the inability of the oocytes to complete the first meiotic division (lack of 1PB extrusion), and subfertility in Myo10-/- full females.

The research topic is interesting and, overall, I consider the manuscript relevant. However, to increase the scientific soundness authors are encouraged to explore the effects of the (partial) interruption of the germ-soma communication on the regulation of meiotic arrest and resumption. This is worth investigating (is optional, but highly recommended) since the lower density of TZPs is associated with an apparent normal meiotic arrest but an abnormal meiotic resumption. At first, the measurement of cGMP and cAMP into oocytes during meiotic arrest and resumption would be a nice try. This will help to shed light on the reasons for the abnormal meiotic progression, indicating if it is the consequence of a direct blockage in the transfer of molecules from follicular cells to the oocyte or an indirect consequence.

Minor points

Lines 53-55: The oocyte does not complete two successive meiotic divisions to generate a mature oocyte ready to be fertilized. Instead, meiosis completion only occurs if fertilization of MII-arrested oocytes takes place. Consider rephrasing to communicate the accurate concept.

Lines 145-153 and Figure S4-F: Authors claim that TZP-deprived oocytes grow up to normal sizes. However, the perimeter of fully grown oocytes is lower in Myo10-/- full oocytes. This is conflicting.

Referees cross-commenting

In addition to the comments made by my own, my colleagues both suggested the inclusion of experiments to determine the functionality of the remaining TZP through dye diffusion assays. I concur with them.

Significance

The manuscript clearly adds to the existing knowledge. I'm convinced that the findings described here will be of interest for readers from the field of reproductive biology, follicle development, and oocyte biology.

Authors are encouraged to better frame their findings as to the existing knowledge. There is at least one another knockout model in mice that leads to TZP density reduction (Zhang et al., 2021; Nature Comm., 12:2523). In this paper, the authors show that the TZPs connecting the GCs and the oocyte support proper oocyte development. Also, its removal results in subfertility. These previous findings should be acknowledged in the current manuscript.

My expertise: researcher in reproductive biology; emphasis on folliculogenesis and oocyte development.

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Reply to the reviewers

Overall comments

We are pleased by the reviewers’ comments and appreciate their suggestions for improvements. In addition to correcting small typos throughout the manuscript, the major changes we did in response to their comments are as follows:

• Changed the title of our paper to reflect the strong evolutionary correlation more accurately between sex chromosomal meiotic drive and gains/losses of SNBP genes in
• New experiments to test the role of the well-conserved, universally retained SNBP, CG30056, in male fertility in * melanogaster*. Although reviewers had suggested we could eliminate this section, we felt that this would add a lot of weight to the unexpectedly inverse relationship between age/retention and fertility functions of SNBP genes. Thus, over the past few months, we have carried out new experiments with increased sample sizes, better controls, and sperm exhaustion. These new results strengthen our earlier analyses.
• Better clarification of the X-Y chromosome fusion, which is a new observation, in the montium group via careful rewriting as partly suggested by Reviewer #2.

• Highlighting that the genetic conflicts hypothesis does not rule out a role for sperm competition or other conflicts in shaping SNBP evolution in a revised Discussion. All changes in response to the reviewer’s comments have been detailed in our point-by-point response (below). You will see that we have addressed almost all the suggestions made, including with new experiments. The only reviewer suggestions (all optional from Reviewer 3), which we did not directly address in our revision are:

• __Branch specific protamine evolution analyses for sex chromosome amplified SNBP genes: __given the state of SNBP gene annotation and the difficulties of assembling these genes in large tandem arrays, this will require considerable work and is beyond the scope of the paper.

• Covariation between SNBP evolution and sperm morphology: We cannot perform these experiments as there is a paucity of sperm morphology data currently. Obtaining this data reliably is a significant undertaking.
• Are SNBP genes more prone to be lost than average in the montium group: We have not comprehensively examined all loss events in the montium group or any other Drosophila This is also a non-trivial analysis, albeit it would be very interesting. However, we believe the more relevant comparison is whether these lost SNBP genes are more likely to be retained in non-montium species, which they are, as we now highlight. We hope you will favorably judge our good faith efforts to address all other reviewers’ comments, and their laudatory comments during the previous round of reviews.

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

Chang and Malik present a comprehensive evolutionary analysis of sperm nuclear basic proteins (SNBPs) in Drosophila. In addition, they provide a preliminary functional characterization of one such protein (CG30056) and describe a newly discovered X-Y chromosomal fusion in the Drosophila montium species group. All of these findings are interesting and important, but the headline from this study is the well-supported possibility that SNBPs, or at least a large fraction of them, function in suppressing X vs. Y chromosome meiotic drive. While this hypothesis is challenging to test experimentally, the authors provide strong correlational evidence that SNBPs are associated with drive by documenting these proteins' rapid evolution. This rapid evolution takes the form of sequence changes (as predicted by coevolution between drivers and suppressors of drive), gene amplification in cases when SNBPs move to sex chromosomes (consistent with the SNBP becoming a potential agent of drive for its new "home chromosome"), and gene loss in species with X-Y chromosome fusions (in which drive is not predicted to occur).

Overall, this is an excellent, comprehensive study. The phylogenetic and genomic analyses are first-rate (and one of the first to make use of the new 101 Drosophila genomes); the logic is very well explained; conclusions are supported by multiple lines of evidence; the writing and figures are clear and accessible; and, the findings are fascinating. It's a good sign that it is easy to imagine several experiments one could do to follow up on this study, but I do not feel any are required in revision, as the manuscript is comprehensive as is. Thus, I have just a few minor points the authors may wish to consider in making revisions and a few suggestions for clarity/typos.

__

We thank the reviewer for their positive comments on our work.

1. I would be interested in whether the authors think that all SNBPs in a given Drosophila species function(ed) in meiotic drive, or whether some fraction may play other roles, such as sexual selection or chromatin compaction, which have been the traditional hypotheses for SNBP function. Relatedly, given the high turnover of SNBPs the authors observe and the fact that some melanogaster-essential SNBPs are younger genes, would they like to comment on whether the subsets of SNBPs involved in drive/suppression vs. chromatin packaging/sperm traits/Wolbachia defense are likely to differ from across fly species? The reviewer raises an excellent point. In our revised discussion, we now speculate that different SNBPs might have distinct functions. For example, the same subset of SNBPs is subject to gene amplification and loss whereas other SNBPs are subject to less turnover. Moreover, even this stable set of SNBPs evolves rapidly, including in the montium group of species that have undergone dramatic SNBP loss. As the reviewer suggests, sperm competition or pressures from Wolbachia toxins might be is a driving force for sperm evolution. We discuss these possibilities and conclude in our discussion: “Our findings do not rule out the possibility that forces other than meiotic drive are also important for driving the rapid evolution and turnover of SNBP genes in Drosophila species.”

What do the authors make of the lower isoelectric points for a few of the SNBPs (e.g., CG31010 with pI = 4.77 in Table 1)? These proteins have identifiable HMG box domains, so is the pI driven lower by other parts of the protein sequence?

We thank the reviewer for raising this point. We found that the pI of HMG domains can range from 6 to 12. Thus, the pI is driven by both HMG domains and other parts of proteins. We now include the pI of the whole SNBP protein and the HMG domain alone in Table 1. We do not have enough biochemical information to speculate on how these differences could alter SNBP function.

__3. For readers less familiar with the field, it may help to spell out (e.g., on p. 6) why the authors consider ProtA/B to be important for fertility. Some of the previous papers on these genes describe them as dispensable - though the present authors are correct that these previous studies do detect fertility defects of various magnitudes under some conditions.

__

We agree with the reviewer. Previous studies are in disagreement about the importance of ProtA/ProtB for male fertility- while no significant effects were seen under standard fertility assays, sperm exhaustion conditions (mating with excess females) did reveal fertility effects. We have now added these references and discussed ProtA/ProtB more fully in our revision.

On p. 9, paragraph 2, the data showing that "six different SNBP genes underwent 11 independent degeneration events in the montium group" are shown in Fig. 6A, not 5A.

Thank you. This has been fixed in our revision.

5. The summary Table 2 is useful, but I wonder whether including relative levels of expression and dN/dS in addition to ordinal rankings might help clarify. For instance, if there were a drop off in mean expression level between the 5th and 6th most highly expressed SNBP, this wouldn't be evident from the table.

We agree with this suggestion and have now added this information.

In Fig. 3, I like the use of the clean CG31010 figure in panel A to illustrate the circular representations. In addition, though, it might be useful to show Prot's graph at this same, larger size, since it's the most complicated and will likely be most closely examined.

We agree with this suggestion and have now amended this figure in line with the reviewer’s suggestion.

In Fig. 4, the end of the legend says that the species tree is shown "on the right," but it's on the left in the figure.

Thank you. This has been fixed in our revision.

__CROSS-CONSULTATION COMMENTS • I agree with both Reviewers 2 and 3 that the title could be changed to be a bit more tentative. I'd had this thought as well.

__

We agree with this suggestion. We have now amended this title to “Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes.”

• I agree with Reviewer 2 that the fertility assay could be conducted with a larger sample size and a better control in order to be better compared with how the authors described other published fertility phenotypes for SNBPs. For the control, crossing the deletion line to y w (or w1118) and using the resulting heterozygotes (KO/+) would be better than using the mutation over the balancer chromosome (KO/CyO). We agree with both suggestions. We now compare fertility between KO/KO and KO/+ males in sperm exhaustion assays. Our more stringent fertility assays find no evidence of CG30056 role in male fertility, strengthening our previous findings. We have now added the motivation for the new assays and the new results to our Revision.

• I agree with Reviewer 3's third bullet point about spending a bit more time on the different possible roles that SNBPs could play in spermatids. (This is a more eloquent version of my review point #1.)

We have now expanded our discussion of other possibilities in our revision.

• I agree in principle with Reviewer 3's first bullet point about examining whether SNBP evolution correlates with changes in sperm morphology, but this feels like it could be a whole, fascinating study on its own, while this manuscript is already packed with data. I'd welcome the authors' thoughts about this in discussion, but wouldn't personally require a formal analysis of this to be added prior to publication.

We also agree that this would be an interesting test. However, we are not able to do the test due to the scarcity of sperm phenotype data in Drosophila. We also think that our original version unintentionally downplayed this possibility. Our revised discussion makes clear that the rapid evolution of some Drosophila SNBP genes may be driven by sperm competition, just as in mammals, and influence the evolution of sperm morphologies.

__Reviewer #1 (Significance (Required)):

This study describes an important conceptual advancing in our understanding of the evolution and potential functions of sperm nuclear basic proteins (SNBPs) in Drosophila, which stands in interesting contrast to the functional roles of equivalent proteins in primates. It should be of broad interest to biologists studying spermatogenesis, meiotic drive, and genome evolution, both in and out of Drosophila. __

We thank the reviewer for their positive appraisal.

__ To contextualize the work, paternal DNA is typically compacted during spermatogenesis. This process involves the replacement of histones with other small, positively charged proteins in a sequential order, ending with protamines that bind DNA in mature sperm. In Drosophila, work over the last two decades (largely from the labs of R. Renkawitz-Pohl, B. Loppin and B. Wakimoto) has identified more than a dozen sperm nuclear basic proteins that localize to condensing/condensed spermatid nuclei. Two interesting observations have been that many of these proteins are dispensable for male fertility, and the proteins vary in their degree of evolutionary conservation. Recent work from Eric Lai's lab (J Vedanayagam et al. 2021, Nat Ecol Evol) showed that in D. simulans and sister species, at least one of these SNBP genes (Prot) underwent gene amplification and now acts in those species as a meiotic driver. This finding suggested the hypothesis, tested thoroughly in the present study, that the rapidly evolving SNBP gene family could be involved in causing or suppressing meiotic drive. Consistent with this idea, the authors here find that SNBP genes expand in copy number more frequently when they move from autosomes to sex chromosomes (consistent with the idea that they may cause or contribute to drive), and that otherwise well-conserved SNBP genes are lost in a group of species in which sex chromosome meiotic drive is not expected to occur. These findings are based on a thorough and well conducted phylogenomic and molecular evolutionary analysis of SNBPs across dozens of Drosophila species. Overall, this work generates exciting new hypotheses about the function of SNBPs and should be widely read both within and outside of the field.

__

We are grateful for the reviewer’s accurate summary of our work and its significance. We share the reviewer’s excitement and expect that more studies will explore the new function of SNBPs in multiple taxa soon.

Keyword describing my field of expertise: Drosophila, molecular evolution, reproduction, genetics, genome evolution.

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

The paper describes interesting patterns on the evolution of Drosophila SNBP genes, and proposes a very interesting explanation, namely, that meiotic drive is the main evolutionary force behind these patterns. Some of these observations have recently been made by other authors in a single case (the Dox genes in D. simulans), but not in the scale and breadth of the present ms. The ms combines an extensive investigation of available genomes with expert analysis, and new experimental data. In particular, the finding that the ancestral Y became incorporated into de X in montium species is very exciting, and may provide a smoking gun for the explanation proposed by the authors. Overall, I think it is a very good paper. I do have several criticisms and suggestions that may help to improve it.

__

We are grateful for the positive comments of the reviewer and for their constructive criticism and suggestions, which we have incorporated into our revision.

__The paper has a speculative side that it almost unavoidable given its novelty and breadth. I do not see this as a problem per se, but I think the uncertain/unsupported/problematic points should be more openly presented to the readers. The main cases I noted are:

1. The title of the ms states that "Genetic conflicts between sex chromosomes drive expansion and loss of sperm nuclear basic protein genes in Drosophila", but the evidence is somewhat circumstantial, and the patterns may be explained also by other known phenomena (e.g., demasculinization of the sex chromosomes; below). I think the tone of the end of the Introduction reflects more faithfully the strength of the evidence ("Thus, we conclude that rapid diversification of SNBP genes might be largely driven by genetic conflicts between sex chromosomes in Drosophila."). I understand the temptation of writing a bold title, but I think it is a bit misleading in the present case. I.e., it would be desirable that the title conveys the uncertainties of the data and their interpretation. __

We agree with this suggestion. We have now amended this title to “Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes.”

However, we also want to highlight that de-masculinization of the X chromosome cannot explain the observed amplification and loss patterns of SNBP genes, except in cases of sex chromosome fusions. We now highlight the de-masculinization hypothesis for the latter case, but still strongly favor the genetic conflicts hypothesis.

"In contrast, we found no instances of pseudogenization or subsequent translocation to the X chromosome of SNBP genes that are still preserved on their original autosomal locations or involved in chromosome fusions between autosomes (0/16). This difference is highly significant (Fig 5 and Table S11; 3:5 versus 0:16, Fisher's exact test, P=0.03). " Readers should be warned that this pattern can also be explained by the well-known demasculinization of X chromosomes (e.g., Sturgill et al. Nature 2007, 450, 238-241)

We agree with this point and thank the reviewer for pointing this out. We now expressly raise the ‘de-masculinization of X chromosomes’ as one potential explanation of the pattern we observe here.

"Indeed, no meiotic drive has been documented in the montium species even though it is rampant in many other Drosophila lineages [38]." Two remarks here: a) the authors should make clear that they are referring to sex-chromosome meiotic drive. b) I think the evidence is much weaker than the sentence implies. Sex-chromosome meiotic drive is known in less than 20 Drosophila species, scattered throughout the phylogeny. As far as I know all cases were discovered by accident, so the sampling is biased towards model species (e.g., the obscura group, which was very popular around 1930-1960). So we do not know the true frequency of sex-ratio meiotic drive among Drosophila species, nor, say, if it is more common in the Drosophila or Sophophora species, if it is suspiciously absent in the montium group (as suggested by the authors), etc. I think these uncertainties should be acknowledged or, perhaps, given the weakness of the argument, the sentence should be deleted or attenuated.

We agree with this comment and have now removed this argument in our revision.

__ "X-Y chromosome fusions eliminate the extent of meiotic drive and may lead to the degeneration of otherwise conserved SNBP genes, whose functions as drive suppressors are no longer required. Thus, unlike in mammals, sex chromosome-associated meiotic drive appears to be the primary cause of SNBP evolutionary turnover in Drosophila species." The authors found that in the montium species the ancestral Y became incorporated into de X chromosome, and that montium species seem to have an inordinate amount of SNBP gene losses. They combine these two observations by suggesting that these SNBP became dispensable or deleterious because they originally were involved in XY meiotic drive. I think many readers will think that males in montium species are X/0, whereas in fact in all of them carry a Y chromosome (just, in most cases, more gene poor than "normal" Y-chromosomes). I do not think this is a fatal flaw for the explanation proposed by the authors, but certainly is a difficulty that should be acknowledged.

__

We agree with this point. It was not our intention to suggest that montium group males are X/O, but this could be misinterpreted as we originally stated. We now add a clarification that montium group males still harbor a Y chromosome, which is missing most ancestrally Y-linked genes.

__Problems/suggestions with experiments and data analysis

1. There is a section titled "CG30056 is universally retained in Drosophila but dispensable for male fertility in D. melanogaster". In this section and in the figures, it is stated, "Although CG30056 is the most conserved SNBP we surveyed, we found no clear difference in offspring number between heterozygous controls and homozygous knockout males (Fig 2B). (...) We found either no or weak evidence of fertility impairments in two different crosses with homozygous CG30056 knockout males.". I think the fertility data are weak for the purpose of the authors, and I strongly suspect that this conclusion is wrong. Let me explain why. At other passages of the ms, the authors classify the SNPB genes in three groups. (i) essential/important for male fertility: "Three genes (Mst77F, Prtl99C and ddbt) are essential for male fertility while knockdown or knockout of two other SNBP genes (ProtA, and ProtB) leads to significant reduction in male fertility [27-30, 32]." (ii) genes that do not appear to impair male fertility at all. (iii) untested. CG30056 was in the last group, and hence the authors produced knockouts, tested their effect in male fertility, and concluded that it belongs to the second group. Now, look at Fig. 3B. The numbers of tested males are too small (it seems to range from 3 to 10), and male fertility is known to be a very noisy phenotype (as shown by the huge scatter in the authors' data). Furthermore, two different knockouts were tested, and both were nominally less fertile than the controls, and in one of them the difference is statistically significant. Taken at the face value, the knockouts seem to be perhaps ~25% less fertile than the controls. Another potentially big problem is that the "control males" actually carry visible dominant mutants (the balancers CyO or SM6) which certainly reduce their fitness, whereas the experimental males are wild-type for these mutants. Without the detrimental effect of these visible mutants in the controls, the difference to the CG30056 knockouts will probably be even larger. Note that the fertility effects of the genes ProtA, and ProtB (a.k.a. "Mst35B") , which the authors put in group "essential/important for male fertility" would not had been detected if assayed as the CG30056 gene: Tirmarche et al (2014; the reference cited by the authors) stated that: "In fact, the impact of Mst35B on male fertility was only revealed when mutant males were allowed to mate with a large excess of virgin females (1 for 10; Figure 3F) but not with a 1:1 sex ratio (not shown). " The authors' fertility test did not used this type of challenge. My general impression is that the fertility effects of CG30056 may actually be similar to ProtA and ProtB. I think the authors should do a proper fertility test of CG30056, or remove this section. Another possibly useful approach would be to classify the SNPB genes in those essential for male fertility and those that are not essential, because "experimentally speaking" this is a safer distinction (e.g., the fertility testes reported by other authors may also had been quick tests). Since these genes only function in sperm and are under purifying selection (otherwise they would have been lost; also, all have dN/dS We are very appreciative of the many important points raised by the reviewer. Rather than removing this conclusion, which is not central to our paper, we have now performed additional, well-controlled experiments to address the reviewer’s concerns, which we summarize below:

2. We agree with the reviewer that it is easier classification to identify SNBP genes that are essential for male fertility versus those that are not.

3. We also agree with the reviewer and now include more details about earlier studies to highlight that ProtA/ProtB fertility effects were only revealed in a sperm exhaustion setting.
4. We agree with the reviewer’s suggestion and have now included sample sizes for all our experiments in a new supplementary Table (Supplementary file 8).
5. We agree with the reviewer that a comparison between KO/KO and KO/Bal males is non-ideal given that Balancer chromosomes carry many deleterious mutations. We now include new experiments in our revision that compare KO/KO and KO/wt chromosomes.
6. We agree with the reviewer that standard fertility assays may be too noisy to detect subtle fertility effects. We therefore now carry out much more stringent fertility assays under sperm exhaustion conditions with a male: female ratio of 1:10 and at least 10 males tested per genotype Despite this higher stringency, we detect no difference in fertility between KO/KO males and KO/wt controls for CG30056 (>10 males were tested for each). Thus, our original conclusion is even stronger that CG30056 has no detectable effect on male fertility. We have not tested the possibility of sperm storage or precedence being affected in our assays. However, we do believe that the finding that one of the best conserved and retained SNBP genes has no detectable effect on male fertility is an important conclusion which greatly increases the impact of our study, especially since most fertility-essential genes are either young or not universally conserved. We hope these changes will satisfy the reviewer's concerns about this section of our paper.

"Our phylogenomic analyses also highlighted one Drosophila clade- the montium group of species (including D. kikkawai)- which suffered a precipitous loss of at least five SNBP genes that are otherwise conserved in sister and outgroup species (Fig 3). (...) Given our hypothesis that autosomal SNBP genes might be linked to the suppression of meiotic drive (above), we speculated that the loss of these genes in the montium group of Drosophila species may have coincided with reduced genetic conflicts between sex chromosomes in this clade." The montium data is an important part of the paper. I think the authors should test the statistical significance of this pattern.

We appreciate the reviewer’s suggestion. However, we are unable to perform the statistical tests suggested for technical reasons. We note that three loss events occurred in the ancestor of D. montium species, while two happened in the ancestors of most D. montium species. Since it’s hard to estimate the evolutionary rates using these internal branches, we can’t directly compare them to other branches using statistics. However, in response to the reviewer’s comments, we now more clearly contrast the fate of SNBPs between D. montium species and other melanogaster group species, noting that three of five genes lost in the montium group are retained in all other melanogaster group species.

__Other points:

1. "The five remaining SNBP genes (Mst33A, CG30056, CG31010, CG34269, and CG42355) remain cytologically uncharacterized [30]." I think it will be interesting if the authors look at other potentially useful resources: Vibranovski et al papers which looked at gene expression in mitotic, meiotic and post-meiotic cells (_https://mnlab.uchicago.edu/sppress/index.php), and the papers by several labs on testis single-cell transcriptomic data (Witt et al 2021 PLOS Genetics. 17(8):e1009728 ; Nat Commun. 2021;12: 892). These may provide additional clues on the function of SNBP genes. There is also a recent report on sperm proteome (doi: _https://doi.org/10.1101/2022.02.14.480191) __

We are grateful to the reviewer for this suggestion. We now add the data from single-cell expression analyses from Witt et al. in Table 1-figure supplement 1. We found most SNBPs are expressed at late spermatocytes and early spermatids, although CG30056 is primarily expressed in late spermatids, whereas CG34269 is expressed earlier in late spermagonia. The data from Vibrranovski et al. also show similar patterns but don’t have four of these genes, including CG34269. The data from Mahadevaraju et al. are from larva testes, and lack some critical stages during spermatogenesis. Thus, we only report the data from Witt et al.

We also surveyed the proteome data as the reviewer suggested, but we only found 3 SNBPs (ProtA, ProtB, and Prtl99C) in the data. This did not include, Mst77F, which is the most highly expressed (see Table 2) and well-studied SNBP, so we suspect the proteomic study might be biased toward proteins from sperm tails. Therefore, we decide not to include this analysis.

____ "Our inability to detect homologs beyond the reported species does not appear to result from their rapid sequence evolution. Indeed, abSENSE analyses [45] support the finding that Prtl99C, Mst77F, Mst33A, Tpl94 and CG42355 were recently acquired in Sophophora within 40 MYA. For example, the probability of a true homolog being undetected for Prtl99C and Mst77F is 0.07 and 0.18 (using E-value=1), respectively (Table S1, Methods)." This should be complemented by synteny analysis.

It may not have been clear from our original version that we did perform synteny analyses for all SNBP genes. We have now restated this more clearly in our revision.

I found the following sentence unclear: "However, we could only ascribe a sex chromosomal linked location for species if no data was available from either BUSCO genes or females (only males and mixed-sex flies)."

We modified the sentence to make it clearer: “However, we could not ascribe a sex-chromosomal linked location of a contig to either the X or Y chromosome in cases where there was no linkage information from BUSCO genes and no read data available from females, only from males and mixed-sex flies.”

"Using the available assemblies with Illumina-based chromosome assignment, we surprisingly found that most ancestrally Y-linked genes are not linked to autosomes as was previously suggested [by Dupim et al 2018] (Fig 6A)."

The new result of X-linkage is exciting, but the sentence is not exact: Dupim et al 2018 made clear that they could only separate X/A from Y-linkage. E.g., the legend of their Fig 3: "Phylogeny and gene content of the Y chromosome in the montium subgroup. "M" means amplification only in males (i.e., Y-linkage), whereas "MF" means amplification in both sexes (autosomal or X-linkage)."

We are grateful to the reviewer for this correction. We now modified the sentence to make clear that Dupim et al had “showed that many ancestrally Y-linked genes are present in females because of possible relocation to other chromosomes in the montium group.”

"The most parsimonious explanation for these findings is a single translocation of most of the Y chromosome to the X chromosome via a chromosome fusion in the ancestor of the montium group of species. Afterward, some of these genes relocated back to the Y chromosome in some species (Fig S6; Supplementary text)." Explanations for this pattern of "return to the Y" have been extensively discussed and tested in Dupim et al 2008 (see their section "Why genes seem to return to the Y chromosome after Y incorporations?" ) The available evidence strongly suggests that it is not a case of relocation to the Y.

We thank the reviewer for raising this point. However, our conclusions disagree slightly with those from Dupim et al. 2018, in part because of additional sequencing in this clade. Dupim et al. suggested the possibility that most Y chromosomal loci duplicated to other chromosomes in the ancestor of the D. montium clade, following which each species degenerated either Y-linked or autosomal copies of genes. If this was the case, Y-linked copies should have diverged from X-linked copies since the ancestor of the D. montium clade. In contrast to this expectation, our phylogenetic analyses found that D. kikkawai Y-linked PRY is more closely related to X-linked PRY in all other related species (Figure 6- figure supplement 1). This result is much less parsimoniously explained by the ancient duplication event proposed by Dupim et al. and is more consistent with a ‘return-to-Y’ that we propose. We also make clear that, unlike PRY, we can’t differentiate the two hypotheses in the case of kl-2.

Fig 6B suggests that the authors assembled the "translocated Y" in D. triauraria. However, no direct data or account for this assembly is provided. Please clarify.

This was not our assembly. We searched all publicly available assemblies in the montium group and found one assembly (NCBI accession GCA_014170315.2) that assembled all ancestral Y-linked regions. We now clarify this in our revision.

__ "Why would meiotic drive only influence Drosophila, but not mammalian, SNBP evolution? One important distinction may arise from the timing of SNBP transcription. In D. melanogaster, SNBP genes are transcribed before meiosis but translated after meiosis [29, 43, 57]. Thus, SNBP transcripts from a single allele, e.g., Xlinked allele, are inherited and translated by all sperm, regardless of which chromosomes they carry. Consequently, they can act as meiotic drivers by causing chromatin dysfunction in sperm without the allele, e.g., Y-bearing sperm." During spermatogenesis Drosophila haploid cells actually are syncytial, which has interesting consequences for the evolution of male genes (Raices et al, Genome Res. 1115-1122, 2019). This may be relevant for the present paper.

__

We thank the reviewer for this suggestion. We now gratefully include this citation in our revision.

__Reviewer #2 (Significance (Required)):

see above __ __Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This manuscript by Chang & Malik consider the evolution of HMG-box-containing sperm nuclear basic proteins (SNBPs) across Drosophila species in phylogenetic context.

Previous work in mammals had highlighted fast evolution of proteins involved in chromatin remodeling during spermatogenesis. Here, the authors provide evidence for widespread positive selection and likely involvement in genetic conflict in a set of proteins with analogous functions in Drosophila. Amongst other findings, the authors highlight biased amplification of SNBP paralogs on sex chromosomes along several Drosophila lineages, a tendency towards loss/pseudogenization following translocation onto a sex chromosome, and an intriguing concerted SNBP loss event in the montium group where parts of the Y chromosome have become fused to the X, thus nullifying the chance that genetic conflicts can play out via distorted segregation of sex chromosomes. The authors suggest that, taken together, their findings support widespread of SNBPs involvement (as instigators and repressors) in meiotic drive. Overall, I found the manuscript to be well written and thorough in its exploration of the evolutionary dynamics of SNBPs in this clade.

__

We thank the reviewer for the accurate summary and the kind comments.

__Below, I have highlighted some aspects that I think would benefit from further attention, none of them major.

• Following their exploration of patterns of SNBP evolution in Drosophila, the authors highlight support of their data for genetic conflict between sex chromosomes. They also rightly acknowledge that other evolutionary drivers such as sperm competition might also play a role in, for example, fast evolution of certain SNBPs. Yet those (not mutually exclusive) alternatives are never pitted directly against each other. The focus is firmly on exploring the support for the sex chromosome genetic conflict model. Given that the authors highlight Drosophila as a great model in part because of its well characterized sperm biology (including comparative morphology), I wondered why the authors had not made an explicit attempt to see if SNBP evolution covaries with aspects of sperm morphology across Drosophila. __

We do agree with the reviewer that it will be very interesting to test whether SNBP evolution covaries with sperm morphology in Drosophila. However, data on sperm morphology is scant in most Drosophila species. Indeed, this trait has only been well studied in clades with heteromorphic (different-sized) sperm but we agree this will be an exciting topic to consider in the future.

We also clarify better in our revised discussion that our analyses do not rule out a role for sperm competition or sperm morphology in driving the evolution of at least some SNBP genes. We note that a subset of SNBP genes undergo gene amplifications and loss, but most SNBP genes evolve rapidly including in species with gene loss. Thus, the meiotic drive hypothesis is not to the exclusion of other hypotheses.

• The most intriguing part of the manuscript for me was the exploration of SNBP fate in the montium group, where the authors find evidence for an ancestral fusion event between the X and parts of the Y chromosome. The loss of SNBPs is certainly consistent with the conflict model but I was wondering to what extent this lineage is characterized more broadly by unusual evolution at the chromosomal level. Is there simply a lot of upheaval in montium, with more frequent gain/loss across the board? How specific is SNBP loss in the context of other orthologous groups? This could be investigated by looking at retention of other genes in other orthologous groups (in montium and some other control group) or perhaps by looking at synteny conservation. This is a good suggestion. Using the same methodology as used in this paper, we found that very few D. melanogaster essential genes (2000) are lost in any single species we surveyed here (unpublished data). However, we have not carried out similar analyses for all genes; given vastly different rates of evolution, this would be a significant undertaking. Thus, we are not able to make a direct comparison between SNBP genes and a control group, that would include other testes-specific or fertility-essential genes. Instead, we highlight the fact that since we identify SNBPs using syntenic analyses, we have known that the neighboring genes of SNBPs are much better conserved than the SNBP genes themselves in the montium group species.

• In introducing SNBPs, the authors focus on their role as packaging agents. Clearly, SNBPs do package the genome in the sense that they bind to DNA and lead to reduced chromosome volume. But is this all packaging for packaging's sake (as portrayed by the sperm shape hypothesis)? Or is the situation a bit more nuanced, where condensation leads to a reduction of volume but also to a shutdown of transcription, protection from DNA damage, etc.? I think the focus on packaging alone is somewhat limiting when it comes to imagining how these proteins might act in the context of genomic conflicts. The authors may want to broaden their description of SNBPs in the Introduction accordingly. We completely agree with the reviewer and are currently exploring these possibilities in follow-up studies on SNBP function. However, it is fair to add that this hypothesis has not been well-recognized, and we, therefore, prefer to include it in our revised Discussion rather than Introduction. However, we also think that SNBP packaging function might be targeted by Wolbachia-encoded toxins, speeding up their evolution (revised Discussion). We think there are many molecular possibilities for SNBPs.

• The authors highlight that some SNBPs are expressed in mature sperm whereas others are transition proteins. The evidence for positive selection chiefly comes from the latter group (and "undefined" proteins that could also be transition proteins). Can the authors comment on whether this is expected/unexpected? Along the same lines, the authors highlight differences between Drosophila and mammals when it comes to the timing of transcription/translation during meiosis, suggesting that meiotic drive can happen in Drosophila because alleles are expressed early and can exert an effect after meiosis regardless of whether the associated locus is present in the gamete. I wonder how this relates, if at all, to the author's finding that transition SNBPs are more likely to be part of conflicts (as indicated by positive selection signals) compared to SNBPs in mature sperm. We thank the reviewer for this comment. We expect that many genes expressed explicitly in spermatogenesis, including SNBP genes, would be under position selection, regardless of whether they are associated with X-Y conflicts. The positive selection signals could come from either X-Y conflicts, sperm competition, or conflicts with Wolbachia; we now discuss all of these in the Discussion.

In contrast, the amplification and loss of a subset of Drosophila SNBPs are more likely associated with X-Y conflicts. We note that known SNBPs retained in mature sperm are more likely to be subject to amplification than known transition proteins.

Regarding the timing of expression, it is true that transition SNBPs act earlier in spermatogenesis than SNBPs retained in mature sperm. However, for the meiotic drive hypothesis to apply, all it requires is for SNBP expression to precede sperm individualization, which it does for most SNBPs, including transition proteins.

• ____ It is not entirely clear from the text (and also e.g. Table S4) how dN and dS (and subsequently dN/dS) where calculated. I presume as a single estimate across the whole phylogeny? If so, how heterogeneous is dN/dS across the phylogeny and can the authors identify specific branches on which selective regimes are different? A branch-level analysis should be better powered than the site-level analysis the authors present, which requires repeated selection on the same set of sites to get a strong enough signal. A branch-specific assessment of evolution would be particularly valuable in combination when combined with the assessment of amplifications/losses. We thank the reviewer for this question. The reviewer is correct. We estimated dN and dS in Supplementary file 4 across the whole phylogeny. We conducted branch tests for the amplification of tHMG only in the Dsim clade (Supplementary file 11).

We are interested in how SNBP amplification happened across species, but we need better gene annotation for their structure in many of these 19 independent cases. Moreover, we hope to combine these with transcriptomic analyses with detailed sequence analyses to reveal how the event happened and how gene conversion, gene duplication, and mutations affect their evolution. Each of these analyses requires extensive additional resources and analyses, and we feel are beyond the scope of this current paper.

• The authors suggest that young SNBPs are more likely to encode essential, non-redundant male fertility functions (p7, third paragraph). I'm not sure whether this generalization is appropriate given the small sample. Tpl94D is as young as Mst77F/Prtl99C, tHMG and CG14835 homologs have been lost along different lineages and most of the events are in a single lineage leading up to D. kikkawai. Do the authors really feel that this generalization is warranted? We agree with the reviewer. However, it is striking that the known fertility essential genes are either young or not universally conserved. We have therefore reworded our conclusion to make this contrast more accurate.__

• How do the sex-chromosomal amplifications differ in sequence from the ancestral autosomal copies? The authors suggest that the sex chromosomal copies might be involved in meiotic drive? Does the sequence offer a function as to how? (e.g. loss of charged residues/DNA-binding capacity?__

These are good questions. We do not know mechanistically how the sex-chromosome amplifications may cause meiotic drive. We did not observe the loss of positive charge or HMG domain in most sex-chromosomal amplified copies (Supplementary file 3). Our current working hypothesis is that they compete for the DNA binding with autosomal SNBP, and might interact with other proteins, e.g., heterochromatin proteins, to disturb sperm function. How they might function to cause meiotic drive is an active area of investigation in our and other labs.

• I think it would be nice to have a final table/figure to summarizing the different lines of evidence for all the genes in Table 1 (i.e. positive selection yes/no, amplification in some lineages yes/no, sex chromosome translocations yes/no), for different lineages, including whether any of the HMG-box genes are unlikely to act as SNBPs. We agree with this suggestion. We have now significantly revised and added to Table 2 to include this added information.

• The evidence the authors present is often consistent with genetic conflicts between sex chromosomes. Is it cogent? Arguably not (since direct tests of the mechanism are provided. I would therefore suggest a more cautious title than one stating that conflicts drive expansion and loss of SNBPs. We agree with all three reviewers and have amended our title to highlight the correlation. We also discuss other possibilities that can drive SNBP evolution in our revised Discussion.

__Typographical errors etc.:

• P3. First paragraph: "One of the driving forces ... " I found this sentence a bit odd in terms of causality (changes in composition being portrayed as a force that leads to selection) __

We thank the reviewer for pointing out the confusing construction. We modified the sentence to “The positive selection of SNBPs results in changes to their amino acid composition.”

- P3. Second paragraph: should be "HMG-box" rather than "HMB-box"

Fixed.

- P3. Fourth paragraph "..., consistent with the observation in mammals". I think "consistent" should be reserved for two observations that speak to the same phenomenon. SNBPs could evolve with no evidence for positive selection in Drosophila and that wouldn't exactly be "inconsistent" with mammals. It would just be different.

Fixed. We changed “consistent with” to “similar to”.

____- P5. Fifth paragraph: should be "in the PAML package" rather than "in PAML package"

Fixed.

- P9. Second paragraph: "... montium group (Fig 5A)...)" should be Fig 6A.

Fixed.

__CROSS-CONSULTATION COMMENTS I have not much to add. The other reviews seem fair and well-informed from my somewhat-outside perspective. I don't know how tricky/time-consuming the suggested additional fly mating experiments are but want to note that, in general, I'm loath to "punish" authors of principally bioinformatic work for including some experiments. If experimental shortcomings can be addressed with appropriate caveats, that should be an option, as should removal of experimental data that - by the experts - would be considered too preliminary.

__

We thank the reviewer for their support. However, we felt that improved experiments on CG30056 role in fertility could broaden the scope of this paper, despite the additional time and labor commitment. We have now finished these experiments and they do reinforce our original conclusions with much greater support.

__It is my policy to sign my reviews.

Tobias Warnecke

Reviewer #3 (Significance (Required)):

I'm not enough of an expert in the field of SNBPs to assess the level of advance provided by this study. __

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

This manuscript by Chang & Malik consider the evolution of HMG-box-containing sperm nuclear basic proteins (SNBPs) across Drosophila species in phylogenetic context.

Previous work in mammals had highlighted fast evolution of proteins involved in chromatin remodeling during spermatogenesis. Here, the authors provide evidence for widespread positive selection and likely involvement in genetic conflict in a set of proteins with analogous functions in Drosophila. Amongst other findings, the authors highlight biased amplification of SNBP paralogs on sex chromosomes along several Drosophila lineages, a tendency towards loss/pseudogenization following translocation onto a sex chromosome, and an intriguing concerted SNBP loss event in the montium group where parts of the Y chromosome have become fused to the X, thus nullifying the chance that genetic conflicts can play out via distorted segregation of sex chromosomes.

The authors suggest that, taken together, their findings support widespread of SNBPs involvement (as instigators and repressors) in meiotic drive.

Overall, I found the manuscript to be well written and thorough in its exploration of the evolutionary dynamics of SNBPs in this clade.

Below, I have highlighted some aspects that I think would benefit from further attention, none of them major.

• Following their exploration of patterns of SNBP evolution in Drosophila, the authors highlight support of their data for genetic conflict between sex chromosomes. They also rightly acknowledge that other evolutionary drivers such as sperm competition might also play a role in, for example, fast evolution of certain SNBPs. Yet those (not mutually exclusive) alternatives are never pitted directly against each other. The focus is firmly on exploring the support for the sex chromosome genetic conflict model. Given that the authors highlight Drosophila as a great model in part because of its well characterized sperm biology (including comparative morphology), I wondered why the authors had not made an explicit attempt to see if SNBP evolution covaries with aspects of sperm morphology across Drosophila.
• The most intriguing part of the manuscript for me was the exploration of SNBP fate in the montium group, where the authors find evidence for an ancestral fusion event between the X and parts of the Y chromosome. The loss of SNBPs is certainly consistent with the conflict model but I was wondering to what extent this lineage is characterized more broadly by unusual evolution at the chromosomal level. Is there simply a lot of upheaval in montium, with more frequent gain/loss across the board? How specific is SNBP loss in the context of other orthologous groups? This could be investigated by looking at retention of other genes in other orthologous groups (in montium and some other control group) or perhaps by looking at synteny conservation.
• In introducing SNBPs, the authors focus on their role as packaging agents. Clearly, SNBPs do package the genome in the sense that they bind to DNA and lead to reduced chromosome volume. But is this all packaging for packaging's sake (as portrayed by the sperm shape hypothesis)? Or is the situation a bit more nuanced, where condensation leads to a reduction of volume but also to a shutdown of transcription, protection from DNA damage, etc.? I think the focus on packaging alone is somewhat limiting when it comes to imagining how these proteins might act in the context of genomic conflicts. The authors may want to broaden their description of SNBPs in the Introduction accordingly.
• The authors highlight that some SNBPs are expressed in mature sperm whereas others are transition proteins. The evidence for positive selection chiefly comes from the latter group (and "undefined" proteins that could also be transition proteins). Can the authors comment on whether this is expected/unexpected? Along the same lines, the authors highlight differences between Drosophila and mammals when it comes to the timing of transcription/translation during meiosis, suggesting that meiotic drive can happen in Drosophila because alleles are expressed early and can exert an effect after meiosis regardless of whether the associated locus is present in the gamete. I wonder how this relates, if at all, to the author's finding that transition SNBPs are more likely to be part of conflicts (as indicated by positive selection signals) compared to SNBPs in mature sperm.
• It is not entirely clear from the text (and also e.g. Table S4) how dN and dS (and subsequently dN/dS) where calculated. I presume as a single estimate across the whole phylogeny? If so, how heterogeneous is dN/dS across the phylogeny and can the authors identify specific branches on which selective regimes are different? A branch-level analysis should be better powered than the site-level analysis the authors present, which requires repeated selection on the same set of sites to get a strong enough signal. A branch-specific assessment of evolution would be particularly valuable in combination when combined with the assessment of amplifications/losses.
• The authors suggest that young SNBPs are more likely to encode essential, non-redundant male fertility functions (p7, third paragraph). I'm not sure whether this generalization is appropriate given the small sample. Tpl94D is as young as Mst77F/Prtl99C, tHMG and CG14835 homologs have been lost along different lineages and most of the events are in a single lineage leading up to D. kikkawai. Do the authors really feel that this generalization is warranted?
• How do the sex-chromosomal amplifications differ in sequence from the ancestral autosomal copies? The authors suggest that the sex chromosomal copies might be involved in meiotic drive? Does the sequence offer a function as to how? (e.g. loss of charged residues/DNA-binding capacity?)
• I think it would be nice to have a final table/figure to summarizing the different lines of evidence for all the genes in Table 1 (i.e. positive selection yes/no, amplification in some lineages yes/no, sex chromosome translocations yes/no), for different lineages, including whether any of the HMG-box genes are unlikely to act as SNBPs.
• The evidence the authors present is often consistent with genetic conflicts between sex chromosomes. Is it cogent? Arguably not (since direct tests of the mechanism are provided. I would therefore suggest a more cautious title than one stating that conflicts drive expansion and loss of SNBPs.

Typographical errors etc.:

• P3. First paragraph: "One of the driving forces ... " I found this sentence a bit odd in terms of causality (changes in composition being portrayed as a force that leads to selection)
• P3. Second paragraph: should be "HMG-box" rather than "HMB-box"
• P3. Fourth paragraph "..., consistent with the observation in mammals". I think "consistent" should be reserved for two observations that speak to the same phenomenon. SNBPs could evolve with no evidence for positive selection in Drosophila and that wouldn't exactly be "inconsistent" with mammals. It would just be different.
• P5. Fifth paragraph: should be "in the PAML package" rather than "in PAML package"
• P9. Second paragraph: "... montium group (Fig 5A)...)" should be Fig 6A.

Referees cross-commenting

I have not much to add. The other reviews seem fair and well-informed from my somewhat-outside perspective. I don't know how tricky/time-consuming the suggested additional fly mating experiments are but want to note that, in general, I'm loath to "punish" authors of principally bioinformatic work for including some experiments. If experimental shortcomings can be addressed with appropriate caveats, that should be an option, as should removal of experimental data that - by the experts - would be considered too preliminary.

It is my policy to sign my reviews.

Tobias Warnecke

Significance

I'm not enough of an expert in the field of SNBPs to assess the level of advance provided by this study.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The paper describes interesting patterns on the evolution of Drosophila SNBP genes, and proposes a very interesting explanation, namely, that meiotic drive is the main evolutionary force behind these patterns. Some of these observations have recently been made by other authors in a single case (the Dox genes in D. simulans), but not in the scale and breadth of the present ms. The ms combines an extensive investigation of available genomes with expert analysis, and new experimental data. In particular, the finding that the ancestral Y became incorporated into de X in montium species is very exciting, and may provide a smoking gun for the explanation proposed by the authors. Overall, I think it is a very good paper. I do have several criticisms and suggestions that may help to improve it.

The paper has a speculative side that it almost unavoidable given its novelty and breadth. I do not see this as a problem per se, but I think the uncertain/unsupported/problematic points should be more openly presented to the readers. The main cases I noted are:

1. The title of the ms states that "Genetic conflicts between sex chromosomes drive expansion and loss of sperm nuclear basic protein genes in Drosophila", but the evidence is somewhat circumstantial, and the patterns may be explained also by other known phenomena (e.g., demasculinization of the sex chromosomes; below). I think the tone of the end of the Introduction reflects more faithfully the strength of the evidence ("Thus, we conclude that rapid diversification of SNBP genes might be largely driven by genetic conflicts between sex chromosomes in Drosophila."). I understand the temptation of writing a bold title, but I think it is a bit misleading in the present case. I.e., it would be desirable that the title conveys the uncertainties of the data and their interpretation.
2. "In contrast, we found no instances of pseudogenization or subsequent translocation to the X chromosome of SNBP genes that are still preserved on their original autosomal locations or involved in chromosome fusions between autosomes (0/16). This difference is highly significant (Fig 5 and Table S11; 3:5 versus 0:16, Fisher's exact test, P=0.03). " Readers should be warned that this pattern can also be explained by the well-known demasculinization of X chromosomes (e.g., Sturgill et al. Nature 2007, 450, 238-241)
3. "Indeed, no meiotic drive has been documented in the montium species even though it is rampant in many other Drosophila lineages [38]." Two remarks here: a) the authors should make clear that they are referring to sex-chromosome meiotic drive. b) I think the evidence is much weaker than the sentence implies. Sex-chromosome meiotic drive is known in less than 20 Drosophila species, scattered throughout the phylogeny. As far as I know all cases were discovered by accident, so the sampling is biased towards model species (e.g., the obscura group, which was very popular around 1930-1960). So we do not know the true frequency of sex-ratio meiotic drive among Drosophila species, nor, say, if it is more common in the Drosophila or Sophophora species, if it is suspiciously absent in the montium group (as suggested by the authors), etc. I think these uncertainties should be acknowledged or, perhaps, given the weakness of the argument, the sentence should be deleted or attenuated.
4. "X-Y chromosome fusions eliminate the extent of meiotic drive and may lead to the degeneration of otherwise conserved SNBP genes, whose functions as drive suppressors are no longer required. Thus, unlike in mammals, sex chromosome-associated meiotic drive appears to be the primary cause of SNBP evolutionary turnover in Drosophila species." The authors found that in the montium species the ancestral Y became incorporated into de X chromosome, and that montium species seem to have an inordinate amount of SNBP gene losses. They combine these two observations by suggesting that these SNBP became dispensable or deleterious because they originally wee involved in XY meiotic drive. I think many readers will think that males in montium species are X/0, whereas in fact in all of them carry a Y chromosome (just, in most cases, more gene poor than "normal" Y-chromosomes). I do not think this is a fatal flaw for the explanation proposed by the authors, but certainly is a difficulty that should be acknowledged.

Problems/suggestions with experiments and data analysis

1. There is a section titled "CG30056 is universally retained in Drosophila but dispensable for male fertility in D. melanogaster". In this section and in the figures, it is stated, "Although CG30056 is the most conserved SNBP we surveyed, we found no clear difference in offspring number between heterozygous controls and homozygous knockout males (Fig 2B). (...) We found either no or weak evidence of fertility impairments in two different crosses with homozygous CG30056 knockout males.". I think the fertility data are weak for the purpose of the authors, and I strongly suspect that this conclusion is wrong. Let me explain why. At other passages of the ms, the authors classify the SNPB genes in three groups.
   • (i) essential/important for male fertility: "Three genes (Mst77F, Prtl99C and ddbt) are essential for male fertility while knockdown or knockout of two other SNBP genes (ProtA, and ProtB) leads to significant reduction in male fertility [27-30, 32]."
   • (ii) genes that do not appear to impair male fertility at all.
   • (iii) untested. CG30056 was in the last group, and hence the authors produced knockouts, tested their effect in male fertility, and concluded that it belongs to the second group. Now, look at Fig. 3B. The numbers of tested males are too small (it seems to range from 3 to 10), and male fertility is known to be a very noisy phenotype (as shown by the huge scatter in the authors' data). Furthermore, two different knockouts were tested, and both were nominally less fertile than the controls, and in one of them the difference is statistically significant. Taken at the face value, the knockouts seem to be perhaps ~25% less fertile than the controls. Another potentially big problem is that the "control males" actually carry visible dominant mutants (the balancers CyO or SM6) which certainly reduce their fitness, whereas the experimental males are wild-type for these mutants. Without the detrimental effect of these visible mutants in the controls, the difference to the CG30056 knockouts will probably be even larger. Note that the fertility effects of the genes ProtA, and ProtB (a.k.a. "Mst35B") , which the authors put in group "essential/important for male fertility" would not had been detected if assayed as the CG30056 gene: Tirmarche et al (2014; the reference cited by the authors) stated that: "In fact, the impact of Mst35B on male fertility was only revealed when mutant males were allowed to mate with a large excess of virgin females (1 for 10; Figure 3F) but not with a 1:1 sex ratio (not shown). " The authors' fertility test did not used this type of challenge. My general impression is that the fertility effects of CG30056 may actually be similar to ProtA and ProtB. I think the authors should do a proper fertility test of CG30056, or remove this section. Another possibly useful approach would be to classify the SNPB genes in those essential for male fertility and those that are not essential, because "experimentally speaking" this is a safer distinction (e.g., the fertility testes reported by other authors may also had been quick tests). Since these genes only function in sperm and are under purifying selection (otherwise they would have been lost; also, all have dN/dS < 1 ), they all most likely affect male fertility to some extent. In case the section on male fertility stays, it will be necessary to provide more details. How many males were crossed for each genotype? In some cases in Fig 2B, it seems that as low as 3, but it may be data superposition in the graph. Please provide the raw data in the supplementary material.
2. "Our phylogenomic analyses also highlighted one Drosophila clade- the montium group of species (including D. kikkawai)- which suffered a precipitous loss of at least five SNBP genes that are otherwise conserved in sister and outgroup species (Fig 3). (...) Given our hypothesis that autosomal SNBP genes might be linked to the suppression of meiotic drive (above), we speculated that the loss of these genes in the montium group of Drosophila species may have coincided with reduced genetic conflicts between sex chromosomes in this clade." The montium data is an important part of the paper. I think the authors should test the statistical significance of this pattern.

Other points:

1. "The five remaining SNBP genes (Mst33A, CG30056, CG31010, CG34269, and CG42355) remain cytologically uncharacterized [30]." I think it will be interesting if the authors look at other potentially useful resources: Vibranovski et al papers which looked at gene expression in mitotic, meiotic and post-meiotic cells (https://mnlab.uchicago.edu/sppress/index.php), and the papers by several labs on testis single-cell transcriptomic data (Witt et al 2021 PLOS Genetics. 17(8):e1009728 ; Nat Commun. 2021;12: 892). These may provide additional clues on the function of SNBP genes. There is also a recent report on sperm proteome (doi: https://doi.org/10.1101/2022.02.14.480191)
2. "Our inability to detect homologs beyond the reported species does not appear to result from their rapid sequence evolution. Indeed, abSENSE analyses [45] support the finding that Prtl99C, Mst77F, Mst33A, Tpl94 and CG42355 were recently acquired in Sophophora within 40 MYA. For example, the probability of a true homolog being undetected for Prtl99C and Mst77F is 0.07 and 0.18 (using E-value=1), respectively (Table S1, Methods)." This should be complemented by synteny analysis.
3. I found the following sentence unclear: "However, we could only ascribe a sex chromosomal linked location for species if no data was available from either BUSCO genes or females (only males and mixed-sex flies)."
4. "Using the available assemblies with Illumina-based chromosome assignment, we surprisingly found that most ancestrally Y-linked genes are not linked to autosomes as was previously suggested [by Dupim et al 2018] (Fig 6A)." The new result of X-linkage is exciting, but the sentence is not exact: Dupim et al 2018 made clear that they could only separate X/A from Y-linkage. E.g., the legend of their Fig 3: "Phylogeny and gene content of the Y chromosome in the montium subgroup. "M" means amplification only in males (i.e., Y-linkage), whereas "MF" means amplification in both sexes (autosomal or X-linkage)."
5. "The most parsimonious explanation for these findings is a single translocation of most of the Y chromosome to the X chromosome via a chromosome fusion in the ancestor of the montium group of species. Afterward, some of these genes relocated back to the Y chromosome in some species (Fig S6; Supplementary text)." Explanations for this pattern of "return to the Y" have been extensively discussed and tested in Dupim et al 2008 (see their section "Why genes seem to return to the Y chromosome after Y incorporations?" ) The available evidence strongly suggests that it is not a case of relocation to the Y.
6. Fig 6B suggests that the authors assembled the "translocated Y" in D. triauraria. However, no direct data or account for this assembly is provided. Please clarify.
7. "Why would meiotic drive only influence Drosophila, but not mammalian, SNBP evolution? One important distinction may arise from the timing of SNBP transcription. In D. melanogaster, SNBP genes are transcribed before meiosis but translated after meiosis [29, 43, 57]. Thus, SNBP transcripts from a single allele, e.g., Xlinked allele, are inherited and translated by all sperm, regardless of which chromosomes they carry. Consequently, they can act as meiotic drivers by causing chromatin dysfunction in sperm without the allele, e.g., Y-bearing sperm." During spermatogenesis Drosophila haploid cells actually are syncytial, which has interesting consequences for the evolution of male genes (Raices et al, Genome Res. 1115-1122, 2019). This may be relevant for the present paper.

Significance

see above

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Referee #1

Evidence, reproducibility and clarity

Chang and Malik present a comprehensive evolutionary analysis of sperm nuclear basic proteins (SNBPs) in Drosophila. In addition, they provide a preliminary functional characterization of one such protein (CG30056) and describe a newly discovered X-Y chromosomal fusion in the Drosophila montium species group. All of these findings are interesting and important, but the headline from this study is the well-supported possibility that SNBPs, or at least a large fraction of them, function in suppressing X vs. Y chromosome meiotic drive. While this hypothesis is challenging to test experimentally, the authors provide strong correlational evidence that SNBPs are associated with drive by documenting these proteins' rapid evolution. This rapid evolution takes the form of sequence changes (as predicted by coevolution between drivers and suppressors of drive), gene amplification in cases when SNBPs move to sex chromosomes (consistent with the SNBP becoming a potential agent of drive for its new "home chromosome"), and gene loss in species with X-Y chromosome fusions (in which drive is not predicted to occur).

Overall, this is an excellent, comprehensive study. The phylogenetic and genomic analyses are first-rate (and one of the first to make use of the new 101 Drosophila genomes); the logic is very well explained; conclusions are supported by multiple lines of evidence; the writing and figures are clear and accessible; and, the findings are fascinating. It's a good sign that it is easy to imagine several experiments one could do to follow up on this study, but I do not feel any are required in revision, as the manuscript is comprehensive as is. Thus, I have just a few minor points the authors may wish to consider in making revisions and a few suggestions for clarity/typos.

1. I would be interested in whether the authors think that all SNBPs in a given Drosophila species function(ed) in meiotic drive, or whether some fraction may play other roles, such as sexual selection or chromatin compaction, which have been the traditional hypotheses for SNBP function. Relatedly, given the high turnover of SNBPs the authors observe and the fact that some melanogaster-essential SNBPs are younger genes, would they like to comment on whether the subsets of SNBPs involved in drive/suppression vs. chromatin packaging/sperm traits/Wolbachia defense are likely to differ from across fly species?
2. What do the authors make of the lower isoelectric points for a few of the SNBPs (e.g., CG31010 with pI = 4.77 in Table 1)? These proteins have identifiable HMG box domains, so is the pI driven lower by other parts of the protein sequence?
3. For readers less familiar with the field, it may help to spell out (e.g., on p. 6) why the authors consider ProtA/B to be important for fertility. Some of the previous papers on these genes describe them as dispensable - though the present authors are correct that these previous studies do detect fertility defects of various magnitudes under some conditions.
4. On p. 9, paragraph 2, the data showing that "six different SNBP genes underwent 11 independent degeneration events in the montium group" are shown in Fig. 6A, not 5A.
5. The summary Table 2 is useful, but I wonder whether including relative levels of expression and dN/dS in addition to ordinal rankings might help clarify. For instance, if there were a drop off in mean expression level between the 5th and 6th most highly expressed SNBP, this wouldn't be evident from the table.
6. In Fig. 3, I like the use of the clean CG31010 figure in panel A to illustrate the circular representations. In addition, though, it might be useful to show Prot's graph at this same, larger size, since it's the most complicated and will likely be most closely examined.
7. In Fig. 4, the end of the legend says that the species tree is shown "on the right," but it's on the left in the figure.

Referees cross-commenting

• I agree with both Reviewers 2 and 3 that the title could be changed to be a bit more tentative. I'd had this thought as well.
• I agree with Reviewer 2 that the fertility assay could be conducted with a larger sample size and a better control in order to be better compared with how the authors described other published fertility phenotypes for SNBPs. For the control, crossing the deletion line to y w (or w1118) and using the resulting heterozygotes (KO/+) would be better than using the mutation over the balancer chromosome (KO/CyO).
• I agree with Reviewer 3's third bullet point about spending a bit more time on the different possible roles that SNBPs could play in spermatids. (This is a more eloquent version of my review point #1.)
• I agree in principle with Reviewer 3's first bullet point about examining whether SNBP evolution correlates with changes in sperm morphology, but this feels like it could be a whole, fascinating study on its own, while this manuscript is already packed with data. I'd welcome the authors' thoughts about this in discussion, but wouldn't personally require a formal analysis of this to be added prior to publication.

Significance

This study describes an important conceptual advancing in our understanding of the evolution and potential functions of sperm nuclear basic proteins (SNBPs) in Drosophila, which stands in interesting contrast to the functional roles of equivalent proteins in primates. It should be of broad interest to biologists studying spermatogenesis, meiotic drive, and genome evolution, both in and out of Drosophila.

To contextualize the work, paternal DNA is typically compacted during spermatogenesis. This process involves the replacement of histones with other small, positively charged proteins in a sequential order, ending with protamines that bind DNA in mature sperm. In Drosophila, work over the last two decades (largely from the labs of R. Renkawitz-Pohl, B. Loppin and B. Wakimoto) has identified more than a dozen sperm nuclear basic proteins that localize to condensing/condensed spermatid nuclei. Two interesting observations have been that many of these proteins are dispensable for male fertility, and the proteins vary in their degree of evolutionary conservation. Recent work from Eric Lai's lab (J Vedanayagam et al. 2021, Nat Ecol Evol) showed that in D. simulans and sister species, at least one of these SNBP genes (Prot) underwent gene amplification and now acts in those species as a meiotic driver. This finding suggested the hypothesis, tested thoroughly in the present study, that the rapidly evolving SNBP gene family could be involved in causing or suppressing meiotic drive. Consistent with this idea, the authors here find that SNBP genes expand in copy number more frequently when they move from autosomes to sex chromosomes (consistent with the idea that they may cause or contribute to drive), and that otherwise well-conserved SNBP genes are lost in a group of species in which sex chromosome meiotic drive is not expected to occur. These findings are based on a thorough and well conducted phylogenomic and molecular evolutionary analysis of SNBPs across dozens of Drosophila species. Overall, this work generates exciting new hypotheses about the function of SNBPs and should be widely read both within and outside of the field.

Keyword describing my field of expertise: Drosophila, molecular evolution, reproduction, genetics, genome evolution.

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Reply to the reviewers

1. General Statements

We thank the reviewers from Review Commons for their thorough reviews of our manuscript entitled, “The role of Limch1 alternative splicing in skeletal muscle function.” We were delighted by the many supportive comments of all three reviewers calling our study a “definite advance in our understanding of developmentally-regulated splice isoform transitions that are disease relevant”, “good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid”, and “The article is well written, and I favor the publication of this [article] with minor revisions.”

The reviews include comments on the interest in identifying the mechanism of action of mLIMCH1 in skeletal muscle function such as “ [The study] presents multiple new tools to study mLimch1 and identifies a possible role for mLIMCH1 in calcium regulation, but stops short of identifying the mechanism by which this regulation occurs.” While we agree that how the skeletal muscle-specific isoform of LIMCH1 affects calcium handling is of interest, we respectfully suggest that this manuscript describe previously unknown biology that will be of interest to investigators in different fields including muscle physiology, alternative splicing regulation, and skeletal muscle pathology in myotonic dystrophy. All experiments in this manuscript are performed in vivo using skeletal muscle tissues from animals lacking the isoform of Limch1 that is expressed only in skeletal muscle and is normally induced after birth. Comparisons were made to age-matched wild-type control animals, often litter mates. The results establish the functional significance of the LIMCH1 protein and particularly the muscle-specific isoform in skeletal muscle through extensive analysis of LIMCH1 localization and the impact of mLIMCH1 knockout on muscle strength, force generation, calcium handling and the disease relevance of this splicing transition in myotonic dystrophy type 1. Please review the comments of all three reviewers who were quite favorable to the significance of the work and overall favorable to its publication. Below, we clarify and describe additional data that has, and will be added to the manuscript to address all comments of the reviewers.

2. Description of the planned revisions

Reviewer 1

“Page 6 - data not shown. The point of conservation is not essential to this story, but the authors should either include a table or panel with that data, or remove the data not shown statement. Given the putative relevance to DM1, it might be preferable to include data to support the developmental transition in human data.”

We have removed the “data not shown” statement as suggested and we highlighted the importance of conservation of the induction of a skeletal muscle isoform of LIMCH1 after birth as a strong indication of functional importance for the isoform. We agree that data showing the conserved LIMCH1 splicing transition in human skeletal muscle development will support this point. We will include RT-PCR analysis of LIMCH1 splicing in fetal and adult human skeletal muscle RNA in Figure 6 to support the reversion of splicing to the fetal pattern observed in DM1. The results will complement the normal Limch1 splicing transition in mice (Figure 1) and the normal and aberrant fetal splicing patterns shown for unaffected and DM1 adult skeletal muscle, respectively (Figure 6).

3. Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer 1

“Figure 4 - The authors do a nice experiment to show the localization of Limch1 and raise an antibody to detect the muscle specific isoform. The data seem to show that the muscle-specific isoform localizes to the sarcolemma, and this staining is largely lost in the mutant mice. By contrast, one could infer that the cytoplasmic signal in the WT comes from the ubiquitous isoform (which accounts for 30-40% of the Limch1 expression). This is consistent with the validation in Fig. 2. However, the authors in the text claim this experiment reveals an increased distribution throughout the myofiber, or a more even signal distribution in the cytoplasm, and that the uLimch1 cannot recapitulate mLimch1 localization. Fig. 2 suggests that total levels of Limch1 are increased (as noted by the authors in the discussion). Given that the muscle-specific isoform localizes to the sarcolemma, and the ubiquitous isoform is presumably sarcoplasmic, it isn't clear to me that there is any change in localization per se. What the authors show is just that the signal at the sarcolemma is lost, and if one compares the intensity in the right-hand plots in Fig. 4B, they are comparable in the sarcoplasmic region. It seems likely there is more of the ubiquitous isoform, and what is seen here is just how that isoform localizes. The quantification the authors perform in D would likely show this strong difference in the localization of the muscle isoform. If the authors redo this quantification, exclude the signal at the sarcolemma and normalize to the average pixel intensity in the fiber, do they still see a difference? I am not convinced that the "clustering" of the signal of the ubiquitous, cytoplasmic isoform is in any way changed. Given the difference in the two proteins, I also would not expect that the ubiquitous isoform could compensate for loss of the muscle isoform, and would not expect it to "recapitulate" the muscle-isoform localization.”

We agree that Figure 4 and the explanation in the text was not clear and we thank the reviewer for pointing this out. We have addressed this concern by modifying the figure as suggested by the reviewer and clarifying the description in the results section. The main point, that is recognized by the reviewer but needed clarification, is that the mLIMCH1 isoform preferentially localizes to the sarcolemma and the uLIMCH1 isoform is preferentially cytoplasmic. In the HOM Limch1 6exKO myofibers, the increased cytoplasmic signal is due to the increased level of uLIMCH1 as shown by the western blot in Figure 2. The reviewer is correct that there is not a “change in localization of isoforms per se”. We clarified this point to highlight the differential localization of the uLIMCH1 and mLIMCH1 isoforms within the sarcolemma vs. the sarcoplasm. The revision of the plot profile in Figure 4B and the analysis of the standard deviation of signal in Figure 4D demonstrates the stark difference in staining observed between the HOM Limch1 6exKO and WT myofibers when stained with a pan-LIMCH1 antibody. The signal intensity plot profile from sarcolemma to sarcolemma (Figure 4B) indicates that the uLIMCH1 isoform is not “mis-localized” upon mLIMCH1 knockout as we originally (mis)-stated. Upon mLIMCH1 knockout, there is increased uLIMCH1 expression compared to WT myofibers. Considering this in combination with the sarcolemma preference of mLIMCH1 (Figure 4E) and the significant loss of signal in the sarcolemma region in Limch1 6exKO myofibers, we conclude that in HOM Limch1 6exKO myofibers, uLIMCH1 is primarily localized throughout the sarcoplasm.

Reviewer 1 (optional)

“Experiments looking more closely at LIMCH1 co-localization with other proteins at the sarcolemma or the sufficiency of the muscle-specific region to localize would also be useful (for example, can the muscle-specific region localize GFP to the membrane in cells?).”

We performed immunofluorescence microscopy of LIMCH1 with several skeletal muscle-relevant proteins but did not observe: (1) disruptions of normal structures in HOM Limch1 6exKO compared to WT myofibers or (2) colocalization that helped clarify any mechanistic role of mLIMCH1 or uLIMCH1. Therefore this data was not included in the original manuscript. In regard to the suggestion on the sufficiency of the muscle-specific region to localize to the sarcolemma region, we had previously generated a plasmid to express a fluorescent protein fused to the protein encoded by the six skeletal muscle-specific exons of LIMCH1 but it failed to localize to the sarcolemma. In collaboration with protein structural experts at Baylor College of Medicine, we analyzed the skeletal muscle-specific region of LIMCH1 and found it to be entirely disordered without known homologs. It appears that this region has no secondary structure but when expressed within the entire LIMCH1 protein which has conserved domains (calponin homology, LIM, coiled-coil regions) and upon protein binding, it is possible for the region to adopt a structure facilitating its binding in the sarcolemma region. Therefore we believe that regions common to both isoforms are required in combination with the muscle-specific region for preferential localization to the sarcolemma.

Reviewer 1 (minor comments)

“In the Figure 3 legend, the order of the descriptions for B-C and D-E is switched. The order of the panels matches the text, but the legend switches the description of the force-frequency curves (shown in B & C but labeled as D & E), with the description of the rate of relaxation and contraction plots (shown in D and E but labeled as B and C in the legend).”

We fixed this error and thank the reviewer for pointing it out.

“The scale in Figure 4, panel B between the top and bottom plots is not the same, so it is difficult to compare, particularly for the panels on the right. See comment above.”

In addition to clarifying uLIMCH1’s localization upon mLIMCH1 knockout within the text, we added figure titles above the plot profile which will clarify the different plot profiles for the reader. In regard to the comment about the scale of the plot profile, we have addressed this by re-scaling the two plot profiles on the right in Figure 4B. These plot profiles now share the same scale, which is advantageous because this plot profile better emphasizes the stark difference in signal observed between the sarcolemma and sarcoplasm in WT myofibers that is lacking in HOM Limch1 6exKO myofibers.

Reviewer 2

“Figure 6A: There is a discrepancy between gene structures and splicing isoforms shown in Fig. 1 vs Fig. 6. There are differences in spacing between exons, and there appear to be six exons in the differentially regulated region in Fig 1, but seven exons in Fig 6. Perhaps this is a difference between human and mouse genes? Does the human gene actually regulate seven exons in this region, rather than six exons in the mouse? In both figures the gene is labeled as Limchi1, and both figures indicate that the ubiquitous isoform lacks exons 9-14. Please clarify.”

The reviewer is correct that the human mLIMCH1 isoform contains seven exons that are skeletal muscle-specific compared with the six exons that are skeletal muscle-specific in the mouse. The seven human exons encode 544 amino acids with 65% homology with the mouse segment. We have clarified this in the figure legend and text. Exons 9-14 are shown in Figure 6B since this diagrams the mouse gene.

“The methods section on RT-qPCR and RNA splicing presumably refers to analysis of mouse tissues. What is the origin of the human DM1 RNA-seq data?”

We obtained adult human DM1-affected and non-affected skeletal muscle autopsy samples from colleagues and the NDRI and performed RNA-sequencing at Baylor College of Medicine. The RNA-seq has not yet been published, but we include the data for LIMCH1 to demonstrate the dramatic change in the alternative splicing pattern in DM1 skeletal muscle tissue. This has been clarified in the methods section.

“Perhaps the word "activity" should be deleted in the following sentence: "The sole study investigating the function of LIMCH1 characterized it as an actin stress fiber associated protein that binds non-muscle myosin 2A (NM2A) activity to regulate focal adhesion formation."

We thank the reviewer for pointing this out and we have removed this word.

Reviewer 3

“The diminution of the muscle force production in Limch16exKO is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend).”

As suggested by Reviewer 3, we have included additional histological data in Supplementary Figure 2. In addition to the histology at 10-12 weeks of age, the new data includes histology of multiple skeletal muscle tissues (quadriceps, EDL, soleus) at one year of age. The histology of Limch1 6exKO tissue at different time points showed no morphological differences (centralized nuclei or fibrosis) consistent with no change in muscle weight which led us to emphasize the significant effect of mLIMCH1 knockout on skeletal muscle function in the absence of muscle loss or overt structural changes. In regard to fiber-type, we have included histology of both the EDL (fast-twitch) and soleus (slow-twitch) and even after one year, we observe no gross morphological differences. Additionally, we analyzed the force production of both the EDL and soleus (Figure 3) with the fiber-type predominance of these tissues in mind and found decreased force generation in both tissues. We included the types of skeletal muscle tissue analyzed and the age of the mice in Supplementary Figure 2 as per the reviewer’s suggestion.

“The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?”

We conducted RNA-sequencing on tissue from HOM Limch1 6exKO and WT controls and the results were disappointing showing minor differences that did not contribute to understanding the phenotype. We used this data only to show the loss of the six exons in Fig. 2B, however, we decided that RT-PCR analysis was the better assay since it shows not only that the exons are not included but also that exons 8 and 15 are spliced correctly, which is not apparent using the RNA-seq displayed on the genome browser.

4. Description of analyses that authors prefer not to carry out

Reviewer 1 (____Both points listed as optional)

“If the authors perform TEM, can they see defects in t-tubules or organization of the sarcoplasmic reticulum, that are not visible by light microscopy?”

We considered conducting TEM to investigate sarcomeric, T-tubule, or sarcolemma changes in myofibers derived from HOM Limch1 6exKO mice, but we concluded that it would most likely be of limited use. We do not think that T-tubule structural changes will be observed via TEM primarily due to the challenges of finding significant changes compared to WT controls in which one can always find abnormal structures. In our experience and the experience of our collaborator (Dr. Rodney) the disruptions must be dramatic to distinguish from the noncanonical structures often observed. Thus, we do not plan on conducting TEM to identify defects in the T-tubules.

“If the muscle-specific isoform is transfected or transduced into differentiated myotubes, how does this affect calcium dynamics in the culture system?”

While an interesting idea, we do not plan on conducting this experiment for multiple reasons. One issue is that all of our data is derived from in vivo analysis or from isolated myofibers and our concern is that the relatively immature state of myotubes in culture will provide a poor comparison to isolated myofibers. Therefore, we believe that it will be difficult to add meaningful data to the calcium data presented in Figure 5 through this experiment. Additionally, we have observed mis-localization of the overexpressed uLIMCH1 and mLIMCH1 in C2C12 cells that we believe would add too many caveats for meaningful interpretation of the results, regardless of the effects on calcium dynamics

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, the authors have generated a knockout mouse model of a skeletal muscle-specific splice variant isoform of Limch1. These KO mice present skeletal muscle force production and calcium handling defects. These results could explain why the deficiency in splicing in myotonic dystrophy1 can lead to skeletal muscle defects.

Overall, this is a good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid. The article is well written, and I favor the publication of this article with minor revisions.

Issues that I think the authors should clarify:

• The diminution of the muscle force production in Limch16exKO, is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend)
• The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?

Significance

In this manuscript, the authors have generated a knockout mouse model of a skeletal muscle-specific splice variant isoform of Limch1. These KO mice present skeletal muscle force production and calcium handling defects. These results could explain why the deficiency in splicing in myotonic dystrophy1 can lead to skeletal muscle defects.

Overall, this is a good comprehensive study with convincing results, the design of the experiments is good, and the conclusions are solid. The article is well written, and I favor the publication of this article EMBO journal with minor revisions.

Issues that I think the authors should clarify:

• The diminution of the muscle force production in Limch16exKO, is not correlated with a change in morphology of the myofibers in H&E and picrosirius stainings (Fig S2). Did the authors look at other skeletal muscles, fiber type, size, or different time points? (The age of the mouse and the name of the skeletal muscle used for the histology could be included in the results sections or figure legend)
• The authors performed RNAseq analysis in the skeletal muscle of the KO mouse (Fig 2B). What is the result of this experiment? Is the KO muscle transcriptome different or similar to control muscles?

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Referee #2

Evidence, reproducibility and clarity

Summary

This manuscript continues the Cooper lab's analysis of the role of alternative splicing in muscle development and function. Here they report an intriguing alternative splicing difference between fetal and adult tissues involving 6 consecutive exons in the LIMCHI1 gene that are included predominantly in adult muscle to encode a longer isoform of the protein. Moreover, by CRISPR/Cas9-mediated deletion of these exons in mouse models, they show that muscle deficient in the longer LIMCHI1 protein isoform exhibits grip strength weakness in vivo and decreased force generation ex vivo. The mechanistic details remain to be investigated, but evidence so far suggests an intrinsic defect in muscle contraction, perhaps related to aberrant calcium handling, without obvious histopathology or muscle loss. Finally, these new findings may have important implications for human patients with myotonic dystrophy type 1 that typically exhibit defects in MBNL-regulated splicing events, because the authors show (1) that patient muscle poorly expresses the muscle isoform of LIMCHI1, due to inappropriate skipping of the exons, and (2) that mice with knockout of MBNL proteins also predominantly skip these exons.

Major comments

1. The major conclusions of the manuscript are clear and convincing -a muscle-specific cluster of 6 exons in the LIMCHI1 gene whose splicing is regulated directly or indirectly by MBNL splicing factor(s); loss of these exons compromises muscle strength; and these exons are poorly spliced in muscle of myotonic dystrophy patients. The data for these conclusions is strong.
2. The authors do consider alternative explanations where appropriate. For example, they speculate in the discussion that muscle defects could be due not only to loss of the muscle-specific isoform, but possibly also due to the corresponding increase in expression of the non-muscle-specific isoform.
3. Figure 6A: There is a discrepancy between gene structures and splicing isoforms shown in Fig. 1 vs Fig. 6. There are differences in spacing between exons, and there appear to be six exons in the differentially regulated region in Fig 1, but seven exons in Fig 6. Perhaps this is a difference between human and mouse genes? Does the human gene actually regulate seven exons in this region, rather than six exons in the mouse? In both figures the gene is labeled as Limchi1, and both figures indicate that the ubiquitous isoform lacks exons 9-14. Please clarify.

Minor comments

1. The methods section on RT-qPCR and RNA splicing presumably refers to analysis of mouse tissues. What is the origin of the human DM1 RNA-seq data?
2. p. 4: Perhaps the word "activity" should be deleted in the following sentence: "The sole study investigating the function of LIMCH1 characterized it as an actin stress fiber associated protein that binds non-muscle myosin 2A (NM2A) activity to regulate focal adhesion formation."
3. Other than the issue raised above regarding LIMCHI1 gene structure, the figures are clearly presented.

Significance

The results in this study could have important implications both regarding muscle function and regulation of alternative splicing. The demonstration of a muscle-specific isoform of LIMCHI1 is a novel finding that suggests previously unknown functions of the protein in muscle contraction. This raises intriguing questions as to how this alternative domain impacts muscle function through cooperation with other domains previously predicted (or shown) to interact with actin and non-muscle myosin. Regarding splicing, co-regulation of exon clusters is a poorly understood phenomenon that could be the subject of future interesting studies. Both issues could be relevant to understanding defects in human patients with myotonic dystrophy type I.

The work would be of interest to scientists studying muscle function as well as those studying alternative splicing. Both groups would probably be intrigued by these results but might consider the results to be relatively preliminary, need more mechanistic details in the future.

Expertise: I have extensive experience in analysis of alternative splicing regulation. My knowledge of specific techniques to evaluate muscle function is more limited. Although the experiments on muscle function seem clear and convincing to me, I admit that I am not an expert on those methods and could have missed an important point.

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Referee #1

Evidence, reproducibility and clarity

Summary

In their paper "The role of Limch1 alternative splicing in skeletal muscle function," Penna and colleagues report a muscle-specific isoform of Limch1 and investigate its function in skeletal muscle. They show that a muscle-specific isoform of Limch1 is expressed preferentially in mature muscle, and demonstrate that animals mutant for this isoform have reduced grip strength and force generation. Notably, although muscle structure and T-tubules are structurally not affected, mutant muscle shows evidence of disrupted calcium handling. Limch1 is also misspliced in DM1 and Mbnl1/2 double mutant mice, suggesting the muscle isoform is disease relevant and regulated by MBNL.

Major comments

Page 6 - data not shown. The point of conservation is not essential to this story, but the authors should either include a table or panel with that data, or remove the data not shown statement. Given the putative relevance to DM1, it might be preferable to include data to support the developmental transition in human data.

Figure 4 - The authors do a nice experiment to show the localization of Limch1, and raise an antibody to detect the muscle specific isoform. The data seem to show that the muscle-specific isoform localizes to the sarcolemma, and this staining is largely lost in the mutant mice. By contrast, one could infer that the cytoplasmic signal in the WT comes from the ubiquitous isoform (which accounts for 30-40% of the Limch1 expression). This is consistent with the validation in Fig. 2. However, the authors in the text claim this experiment reveals an increased distribution throughout the myofiber, or a more even signal distribution in the cytoplasm, and that the uLimch1 cannot recapitulate mLimch1 localization. Fig. 2 suggests that total levels of Limch1 are increased (as noted by the authors in the discussion). Given that the muscle specific isoform localizes to the sarcolemma, and the ubiquitous isoform is presumably sarcoplasmic, it isn't clear to me that there is any change in localization per se. What the authors show is just that the signal at the sarcolemma is lost, and if one compares the intensity in the right-hand plots in Fig. 4B, they are comparable in the sarcoplasmic region. It seems likely there is more of the ubiquitous isoform, and what is seen here is just how that isoform localizes. The quantification the authors perform in D would likely show this strong difference in the localization of the muscle isoform. If the authors redo this quantification, exclude the signal at the sarcolemma and normalize to the average pixel intensity in the fiber, do they still see a difference? I am not convinced that the "clustering" of the signal of the ubiquitous, cytoplasmic isoform is in any way changed. Given the difference in the two proteins, I also would not expect that the ubiquitous isoform could compensate for loss of the muscle isoform, and would not expect it to "recapitulate" the muscle-isoform localization.

OPTIONAL: It would be interesting to examine how loss of the muscle-specific Limch1 isoform results in disrupted calcium handling. This is the mechanism that is not addressed in the paper, as the authors note in the discussion. If the authors perform TEM, can they see defects in t-tubules or organization of the sarcoplasmic reticulum, that are not visible by light microscopy? Experiments looking more closely at LIMCH1 co-localization with other proteins at the sarcolemma or the sufficiency of the muscle-specific region to localize would also be useful (for example, can the muscle-specific region localize GFP to the membrane in cells?). If the muscle-specific isoform is transfected or transduced into differentiated myotubes, how does this affect calcium dynamics in the culture system? As the authors note in the discussion, identification of mLimch1 versus uLimch1 interactors would be particularly interesting, and provide insight into how this protein can affect calcium handling without impacting structure.

Minor comments

• a. In the Figure 3 legend, the order of the descriptions for B-C and D-E is switched. The order of the panels matches the text, but the legend switches the description of the force-frequence curves (shown in B & C but labeled as D & E), with the description of the rate of relaxation and contraction plots (shown in D and E but labeled as B and C in the legend).
• b. The scale in Figure 4, panel B between the top and bottom plots is not the same, so it is difficult to compare, particularly for the panels on the right. See comment above.

Significance

This is a well-written study identifying the function of a muscle-specific isoform of LIMCH1, as well as implicating a switch in Limch1 isoform expression in DM1 models as a target of MBNL regulation. It presents multiple new tools to study mLimch1, and identifies a possible role for mLIMCH1 in calcium regulation, but stops short of identifying the mechanism by which this regulation occurs. The study is a definite advance in our understanding of developmentally-regulated splice isoform transitions that are disease relevant. The work would be of interest to scientists with specialized interests in muscle development and isoform-specific function in myogenesis, as well as more broadly of interest to clinical scientists for the possible connection to DM1.

I am an expert in RNA regulation and muscle development.

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Reply to the reviewers

Manuscript number: #RC-2022-01697

Corresponding author(s): William Roman; Edgar R. Gomes

[The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

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1. General Statements [optional]

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

We would like to thank the reviewers for their careful evaluation of our study. The goal of this work is to demonstrate that fiber type composition can be altered with exercise of in vitro muscle cultures. These findings provide an additional strategy to better mimic muscle in vitro for biological investigation and disease modelling. The reviewers’ comments will strengthen the conclusions of our study.

In this point-by-point answer, we also include a statement on the feasibility of each comment based on preliminary work we have performed since receiving the reviews. We expect experiments can be achieved within 2 – 3 months.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

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

The manuscript by Henning et al describes a method to induce myofiber subtype specification in vitro based on optogenetics and particle image velocimetry. The work is well performed and the manuscript is clear. The findings might be useful to the muscle community, but there are some issues which should be addressed in order to improve the quality and impact of the manuscript.

My main concern is that the whole work is performed in murine cells. Although I appreciate that the authors have used primary myoblasts rather than cell lines, I also think that the key advantage of such in vitro platforms is the possibility to "humanise" the experiments as much as possible. In this context, the key findings of this work should be reproduced using human myoblasts. This will significantly enhance the relevance of the work. *

Point 1.1) We thank the reviewer for his suggestion and have already performed some pilot experiments to “humanize” experiments. We infected hiPSC-derived myotubes (van der Wal et al., 2018) and human immortalized myotubes (Mamchaoui et al., 2011) with AAV9-pACAGW-ChR2-Venus-AAV. After infection, human immortalized myotubes did not express ChR2, not permitting optogenetic training on these cultures. For hiPSC-derived myotubes, the infection rate was very low and insufficient to perform a bulk analysis to evaluate the effect of long term intermittent light stimulation. Moreover, the contractile behavior of hiPSC-derived myotubes expressing ChR2 significantly differed from primary mouse myotubes. They underwent a single and slow contraction when compared to the cyclic contractions observed in mouse myotubes. This suggests that the maturation of the contractile apparatus of 2D hiPSC-derived myotubes is insufficient to perform consistent in vitro training studies.

As such, we agree with the reviewer that reproducing our key findings with human cells would improve the relevance of this work. However, due to the experimental limitations described above, significant improvements in human myotube maturation in vitro are required to perform such experiments. We will attempt to increase infection efficiency by using another AAV serotype in hiPSC-derived myotubes but this has a low probability of solving all the technical limitations. Our work is a proof of principal that fiber type composition can be influenced in vitro through contraction stimulation. We expect these findings to be the translated to human cultures when the field has discovered the necessary protocols to push human myotube maturation.

Feasibility: run additional tests but probability of success is low due to technical limitations.

*Other issues: *

1) From a methodological perspective, I think some clarifications are needed on the western blots shown in Fig 4K-L, as the pattern of Myh3 and Myh8 in both panels appear very similar. This could easily be ruled out by providing raw data/images. Please accept my apologies if this is simply caused by similar migration patterns in the gels (worth checking).

Point 1.2) The very similar appearance of both patterns is due to the same molecular weight (220 kDA) of distinct myh isoforms. After an initial staining of western blot membranes, primary and secondary antibodies were stripped off and the membrane was subsequently re-probed using a primary and secondary antibody. We incubated stripped membranes with secondary antibodies only and observed no signal, confirming the stripping was efficient. We have updated the representative images of the Western Blot membranes in Figure 4 and included the α-actinin loading controls on which the bands are normalized to account for sarcomerogenesis (Figure 4 K-M).

Feasibility: Accomplished

*2) Figure 3K-L (BTX): better imaging should be performed to assess morphology of NMJ (eg. pretzel-shaped as in mature/adult NMJ?) *

Point 1.3) We agree with the point raised by the reviewer. However, a morphological assessment of the NMJ is difficult in this in vitro system due to our inability to generate mature muscle end plates as seen in in vivo adult NMJs. We will nevertheless perform a more quantitative evaluation of BTX stainings imaged with high spatial resolution by measuring the size and shape of the AChR clusters. The technical pipeline to do this quantitative approach is already established.

Feasibility: will be accomplished

*3) Figure 3 N-P: Why did the authors used a relatively complex techniques such as smFISH to answer a question more simply addressable with more conventional (and perhaps less operator dependent) techniques such quantitative PCR?

*

Point 1.4) We agree with the reviewer that the more conventional qPCR technique would highlight similar results to the smFISH quantifications. Due to the heterogeneity of our primary myotube cultures (presence of non-muscle cell types and varying degrees of muscle cell maturation), we opted to monitor AChR expression by conserving a spatial dimension. This allows us to observe ChrnE and ChrnG expression in mature muscle cells selected to perform the contraction analysis. Nevertheless, performing a bulk RNA expression analysis would be informative to show a significant increase in AChR expression across the culture. This point will be fully addressed by qPCR assays of ChrnE and ChrnG.

Feasibility: will be accomplished

*Reviewer #1 (Significance (Required)):

Nature and significance: as mentioned in the previous section, the work can be very significant if expanded to human myoblasts/myotubes, which can have different slow/fast myosin expression pattern. The work is clearly methodological/descriptive, so showing an application of this technique using diseased/mutant cells may increase its relevance even more (but I do not believe it is a key barrier to publication). *

We thank the reviewer for his comments as the “other issues” raised will significantly improve the manuscript and will all be tackled. With regards to using human myotubes, we will attempt a few more strategies to translate our findings to human cultures, but our preliminary data suggests that many technical barriers need to be overcome to perform such experiments. Nevertheless, it is our opinion that the main contribution of this manuscript is to show that fiber switching can be achieved in vitro and that this will be routinely used in the next generation of human in vitro muscle systems.

*

*

*Comparison with other methods: Similar methods have been published but not with this level of resolution.

Expertise: muscle disease and regeneration, in vitro and in vivo models.*

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

* The work presented shows that muscle stem cells isolated from 5-day-old mice can be transduced with a DNA coding for a Channelrhodopsin2-Venus which will allow the muscle cell to be excited by a light beam (475nm) and to induce the contraction of myotubes. The authors measure the speed of contraction, relaxation and fatigability of such cells as a function of a more or less long excitation time. In particular, they show that myotubes in culture, excited at a frequency of 5 Hz, 8 hours per day for 7 days are larger than unstimulated myotubes and are more resistant to fatigue. Surprisingly, they show that myotubes stimulated at the low frequency of 5Hz express the neonatal Myosin heavy chain more than the slow Myh whose expression is known in adult muscle to be specifically strong in muscle fibers stimulated at low frequency. As the authors do not apply a high stimulation frequency (100Hz) to their culture, it is difficult to conclude whether the stimulation frequency applied in the study induces a specific phenotypic specialization of the myofiber, or a more general role. In this respect, the size of the myotubes obtained after training seems to be increased, showing a hypertrophic effect on the cultured myotubes. This study does not allow us to conclude, beyond the expression of the Myh8 gene, on the “gain” of the fast-twitch specialization of the myofiber by repeated stimulation over several days. A complementary study would certainly provide elements to better understand the role of muscle fiber stimulation, apart from the trophic contribution provided in vivo by the motoneuron. If the study is well conducted, some points are nevertheless important to address before publication.*

*Reviewer #2 (Significance (Required)): *

* - Figures 4F/G are difficult to understand: the Myh7 signal seems much higher in trained myonuclei (F), but the histogram shows the opposite (G).*

__Point 2.1) __We apologize for the confusion. The apparent higher Myh7 signal in trained cells in Figure 4F is due to background noise in the image. When mRNA is expressed, the smFISH probes are visible as small round dots. For clarity, we updated the representative images for the smFISH probes and highlighted the smFISH dots with arrows. We also adapted the y-axis of each graph to better represent the analysis of mRNA counts per myonuclei.

Feasibility: Accomplished

*- Figures 4L, the western blot shows the same increase in Myh3 and Myh8 at day 4, while the graph shows an increase at d4 only in Myh8, why? *

Point 2.2) We have chosen another western blot to better reflect the quantification. It is important to note that we have normalized the band intensity to a-actinin instead of a house keeping gene to account for changes in sarcomerogenesis over the lifetime of the cultures. As such, although we observe an increase in Myh3 intensity, it is counter balanced by an increase in a-actinin expression. We have now added the a-actinin bands.

- For immunocytochemistry against fMyh (Fig4 H, I) as well as for Western blots (Fig 4M, N), the authors have to provide arguments regarding the specificity of the antibodies used: some fMyh-specific antibodies recognize, Myh 3, 8, 1, 2, and 4, some only Myh 8, 1, 2, and 4, so it is quite difficult to conclude on the experiments using sc-32732 antibodies, (clone F59) which Myh are actually recognized in Western blot or immunocytochemistry.

Point 2.3) According to the manufacturer, the sc-32732 antibody is specific for fast Myh (Myh1, 2, 4 and 6). Nevertheless, we will ensure the specificity of the sc-32732 antibody against fast Myosins by staining neonatal and adult TA/EDL muscle sections with anti-Myh3 (embryonic), anti-Myh8 (neonatal) and anti-fMyh antibodies.

Feasibility: will be accomplished

While 10Hz stimulation is known in vivo to increase the slow program, and Myh7 expression in adult muscles, the authors show that ex vivo this is not the case with primary myotubes, with Myh7 protein level not being upregulated in the 7 day stimulation paradigm, while on the contrary Myh8 expression is upregulated. I think it would be important to quantify the mRNA of each of the Myh genes to be sure that there is no problem with the antibodies, which could recognize several Myh proteins, in the absence of a resolving acrylamide gel allowing visualization and relative level of each isoform according to its migration. Nevertheless, this is an interesting observation that could be related to the early phases of muscle contraction in vivo. Indeed, it has been shown in rats that early postnatal development animals are essentially sedentary and whose muscles (Sol and EDL) are stimulated by short intermittent bursts similar to 10Hz (doi: 10.1111/j.0953-816X.2004.03418.x) during the first 2-3 weeks of life. This should be compatible with Myh8 expression. It would be relevant in this idea to verify that the paradigm presented leads to myotubes with a "neonatal" phenotype. Quantification of the expression level of *genes specifically expressed during the neonatal period, compared with those expressed in adult slow or fast myofibers, would enhance the conclusions drawn by the authors. *

Point 2.4) The reviewer raises an important technical limitation of observing Myh proteins to identify fiber types due to the cross-reactivity of antibodies. Despite our best efforts to select the appropriate antibodies, we agree that investigating mRNA expression of individual Myh isoforms would strengthen the conclusion of our study. We will design specific primers and perform qPCR for distinct Myh isoforms on untrained and trained cultures.

With regards to the “neonatal” phenotype of these in vitro cultures, this does indeed seem to be the case as the cultures transition from embryonic and neonatal myosins to adult myosins during the lifetime of the cultures.

Feasibility: will be accomplished

*Should we also be cautious about bulk analysis since, as shown in Figure S1, not all myotubes express ChR2? *

Point 2.5) Although 10% of myotubes do not express ChR2, we believe that 90% of infected myotubes is sufficient for bulk analysis. We nevertheless combine in our study bulk analysis with single cell assays such as smFISH and immunofluorescence, which are in line with the bulk analyses.

Feasibility: Accomplished

May the authors correlate the ex vivo neonatal phenotype observed with the neonatal muscles they used to prepare myogenic stem cells?

Point 2.6) We understand from this that the reviewer would like us to check the expression of distinct Myh isoforms in our in vitro system and compare it to neonatal muscle. We will perform Myh staining of muscle sections from 6-day old mouse pups (time of myogenic stem cell isolation) and compare the expression of Myosin heavy chains with what we observe in our in vitro cultures.

Feasibility: will be accomplished

Overall, we will address all the points of the reviewer. Those ensuring the specificity of antibodies used are particularly relevant. With regards to the comparison between our in vitro cultures with neonatal muscle, we believe this will help contextualize our findings with the literature.

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

*Summary: *

*In this work, the authors propose an in vitro model describing a strategy to alter fiber type composition of myotubes with a long-term, intermittent mechanical training. The authors present a model of myotubes transfected with an adenovirus, which makes them photosensitive; in this way, fibers contraction can be induced upon stimulation with blue LEDs. *

*Even though ChR2 expressing myotubes have previously been used by other groups (Asano T, Ishizua T, Yawo H. Optically controlled contraction of photosensitive skeletal muscle cells. Biotechnol Bioeng. 2012 Jan;109(1):199-204), no one has ever used it in the way proposed by the authors. For this reason, this work opens new perspectives on the possible use for clinical and therapeutic purposes for this in vitro muscle system. *

*Major comments: *

*I believe that the authors have presented their results, conclusion and methods in a fair and clear way, so that the experiment could also be reproduced. *

*However, I think there are some adjustments that could be done in order to improve and strengthen the quality of this work: *

*- The authors have analysed the expression of different myosin heavy chain isoforms, both regarding the slow and fast twitch fibers. Though, I think it would be interesting to investigate also the expression of Myh4, which is mainly expressed in type IIB fast twitch fibers; *

Point 3.1) We agree with the reviewer’s comment. We will add the analysis for Myh 4 (western blots and qPCR) to our manuscript.

Feasibility: will be accomplished

The authors have observed a switch in the fiber type upon prolonged intermittent stimulation with blue LEDs, which translates into a higher number of type II fibers. It is known that exercise helps rescuing the loss of type II fibers, which is typical of age-related physiological processes, such as sarcopenia (Brunner F, Schmid A, Sheikhzadeh A, Nordin M, Yoon J, Frankel V. Effects of aging on Type II muscle fibers: a systematic review of the literature. J Aging Phys Act. 2007 Jul;15(3):336-48). However, I believe that providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic) could be helpful in order to have a clearer view on the specific subset of fibers that are generated with the given experimental conditions;

Point 3.2) We agree with the reviewer's suggestion that an additional metabolic analysis would strengthen our observation. We propose to perform lactate measurements in cell lysate and supernatant to monitor a switch from oxidative to glycolytic metabolism. Specific inhibitors of the glycolytic pathway (2-DG, UK5099, Rotenone and AntimycinA) will be used as a control to prevent trained cells to shift towards a fast fiber type.

Alternatively, we will assess the protein expression levels of key metabolic proteins involved in oxidative phosphorylation and in pyruvate and lactate production (e.g. OxPhos, …). All these techniques are routinely performed in an adjacent laboratory and we foresee no technical limitations.

Feasibility: will be accomplished

*Minor comments: *

*The text and the figures are clear and well written, and help to explain better the experimental setup and procedures. Still, I would suggest some minor adjustments: *

- I would suggest providing more information on the pH used for the experiments, since it plays a pivotal role in regulating myosin ATPase activity and, thus, muscular contractility. This would improve the replicability of your experiment.

We thank the reviewer for this comment. We will provide information regarding the pH and add it in the method and materials section.

Feasibility: will be accomplished

The caption of Figure 1 is missing a description of panel E, even if it has been addressed in the text.

Point 3.3.) We apologize for this mistake. We added the missing description of Fig. 1E.

Feasibility: Accomplished

*Reviewer #3 (Significance (Required)): *

*This model opens new perspectives on in vitro muscle systems for the study of pathologies. The authors have been able to assess that myofibers contraction is able to induce a shift towards type II fibers, reproducing in vitro what is also known in vivo. For this reason, I believe that this model could be useful for further clinical approaches. It is important, though, to keep in mind that muscular disorders are not all characterized by a loss of type II fibers; for instance, myotonic dystrophies type I and type 2 exhibit similar phenotypes, even if different types of muscle fibers are affected. *

*For this reason, it would be interesting to investigate the versatility of this model in terms of giving rise to different fiber types. *

Point 3.4.) We added a sentence in the introduction that highlights an example of muscle disorders in which slow muscle fibers are predominately affected. Concerning the versatility of the model, we will add a paragraph to the discussion elaborating on how different stimulus frequency and durations could influence the specialization of fiber types.

Feasibility: Accomplished

Overall, we will address all major and minor comments from the reviewer. We have identified the experiments required for the metabolic analysis and agree that it will bolster our findings.

Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

We have already carried out the following changes in the manuscript, which were proposed by the reviewers:

Point 1.2: pattern of Myh3 and Myh8 in both panels appear very similar - We updated the representative images of Myh 3 and Myh8 in Figure 4 K-N __and included the loading controls Myh 8 and fMyh images in __Figure 4K-N __and to __supplementary Figure 4 A, B.

Point 2.1: Figures 4F/G: representative images of Myh7 smFISH probe and the graph showing opposite trends – We have updated the representative images of Figure 4F and we have changed the x-axis of the graph in Figure 4E and G.

__Point 2.5: __caution around bulk analysis – we consider that based on the high percentage of contracting cells in response to blue light (~90%), this concern is not warranted.

Point 3.3: caption of Figure 1 is missing a description of panel E – We have added the missing description to the manuscript (Figure 1E).

Point 3.4: muscular disorders are not all characterized by a loss of type II fibers – we have added an example of a muscle disorder, in which slow fibers are predominantly affected, to the introduction (line 42-44) of the manuscript.

investigate the versatility of this model in terms of giving rise to different fiber types – we added a paragraph to the discussion elaborating on how different stimulus frequency can lead to different fiber types (line 264-275).

3. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

Point 1.1: Reproducing our key findings with human cells – we ran pilot experiments on immortalized human cell lines and human iPSC-derived myotubes but were not able to mature these cells sufficiently nor infect them to allow long-term in vitro training. Increased maturation of myotubes derived from hiPSCs is an endeavor currently undertaken by many laboratories. Although we will attempt a few more trials, we believe the technical limitations are too important to address this point.

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Referee #3

Evidence, reproducibility and clarity

Summary:

In this work, the authors propose an in vitro model describing a strategy to alter fiber type composition of myotubes with a long-term, intermittent mechanical training. The authors present a model of myotubes transfected with an adenovirus, which makes them photosensitive; in this way, fibers contraction can be induced upon stimulation with blue LEDs. Even though ChR2 expressing myotubes have previously been used by other groups (Asano T, Ishizua T, Yawo H. Optically controlled contraction of photosensitive skeletal muscle cells. Biotechnol Bioeng. 2012 Jan;109(1):199-204), no one has ever used it in the way proposed by the authors. For this reason, this work opens new perspectives on the possible use for clinical and therapeutic purposes for this in vitro muscle system.

Major comments:

I believe that the authors have presented their results, conclusion and methods in a fair and clear way, so that the experiment could also be reproduced.

However, I think there are some adjustments that could be done in order to improve and strengthen the quality of this work: - The authors have analysed the expression of different myosin heavy chain isoforms, both regarding the slow and fast twitch fibers. Though, I think it would be interesting to investigate also the expression of Myh4, which is mainly expressed in type IIB fast twitch fibers; - The authors have observed a switch in the fiber type upon prolonged intermittent stimulation with blue LEDs, which translates into a higher number of type II fibers. It is known that exercise helps rescuing the loss of type II fibers, which is typical of age-related physiological processes, such as sarcopenia (Brunner F, Schmid A, Sheikhzadeh A, Nordin M, Yoon J, Frankel V. Effects of aging on Type II muscle fibers: a systematic review of the literature. J Aging Phys Act. 2007 Jul;15(3):336-48). However, I believe that providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic) could be helpful in order to have a clearer view on the specific subset of fibers that are generated with the given experimental conditions;

Minor comments:

The text and the figures are clear and well written, and help to explain better the experimental setup and procedures. Still, I would suggest some minor adjustments:<br /> - I would suggest providing more information on the pH used for the experiments, since it plays a pivotal role in regulating myosin ATPase activity and, thus, muscular contractility. This would improve the replicability of your experiment; - The caption of Figure 1 is missing a description of panel E, even if it has been addressed in the text.

Significance

This model opens new perspectives on in vitro muscle systems for the study of pathologies. The authors have been able to assess that myofibers contraction is able to induce a shift towards type II fibers, reproducing in vitro what is also known in vivo. For this reason, I believe that this model could be useful for further clinical approaches. It is important, though, to keep in mind that muscular disorders are not all characterised by a loss of type II fibers; for instance, myotonic dystrophies type I and type 2 exhibit similar phenotypes, even if different types of muscle fibers are affected.

For this reason, it would be interesting to investigate the versatility of this model in terms of giving rise to different fiber types.

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 #2

Evidence, reproducibility and clarity

The work presented shows that muscle stem cells isolated from 5-day-old mice can be transduced with a DNA coding for a Channelrhodopsin2-Venus which will allow the muscle cell to be excited by a light beam (475nm) and to induce the contraction of myotubes. The authors measure the speed of contraction, relaxation and fatigability of such cells as a function of a more or less long excitation time. In particular, they show that myotubes in culture, excited at a frequency of 5 Hz, 8 hours per day for 7 days are larger than unstimulated myotubes and are more resistant to fatigue. Surprisingly, they show that myotubes stimulated at the low frequency of 5Hz express the neonatal Myosin heavy chain more than the slow Myh whose expression is known in adult muscle to be specifically strong in muscle fibers stimulated at low frequency. As the authors do not apply a high stimulation frequency (100Hz) to their culture, it is difficult to conclude whether the stimulation frequency applied in the study induces a specific phenotypic specialization of the myofiber, or a more general role. In this respect, the size of the myotubes obtained after training seems to be increased, showing a hypertrophic effect on the cultured myotubes. This study does not allow us to conclude, beyond the expression of the Myh8 gene, on the "gain" of the fast-twitch specialization of the myofiber by repeated stimulation over several days. A complementary study would certainly provide elements to better understand the role of muscle fiber stimulation, apart from the trophic contribution provided in vivo by the motoneuron.

If the study is well conducted, some points are nevertheless important to address before publication.

Significance

• Figures 4F/G are difficult to understand: the Myh7 signal seems much higher in trained myonuclei (F), but the histogram shows the opposite (G).
• Figures 4L, the western blot shows the same increase in Myh3 and Myh8 at day 4, while the graph shows an increase at d4 only in Myh8, why?
• For immunocytochemistry against fMyh (Fig4 H, I) as well as for Western blots (Fig 4M, N), the authors have to provide arguments regarding the specificity of the antibodies used: some fMyh-specific antibodies recognize, Myh 3, 8, 1, 2, and 4, some only Myh 8, 1, 2, and 4, so it is quite difficult to conclude on the experiments using sc-32732 antibodies, (clone F59) which Myh are actually recognized in Western blot or immunocytochemistry.
• While 10Hz stimulation is known in vivo to increase the slow program, and Myh7 expression in adult muscles, the authors show that ex vivo this is not the case with primary myotubes, with Myh7 protein level not being upregulated in the 7 day stimulation paradigm, while on the contrary Myh8 expression is upregulated. I think it would be important to quantify the mRNA of each of the Myh genes to be sure that there is no problem with the antibodies, which could recognize several Myh proteins, in the absence of a resolving acrylamide gel allowing visualization and relative level of each isoform according to its migration. Nevertheless, this is an interesting observation that could be related to the early phases of muscle contraction in vivo. Indeed, it has been shown in rats that early postnatal development animals are essentially sedentary and whose muscles (Sol and EDL) are stimulated by short intermittent bursts similar to 10Hz (doi: 10.1111/j.0953-816X.2004.03418.x) during the first 2-3 weeks of life. This should be compatible with Myh8 expression. It would be relevant in this idea to verify that the paradigm presented leads to myotubes with a "neonatal" phenotype. Quantification of the expression level of genes specifically expressed during the neonatal period, compared with those expressed in adult slow or fast myofibers, would enhance the conclusions drawn by the authors.
• Should we also be cautious about bulk analysis since, as shown in Figure S1, not all myotubes express ChR2?
• May the authors correlate the ex vivo neonatal phenotype observed with the neonatal muscles they used to prepare myogenic stem cells?

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The manuscript by Henning et al describes a method to induce myofiber subtype specification in vitro based on optogenetics and particle image velocimetry. The work is well performed and the manuscript is clear. The findings might be useful to the muscle community, but there are some issues which should be addressed in order to improve the quality and impact of the manuscript.

My main concern is that the whole work is performed in murine cells. Although I appreciate that the authors have used primary myoblasts rather than cell lines, I also think that the key advantage of such in vitro platforms is the possibility to "humanise" the experiments as much as possible. In this context, the key findings of this work should be reproduced using human myoblasts. This will significantly enhance the relevance of the work.

Other issues:

1. From a methodological perspective, I think some clarifications are needed on the western blots shown in Fig 4K-L, as the pattern of Myh3 and Myh8 in both panels appear very similar. This could easily be ruled out by providing raw data/images. Please accept my apologies if this is simply caused by similar migration patterns in the gels (worth checking).
2. Figure 3K-L (BTX): better imaging should be performed to assess morphology of NMJ (eg. pretzel-shaped as in mature/adult NMJ?)
3. Figure 3 N-P: Why did the authors used a relatively complex techniques such as snFISH to answer a question more simply addressable with more conventional (and perhaps less operator dependent) techniques such quantitative PCR?

Significance

Nature and significance: as mentioned in the previous section, the work can be very significant if expanded to human myoblasts/myotubes, which can have different slow/fast myosin expression pattern. The work is clearly methodological/descriptive, so showing an application of this technique using diseased/mutant cells may increase its relevance even more (but I do not believe it is a key barrier to publication).

Comparison with other methods: Similar methods have been published but not with this level of resolution.

Expertise: muscle disease and regeneration, in vitro and in vivo models.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

2. Point-by-point description of the revision

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

*The paper titled "Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1" by the Jagla group studied the effect of down-regulation of miR-1 in myotonic dystrophy type 1 (DM1) using fly as the disease model. The study is based on previous findings that in DM1 MBNL1 is sequestered, CELF1 is stabilized, and miR-1 is down regulated. The authors further identified Multiplexin to be the target effector of miR-1 in the fly heart and studied its function with a series of gain- and loss-of-function and rescue experiments. The authors' findings represent a significant advance in understanding the genetic mechanisms that can explain the pathogenic causes of dilated cardiomyopathy associated with DM1. Overall, this paper is well written and organized, with well-designed experiments and a clear model. A few additional experiments are suggested to further strengthen the conclusion. *

Answer: We are grateful to the Reviewer for appreciating quality and significance of our work.

2. Wild-type and Hand-Gal4 controls are missing for all experiments (only UAS lines outcrossed to w1118 are displayed). Also, Hand-Gal4 driver lines can cause mild dilation by itself, which would influence interpretation and statistics of data. * Answer: We agree and include the Hand-Gal4/+ control condition to all main and supplemental figures showing heart parameters. The differences in data statistics using Hand-Gal4/+ compared to UAS/+ control lines reinforce our data interpretation.

They are listed below:

• increase in diastolic diameter and reduction of fractional shortening become statistically significant in Hand>miR-1sponge hearts at 5 weeks (Fig. 1D, F);
• reductions of fractional shortening become significant in Hand>Bru3 (Fig. 2C) and Hand>mblRNAi (Fig. 2F) contexts at 1 week;
• increase in diastolic diameter of Hand>mblRNAi hearts at 5 weaks becomes statistically significant (Fig. 2D);
• increase in diastolic diameter of Hand>Mp hearts at 5 weeks (Fig. 4E) becomes statistically significant ;
• reduction of fractional shortening becomes statistically significant in Hand>Mp context at 1 week (Fig. 4G);
• increase in diastolic diameter in Hand>960CTG context at 5 weeks becomes statistically significant (Fig. S2A). *Also, the authors should consider to confirm the miR-1 phenotype obtained with the sponge with a miR mutant, and also combine miR-1 het with miR sponge (worsening of phenotype?). Alternatively, knockdown efficiency of miR should be tested by qPCR or HCR/smFISH. *

Answer: We are grateful for these comments. Below we refer to published data and to performed additional experiments that are in support of miR-1 sponge phenotypes:

• UAS-miR-1 sponge line we used was generated and tested by Fulga et al., (Nat Comm, 2015). Fulga and colleagues apply UAS-miR1sponge line to attenuate miR-1 function in muscles and obtain miR-KO-like muscle phenotypes.
• Here, we identify Mp as a new direct miR-1 target. To test whether miR-1 sponge attenuates miR-1 function we analyzed Mp protein levels in the hearts from wt and Hand>miR-1sponge flies. Mp expression is highly increased in Hand>miR-1sponge context indicating attenuation of miR-1 by the sponge transgene. These data are presented in new Fig. S5J;

• We tested whether heterozygous dmiR-1 KO -/+ flies (homozygous dmiR-1 mutants are lethal) develop Hand>miR-1sponge-like heart phenotype. Indeed, at 5 weeks of age dmiR-1 KO -/+ flies show significantly increased diastolic and systolic heart diameters. Thus, in old flies loss of one copy of miR-1 mimics heart dilation observed in Hand>dmiR-1sponge context. Heart contractility remains unaffected in dmiR-1 KO -/+ flies, suggesting that loss of one copy of miR-1 has a weaker impact on heart function than heart-targeted miR-1sponge. These data are shown in a new supplemental figure (Fig. S4A-C).

• It is surprising that one of the DM1 fly models, overexpression of 960 CTG repeats, did not show DCM, considering it is the primary cause of DM1 in humans due to excessive CTG repeats. It should be discussed why Hand>960 CTG does not lead to DCM, since the authors claim that this model with high number of CTG repeats shows a strong phenotype. Are Hand>bru3 and Hand>mbl stronger? *

Answer: We thank Reviewer for pointing this out.

Heart and muscle-specific DM1 models we established and tested (Hand> or Mef>960CTG, Hand> or Mef>mblRNAi and Hand> or Mef>Bru3) all develop the majority of DM1 phenotypes (Picchio et al., 2013 ; Picchio et al., 2018 ; Auxerre-Planté et al., 2019). However, some cardiac DM1 phenotypes such as conduction defects (Auxerre-Plantié et al., 2019) and described here DCM are only observed in Hand>Bru3 and Hand>mblRNAi contexts. We previously observed that the down-regulation of sarcomeric genes is more important in Mef>Bru3 than in Mef>960CTG context (Picchio et al., 2018). This could result from a milder effect of 960CTG repeats on Bru3 and Mbl levels when compared with Gal4-driven overexpression of Bru3 and RNAi-knockdown of mbl. We add a comment to Results section (page 5) to discuss this point: “The Hand>960CTG line shows cardiac dilation at 5 weeks of age characterized by significant increase in diastolic and systolic diameters but with normal cardiac contractility (Fig. S2A,B,C). We hypothesise that non-affected contractility in this DM1 line is due to a milder effect of 960CTG repeats on Bru3 and Mbl levels compared to GAL4-driven overexpression of Bru3 and RNAi-knockdown of mbl.”

*Is miR-1 (and Mp) unaltered in these flies with 960 CTG repeats? *

Answer: In Hand>960CTG context a reduced level of miR-1 and an increase in Mp are also observed (not shown). Hand>960CTG flies do not develop DCM but at 5 weeks of age show a significant increase in diastolic and systolic heart diameters. One possibility we favor is that deregulation of miR-1 and Mp in Hand>960CTG is under the level that induces DCM. By analogy, only DM1 patients with a high increase in Col15A1 develop DCM (Fig. 5).

It would be interesting to overexpress 960 CTG in a miR-1 or mbl heterozygous mutant background, which may produce DCM.

Answer: To test additional genetic context in which miR-1 is reduced we used miR-1 heterozygous KO flies. We found that in 5 weeks-old flies loss of one copy of miR-1 leads, like in Hand>miR-1sponge flies, to heart dilation (Fig. S4A-C), however mir-1KO +/- flies do not show affected contractility.

We agree that combining Hand>960CTG with mir-1 heterozygous mutants would potentially result in an additional DCM producing context. As we are focusing on our DCM developing DM1 models (Hand>Bru3 and Hand>mblRNAi) and on conserved deregulation of miR-1 and its target Mp/Col15A1, we didn’t follow this suggestion.

• In Figure 2H, the mean intensity is displayed as the readout of the smFISH quantification of miR-1 levels. If understood correctly, this is the wrong readout since smFISH detects single molecule fluorescence of transcripts, so the number of transcripts should be quantified. *

Answer: The miR-1 quantification was done using FISH with miR-1-specific LNA probe (Qiagen miRCURY system). This is highly sensitive ISH but the resolution is not at a single molecule level. The Imaris-generated spots in Fig. 2G and 2G’ represent (for each of them) several miR-1 molecules. The mean intensity of the fluorescent signal for a given spot is proportional to the number of miR-1 molecules and the average of the mean intensities of all spots illustrates the level of miR-1 expression (Fig. 2H). We remove “sm” abbreviations from the text and Figure legend and provide a more detailed description of miR-1 quantification in the Method section.

Furthermore, Hand-Gal4 is not expressed in the ventral longitudinal muscles (VLM). As a proof of principle, miR-1 levels should be quantified in VLM (no change in transcripts levels expected).

Answer: When cross with UAS-GFP the Hand-Gal4 expression could be detected in VLMs even if VLM associated GFP signal is much lower than in cardioblasts (CB) and pericardial cells (PC). Representative views of Hand>GFP hearts labeled with anti-GFP are shown below.

• Fig. 5: Since DCM- DM1 patients still show elevated COL15A1 levels but no DCM, it would be interesting to know if DCM phenotypes are COL15A1-dosage dependent. This could be easily tested in the fly model by testing UAS-Mp overexpression at different temperatures. *

Answer: Heart parameters are to some extent temperature-sensitive (our observations) thus in our view increasing targeted Mp expression by elevating temperature is not appropriate for heart physiology experiments.

Presented in the manuscript data on Mp overexpression at 25°C already provide some indication for Mp dose-dependent effect in the fly model. We observe that DCM is induced at both 1 and 5 weeks of age, but the cardiac tube dilation is less important in 1 than in 5 weeks-old flies (Fig. 4). Also, when analysing Hand>960CTG DM1 model we observed that young flies with a low Mp levels do not show cardiac dilation while aged Hand>960CTG flies display an increase in diastolic and systolic heart diameters concomitant with a higher Mp.

• The authors elegantly show rescue of Hand>Bru3 flies by Mp RNAi. Their model would be further strengthened if a similar rescue can be shown with Hand>mblRNAi. *

Answer: So far we were unsuccessful in generation of recombined mblRNAi ;MpRNAi line most probably because of incompatibility in chromosomal transgene locations. Thus, we were unable to perform this experiment.

Because gene deregulations and DCM phenotypes we describe are highly similar in Hand>Bru3 and Hand>mblRNAi context we believe that rescue experiment we provide is representative for both DM1-associated DCM contexts.

Minor points:

*Fig. 1A,B: Ventral longitudinal muscles are covering the hearts on these images, so it's difficult to see the heart dimensions. This holds true for images throughout the manuscript. Where were the diameters measured (by the valves)? A better description and illustration would help the reader understand the situation. *

Answer: In the lateral heart views as in Fig. 1A and B it is indeed difficult to appreciate heart dimensions. For this reason we always show transversal sections derived from 3D reconstruction (as in Fig. 1A’ and B’). In this context differences in the internal heart diameters could be appreciated (white lines). All diastolic and systolic heart diameter measures presented in the graphs are extracted from the SOHA registrations (see Methods).

*Fig. 1 A',B': White line does not reflect the location where SOHA data are measured and should be horizontal for consistency. Where is ventral vs. dorsal? *

Answer: We agree. We indicate where is ventral and dorsa. For consistency we remove white lines from panels 1A and 1B and maintain orientations of white lines in panels 1A’ and 1B’.

Fig. 1D-F: Annotate 1 and 5 weeks in Figure, please. Also, why were 1 and 5 weeks tested? Is there an age-component in DM1 phenotype severity?

Answer: We add 1 and 5 weeks indications to the figures and discuss in the text (Results section page 4) that 1 and 5 weeks analyses were applied because the severity of cardiac phenotypes increases with age.

*Fig. 3A: Transcriptional analysis was done at which stage of development? *

Answer: It was done at 5 weeks of age. We add information to the figure legend.

*Fig. 3: It is not clear, in which set the authors looked for miR-1 bindings sites (144 genes or the whole set)? Not well annotated. What is meant by 'heart-targeted'? *

Answer: In silico search was performed on the whole set of genes. We provide more precisions on in silico screen in Method section.

*Fig. 4C,D: It looks like they are not shown in the same dorsal-ventral orientation. Also, it looks Mp is overexpressed in the VLM, but Hand-Gal4 only drives in the cardiomyocytes and pericardial cells? How was quantification done? *

Answer: We are thanking for pointing this out. We revised heart orientations in panel 4C and 4D. As previously mentioned Hand-Gal4 is also expressed in the VLM. We present a more representative view in 4D with a lower Mp signal in VLMs. Quantification of Mp expression is not presented here but performed like in Fig. 3G.

*Fig. 4I: Why are some myofibers indicated in red in the model? *

Answer: In red are indicated additional actin filaments that form in the case of heart dilation. As we do not discuss this aspect we modify drawing in the model.

Fig. 5 D-E: Genotypes need to be better indicated in the graphs.

Answer: We provide now more complete genotypes.

*Did the authors control for multiple UAS sites? Is UPRT a UAS control? *

Answer: Yes, UPRT is the UAS line.

*In the first paragraph of Result 3, the last sentence seems unfinished. "We identified a set of candidate genes, of which Multiplexin (Mp)" *

Answer: We revise this sentence.

*In Method, the in silico screening for miR-1 target should be explained in more detail. *

Answer: We provide a more detailed in silico screening protocol in Method section.

*Reviewer #1 (Significance (Required)): *

* The presented data is a significant advance our knowledge of our understanding of the molecular mechanisms involved in DM1. I expect that scientists in the muscular disease field and beyond will find this work of high interest. *

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

*Summary: This well-written manuscript utilizes the Drosophila model system to demonstrate that reduction of micro-RNA miR-1 and the resulting increase in one of its down-regulated proteins (Multiplexin) contribute to a dilated cardiomyopathy (DCM) phenotype. This is of interest in that this particular micro-RNA is downregulated in myotonic dystrophy type 1 (DM1), and this correlates with the DCM phenotype observed in patients. Further, the authors show that the human ortholog of Multiplexin is enriched in human DM1-DCM hearts and that downregulation of this protein in Drosophila DM1 models improves the DCM phenotype. Hence, the work demonstrates a potential mechanism for disease development and its amelioration. *

Answer: We are grateful to the Reviewer for appreciating our work and pointing out potential impact of our findings.

Major comments:

2. As pericardial cells are probed and mentioned substantially in this paper, the authors should explain what these cells do in flies. While affiliated with the heart, they are not myocytes and are probably not particularly relevant to the human heart. In this regard, it is possible that the phenotypes observed in the heart are partially or completely the result of Hand-driven expression of transgenes in the pericardial cells. Although unlikely, this issue should be mentioned as well. * Answer: Hand-Gal4 driver is the most commonly used Drosophila cardiac driver. Regarding influence of Hand-Gal4 driven expression in pericardial cells on the heart phenotypes, we previously tested all our DM1 models using cardioblast-specific Tin-GAL4 driver. All cardiac phenotypes including DCM are also observed when using Tin-Gal4 driver (as shown for conduction defects in Fig.5B Auxerre-Plantié et al., Elife 2019) indicating that the phenotypes are mainly due to gene deregulations within the cardioblasts.

*Does human miR-1 target Col15A1 transcripts based upon in silico analysis? This issue should be mentioned and discussed. *

Answer: In silico analysis (new supplemental Fig.S4G) reveals that Col15A1 transcripts carry a perfect miR-1 seed site in 3’UTR region.

Minor comments:

2. The abstract should explicitly state that Multiplexin is a form of collagen.* Answer: We mention this in the abstract.

*More information on the identity between the Drosophila and human forms of miR-1 would be helpful to establish that they are conserved. What is the percent identity and are the sequences that target mRNAs homologous? *

Answer: Mature Drosophila and human miR-1 are highly homologous. We provide their sequences in new supplemental Fig. S4F.

• In Figure 1C, it appears that there is an increased heartbeat frequency and arrhythmicity. Are these mutant phenotypes as well? *

Answer: We check it again and do not observe any significant change in heart period or in arrhythmia index in Hand>miR1 sponge context in both young and old flies. We show a new more representative view of M-modes in panel 1C.

• Incomplete sentence (page 5): We identified a set of candidate genes, of which Multiplexin (Mp) *

Answer: This sentence was revised.

*What is the basis for studying Multiplexin function as opposed to other candidates that were identified? It would be useful to mention this in the Results, although it is mentioned in the Discussion ("We top-ranked Mp because of its known role in setting the size of the cardiac lumen"). *

Answer: We add following sentences to Results section to clarify this point earlier in the manuscript.

“Mp overexpression in the developing embryonic heart leads to an enlargement of heart lumen and is sufficient to promote an increase of the embryonic aorta diameter to that of the heart proper (Harpaz et al., 2013). We thus reasoned that Mp could be involved in DM1-associated DCM.”

• "Mp was detected on the luminal and external surfaces of the cardiomyocytes ensuring cardiac contractions" Why does this ensure cardiac contractions? *

Answer: We are grateful for pointing this out. Mp is not ensuring but could influence cardiac contractions. We revise this sentence by deleting its second part “ensuring cardiac contractions”.

• Need to state in the text that the increased level of Col15A1 transcript expression in DM1 patients was not statistically significant. *

Answer: We state this in the text.

• Need a magnification bar for Figures 5F-H. *

Answer: Scale bar is added.

*Please speculate as to why the third DM1 model does not recapitulate the cardiac phenotypes. *

Answer: Heart and muscle-specific DM1 models we established and tested (Hand> or Mef>960CTG, Hand> or Mef>mblRNAi and Hand> or Mef>Bru3) all develop the majority of DM1 phenotypes (Picchio et al., 2013 ; Picchio et al., 2018 ; Auxerre-Planté et al., 2019). However, some cardiac DM1 phenotypes such as conduction defects (Auxerre-Plantié et al., 2019) and described here DCM are only observed in Hand>Bru3 and Hand>mblRNAi contexts. We previously observed that downregulation of sarcomeric genes is higher in Mef>Bru3 than in Mef>960CTG contexts (Picchio et al., 2018). This could result from a milder effect of 960CTG repeats on Bru3 and Mbl levels when compared with Gal4-driven overexpression of Bru3 and RNAi-knockdown of mbl. We add a comment in Results section (page 5) to discuss this point: “The Hand>960CTG line shows cardiac dilation at 5 weeks of age characterized by significant increase in diastolic and systolic diameters but with normal cardiac contractility (Fig. S2A,B,C). We hypothesise that non-affected contractility in this DM1 line is due to a milder effect of 960CTG repeats on Bru3 and Mbl levels compared to GAL4-driven overexpression of Bru3 and RNAi-knockdown of mbl.”

• Did the confocal studies indicate whether there was myofibrillar disarray in the heart tubes? *

Answer: Thank you for this comment. Yes, we observe myofibrillar disarray. We show disarray phenotypes in all DCM developing contexts in a new supplementary figure (Fig.S1).

• For the statistical comparisons in the figures, please indicate in the legends that statistically significant differences (p

Answer: We provide this precision in Figure legends.

*Please more thoroughly explain the UPRT control line. *

Answer: We provide information about UPRT line in the Results section (p.8).

*Figure S1 legend: "(red) and 5 (darck)"; the latter should read "(black)" *

Answer: Revised.

*Figure S2 panels J and K: it would be helpful to indicate what is being measured on the Y axis, e.g., Mean intensity of dmiR-1 levels. This is true for the various panels in other figures labeled CTCF on their Y axes. *

Answer: Revised as suggested by the Reviewer.

*CROSS-CONSULTATION COMMENTS All reviewers agree that this is a well-designed study and that the manuscript is well written. The missing Hand-Gal4 control mentioned by Reviewers 1 and 3 seems an important element that is missing. These reviewers also call into question the FISH quantification methodology. These two issues seem the most critical to resolve. The other additional experiments suggested deserve input from the authors as to whether they already have relevant data that can be cited, whether they are important to pursue or if they go beyond the scope of the current study. Reviewers 1 and 2 agree that further discussion of the fly model that does not show DCM should be provided. The question on fibrosis in the fly models is germane (Reviewer 3), although it might be indirectly addressed by the fact that a collagen molecule is upregulated here (a major player in fibrosis). All of the minor comments are reasonable and should be addressed by the authors. *

Answer: We provide answer to all these comments.

*Reviewer #2 (Significance (Required)): *

* This paper is significant in that it draws a more direct connection between the reduction in a microRNA that occurs in myotonic dystrophy and dilated cardiomyopathy that is affiliated with this disease. It shows that a form of collagen that is overexpressed in both Drosophila models and humans with DM1-caused DCM is causative/correlated with the increased heart diameters. Thus, the fly model provides important insights into the link between the mutant gene and the cardiac phenotype. This work will be of interest to those studying skeletal and cardiac muscle disease and scientists interested in developing potential therapeutics for treating DM1-caused DCM. Note that my expertise is in producing and studying skeletal muscle and cardiac disease models in the Drosophila system, which is relevant to evaluating this paper and defining its significance in the field. *

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

*A current study demonstrates that miR-1 targets the newly identified heat-specific target Multiplexin, which, when upregulated, exhibits similar phenotypes observed for the Drosophila DM1 model. Furthermore, the authors additionally confirm some of their results using samples derived from DM1 patients and support the data obtained in flies. Overall, it is a good study with well-performed experiments. The data presented in the paper are convincing and most of the claims the authors provide are supported by their findings. Manuscript is clearly written and easy to read and understand. Statistical analysis and the description of the methods are appropriate. It is an interesting paper and I would highly support it to be accepted for publication; however, I few comments I would like authors to address. *

Answer: We are grateful to the Reviewer for his enthusiastic and supportive comments on our work.

Major comments:

2. The cardiac dilation/fractional shortening phenotype in Hand>dmiR-1-KD flies is only observed in young flies but not in old flies. However, heart-targeted Mp overexpression leads to DCM in aged flies. Could authors comment on this? * Answer: We speculate that attenuation of miR-1 in the heart could lead to more drastic pro-DCM alterations thus leading to earlier phenotypes than in the case of Mp overexpression.

To better assess DCM phenotypes we included additional Hand-Gal4/+ control context and more heart samples from 5 weeks-old flies. These additional analyses presented in revised Fig. 1D-F reveal that old Hand>dmiR-1KD flies, like the young one, also develop DCM phenotype.

• Since the HAND-Gal4 line was used to drive multiple transgenes, it would be important to have the cardiac dilation/fractional shortening phenotype measured in this line as a control. *

Answer: We performed these control experiments and suggested by the reviewer they are now included to the graphs.

• The use of a sponge line is highly appreciated as it allows for tissue-specific downregulation of miRNA. However, to corroborate the data, I would recommend including the knockdown mutant that is available in Bloomington as additional confirmation since no qPCR is provided for the efficacy of the sponge line. This line could also be used in combination with reporter lines to perform a targeting experiment. *

Answer: We are grateful for these comments. Below we refer to performed additional experiments:

• We tested whether heterozygous dmiR-1 KO -/+ flies (homozygous dmiR-1 mutants are lethal) develop Hand>miR-1sponge-like heart phenotype. Indeed, at 5 weeks of age dmiR-1 KO -/+ flies show significantly increased diastolic and systolic heart diameters. Thus, in old flies loss of one copy of miR-1 mimics heart dilation observed in Hand>dmiR-1sponge context. Heart contractility remains unaffected in dmiR-1 KO -/+ flies, suggesting that loss of one copy of miR-1 has a weaker impact on heart function than heart-targeted miR-1sponge. These data are shown in a new supplemental figure (Fig. S4A-C).

• Here, we identify Mp as a new direct miR-1 target. To test whether miR-1 sponge attenuates miR-1 function we analyzed Mp protein levels in the hearts from wt and Hand>miR-1sponge flies. Mp expression is highly increased in Hand>miR-1sponge context indicating attenuation of miR-1 by the sponge transgene. These data are presented in new Fig. S5J;

• The authors state that the reduced miR-1 levels have already been shown in DM1 patients. It would be a stronger argument if similar downregulation was shown in patient samples used in this manuscript (qPCR would be sufficient). *

Answer: We performed suggested by the reviewer analyses of miR-1 in patient samples. We show that miR-1 is indeed down regulated. These new data supporting conserved pro-DCM deregulation of miR-1 and its target Mp/Col15A1 are shown in new Fig 5C.

• Because fibrosis is a hallmark of myotonic dystrophy, do the authors have some makers or other methods to test whether observed phenotypes are due to fibrosis? *

Answer: Fibrosis (replacement of muscle by fibrotic tissue) has not been reported in Drosophila and is not associated with degeneration of body wall or cardiac Drosophila muscles in so far described fly models of human muscular dystrophies. However, one could speculate that increase in Mp/Col15A1 levels within the ECM of diseased DM1 cardiac cells we observe, could have, a fibrotic-like, negative effect on cardiac function.

• The explanation of the observation that pre-miR-1 levels are down-regulated only in young flies, whereas old flies show an opposite tendency, is missing. *

Answer: Accumulation of pre-miR-1 in old flies is most probably due to the affected processing mediated by mbl. This is correlated with the reduction of the mature miR-1.

The authors suggested that this is due to "impaired processing". To corroborate this interesting hypothesis, the authors performed only the smFISH intensity analyses, which are somewhat difficult to decipher. I would recommend, in addition to the pre-miRNA levels, to test and compare the mature miRNA expression using TaqMan qPCR.

Answer: Impaired miR-1 processing is supported by the previous studies in human cells (Rau et al., 2011) and in Drosophila models (Fernandez-Costa et al., 2013). We believe that our method of quantification of miR-1 expression via highly sensitive miRCURY LNA FISH is a well-adapted method. It was performed with all necessary controls. In the method section we provide now more details for the LNA FISH based miR-1 quantification approach.

In parallel, TaqManPCR-based miR-1 quantification was performed for human cardiac samples from DM1 patients.

• The relationship between Bru3 and miR-1 shown in the schematic is not well-defined and would rather require a question mark or dotted line, as the authors provide no evidence that Bru3 can be directly involved in miR-1 processing. The authors suggest that CELF1 may bind UG-rich miRNAs and mediate their degradation by recruiting poly(A)-specific ribonuclease (PARN), but this is only a hypothesis and does not justify the placement of a direct line of repression on the schematic in the last figure. *

Answer: We agree and modify scheme accordingly.

• I also feel that the authors did not clearly explain the cardiac phenotypes in terms of systolic and diastolic diameter measurements. Which parameters clearly represent the DM1 model, specifically higher or lower diameters of systole and diastole? Results should be clearly indicated in figure legends. *

Answer: We provide appropriate precisions in figure legends.

Minor comments:

2. The full name of CELF1 on page 2: CUGBP Elav-like family member 1 should be added*. Answer: Revised

3. For better readability of the text and corresponding figures, consistent use of UAS-Mp or UAS-3HNC1 is recommended, but not a mixture of both. *

Answer: We consistently use UAS-Mp in the revised version

• Why is the Multiplexin overexpression line called UAS-3HNC1? *

Answer: This is the name that resumes protein Mp domains: Collagen tripple helix and trimerization region (3H) and NC1 domain (C-terminal non-triple helical domain) comprising Endostatin domain. We provide this information in Methods section.

• For all figures, it would be better if the genotypes were indicated in the panels and the graphs had the age of the flies instead of color coding. *

Answer: We revised these points as suggested.

• Figure 5. Were technical replicates performed for the western blot shown in 5B? *

Answer: We didn’t perform technical replicates because of limited human sample amounts

• Figure S1-S2. Why the data for qPCR of miR-1 is in figure S1 and not S2? *

Answer: In the revised version all supplemental analyses on miR1 are included to the new Fig. S4

• Figure S4. Misspelling in figure legend: Scale "barre" instead of scale "bar".*

Answer:Revised

Reviewer #3 (Significance (Required)):

* The manuscript "Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1" by Souidi et al., reports a novel role of identified Multiplexin (Mp) as a new cardiac miR-1 target involved in myotonic dystrophy type 1 (DM1) using Drosophila as a model system. Myotonic dystrophy type 1 (MD1) is a severe disease that results in a multisystem disorder affecting the skeletal and smooth muscles as well as the eye, heart, endocrine system, and central nervous system. At the moment, no appropriate treatment has been identified to prevent it. Previous studies have also shown that heart-specific miR-1 levels are reduced in patients with DM1, but the role and targets of this miRNA in the heart have not been analyzed. Research presented in this paper is of a broad interest and provide new evidence that will help to better understating DM1 on molecular level. It will be interesting not only to scientists from the Drosophila field but will also contribute to medical research field.*

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

A current study demonstrates that miR-1 targets the newly identified heat-specific target Multiplexin, which, when upregulated, exhibits similar phenotypes observed for the Drosophila DM1 model. Furthermore, the authors additionally confirm some of their results using samples derived from DM1 patients and support the data obtained in flies. Overall, it is a good study with well-performed experiments. The data presented in the paper are convincing and most of the claims the authors provide are supported by their findings. Manuscript is clearly written and easy to read and understand. Statistical analysis and the description of the methods are appropriate. It is an interesting paper and I would highly support it to be accepted for publication; however, I few comments I would like authors to address.

Major comments:

1. The cardiac dilation/fractional shortening phenotype in Hand>dmiR-1-KD flies is only observed in young flies but not in old flies. However, heart-targeted Mp overexpression leads to DCM in aged flies. Could authors comment on this?

2. Since the HAND-Gal4 line was used to drive multiple transgenes, it would be important to have the cardiac dilation/fractional shortening phenotype measured in this line as a control.

3. The use of a sponge line is highly appreciated as it allows for tissue-specific downregulation of miRNA. However, to corroborate the data, I would recommend including the knockdown mutant that is available in Bloomington as additional confirmation since no qPCR is provided for the efficacy of the sponge line. This line could also be used in combination with reporter lines to perform a targeting experiment.

4. The authors state that the reduced miR-1 levels have already been shown in DM1 patients. It would be a stronger argument if similar downregulation was shown in patient samples used in this manuscript (qPCR would be sufficient).

5. Because fibrosis is a hallmark of myotonic dystrophy, do the authors have some makers or other methods to test whether observed phenotypes are due to fibrosis?

6. The explanation of the observation that pre-miR-1 levels are down-regulated only in young flies, whereas old flies show an opposite tendency, is missing. The authors suggested that this is due to "impaired processing". To corroborate this interesting hypothesis, the authors performed only the smFISH intensity analyses, which are somewhat difficult to decipher. I would recommend, in addition to the pre-miRNA levels, to test and compare the mature miRNA expression using TaqMan qPCR.

7. The relationship between Bru3 and miR-1 shown in the schematic is not well-defined and would rather require a question mark or dotted line, as the authors provide no evidence that Bru3 can be directly involved in miR-1 processing. The authors suggest that CELF1 may bind UG-rich miRNAs and mediate their degradation by recruiting poly(A)-specific ribonuclease (PARN), but this is only a hypothesis and does not justify the placement of a direct line of repression on the schematic in the last figure.

8. I also feel that the authors did not clearly explain the cardiac phenotypes in terms of systolic and diastolic diameter measurements. Which parameters clearly represent the DM1 model, specifically higher or lower diameters of systole and diastole? Results should be clearly indicated in figure legends.

Minor comments:

1. The full name of CELF1 on page 2: CUGBP Elav-like family member 1 should be added.

2. For better readability of the text and corresponding figures, consistent use of UAS-Mp or UAS-3HNC1 is recommended, but not a mixture of both.

3. Why is the Multiplexin overexpression line called UAS-3HNC1?

4. For all figures, it would be better if the genotypes were indicated in the panels and the graphs had the age of the flies instead of color coding.

5. Figure 5. Were technical replicates performed for the western blot shown in 5B?

6. Figure S1-S2. Why the data for qPCR of miR-1 is in figure S1 and not S2?

7. Figure S4. Misspelling in figure legend: Scale "barre" instead of scale "bar".

Significance

The manuscript "Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1" by Souidi et al., reports a novel role of identified Multiplexin (Mp) as a new cardiac miR-1 target involved in myotonic dystrophy type 1 (DM1) using Drosophila as a model system. Myotonic dystrophy type 1 (MD1) is a severe disease that results in a multisystem disorder affecting the skeletal and smooth muscles as well as the eye, heart, endocrine system, and central nervous system. At the moment, no appropriate treatment has been identified to prevent it. Previous studies have also shown that heart-specific miR-1 levels are reduced in patients with DM1, but the role and targets of this miRNA in the heart have not been analyzed. Research presented in this paper is of a broad interest and provide new evidence that will help to better understating DM1 on molecular level. It will be interesting not only to scientists from the Drosophila field but will also contribute to medical research field.

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Referee #2

Evidence, reproducibility and clarity

Summary:

This well-written manuscript utilizes the Drosophila model system to demonstrate that reduction of micro-RNA miR-1 and the resulting increase in one of its down-regulated proteins (Multiplexin) contribute to a dilated cardiomyopathy (DCM) phenotype. This is of interest in that this particular micro-RNA is downregulated in myotonic dystrophy type 1 (DM1), and this correlates with the DCM phenotype observed in patients. Further, the authors show that the human ortholog of Multiplexin is enriched in human DM1-DCM hearts and that downregulation of this protein in Drosophila DM1 models improves the DCM phenotype. Hence, the work demonstrates a potential mechanism for disease development and its amelioration.

Major comments:

1. As pericardial cells are probed and mentioned substantially in this paper, the authors should explain what these cells do in flies. While affiliated with the heart, they are not myocytes and are probably not particularly relevant to the human heart. In this regard, it is possible that the phenotypes observed in the heart are partially or completely the result of Hand-driven expression of transgenes in the pericardial cells. Although unlikely, this issue should be mentioned as well.

2. Does human miR-1 target Col15A1 transcripts based upon in silico analysis? This issue should be mentioned and discussed.

Minor comments:

1. The abstract should explicitly state that Multiplexin is a form of collagen.

2. More information on the identity between the Drosophila and human forms of miR-1 would be helpful to establish that they are conserved. What is the percent identity and are the sequences that target mRNAs homologous?

3. In Figure 1C, it appears that there is an increased heartbeat frequency and arrhythmicity. Are these mutant phenotypes as well?

4. Incomplete sentence (page 5): We identified a set of candidate genes, of which Multiplexin (Mp)

5. What is the basis for studying Multiplexin function as opposed to other candidates that were identified? It would be useful to mention this in the Results, although it is mentioned in the Discussion ("We top-ranked Mp because of its known role in setting the size of the cardiac lumen").

6. "Mp was detected on the luminal and external surfaces of the cardiomyocytes ensuring cardiac contractions" Why does this ensure cardiac contractions?

7. Need to state in the text that the increased levels of Col15A1 transcript expression in DM1 patients was not statistically significant.

8. Need a magnification bar for Figures 5F-H.

9. Please speculate as to why the third DM1 model does not recapitulate the cardiac phenotypes.

10. Did the confocal studies indicate whether there was myofibrillar disarray in the heart tubes?

11. For the statistical comparisons in the figures, please indicate in the legends that statistically significant differences (p<0.05) are shown.

12. Please more thoroughly explain the UPRT control line.

13. Figure S1 legend: "(red) and 5 (darck)"; the latter should read "(black)"

14. Figure S2 panels J and K: it would be helpful to indicate what is being measured on the Y axis, e.g., Mean intensity of dmiR-1 levels. This is true for the various panels in other figures labeled CTCF on their Y axes.

CROSS-CONSULTATION COMMENTS

All reviewers agree that this is a well-designed study and that the manuscript is well written. The missing Hand-Gal4 control mentioned by Reviewers 1 and 3 seems an important element that is missing. These reviewers also call into question the FISH quantification methodology. These two issues seem the most critical to resolve. The other additional experiments suggested deserve input from the authors as to whether they already have relevant data that can be cited, whether they are important to pursue or if they go beyond the scope of the current study. Reviewers 1 and 2 agree that further discussion of the fly model that does not show DCM should be provided. The question on fibrosis in the fly models is germane (Reviewer 3), although it might be indirectly addressed by the fact that a collagen molecule is upregulated here (a major player in fibrosis). All of the minor comments are reasonable and should be addressed by the authors.

Significance

This paper is significant in that it draws a more direct connection between the reduction in a microRNA that occurs in myotonic dystrophy and dilated cardiomyopathy that is affiliated with this disease. It shows that a form of collagen that is overexpressed in both Drosophila models and humans with DM1-caused DCM is causative/correlated with the increased heart diameters. Thus, the fly model provides important insights into the link between the mutant gene and the cardiac phenotype. This work will be of interest to those studying skeletal and cardiac muscle disease and scientists interested in developing potential therapeutics for treating DM1-caused DCM. Note that my expertise is in producing and studying skeletal muscle and cardiac disease models in the Drosophila system, which is relevant to evaluating this paper and defining its significance in the field.

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Referee #1

Evidence, reproducibility and clarity

The paper titled "Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1" by the Jagla group studied the effect of down-regulation of miR-1 in myotonic dystrophy type 1 (DM1) using fly as the disease model. The study is based on previous findings that in DM1 MBNL1 is sequestered, CELF1 is stabilized, and miR-1 is down regulated. The authors further identified Multiplexin to be the target effector of miR-1 in the fly heart and studied its function with a series of gain- and loss-of-function and rescue experiments. The authors' findings represent a significant advance in understanding the genetic mechanisms that can explain the pathogenic causes of dilated cardiomyopathy associated with DM1. Overall, this paper is well written and organized, with well-designed experiments and a clear model. A few additional experiments are suggested to further strengthen the conclusion.

Major points:

1. Wild-type and Hand-Gal4 controls are missing for all experiments (only UAS lines outcrossed to w1118 are displayed). Also, Hand-Gal4 driver lines can cause mild dilation by itself, which would influence interpretation and statistics of data. Also, the authors should consider to confirm the miR-1 phenotype obtained with the sponge with a miR mutant, and also combine miR-1 het with miR sponge (worsening of phenotype?). Alternatively, knockdown efficiency of miR should be tested by qPCR or HCR/smFISH.

2. It is surprising that one of the DM1 fly models, overexpression of 960 CTG repeats, did not show DCM, considering it is the primary cause of DM1 in humans due to excessive CTG repeats. It should be discussed why Hand>960 CTG does not lead to DCM, since the authors claim that this model with high number of CTG repeats shows a strong phenotype. Are Hand>bru3 and Hand>mbl stronger? Is miR-1 (and Mp) unaltered in these flies with 960 CTG repeats? It would be interesting to overexpress 960 CTG in a miR-1 or mbl heterozygous mutant background, which may produce DCM.

3. In Figure 2H, the mean intensity is displayed as the readout of the smFISH quantification of miR-1 levels. If understood correctly, this is the wrong readout since smFISH detects single molecule fluorescence of transcripts, so the number of transcripts should be quantified. Furthermore, Hand-Gal4 is not expressed in the ventral longitudinal muscles (VLM). As a proof of principle, miR-1 levels should be quantified in VLM (no change in transcripts levels expected).

4. Fig. 5: Since DCM- DM1 patients still show elevated COL15A1 levels but no DCM, it would be interesting to know if DCM phenotypes are COL15A1-dosage dependent. This could be easily tested in the fly model by testing UAS-Mp overexpression at different temperatures.

5. The authors elegantly show rescue of Hand>Bru3 flies by Mp RNAi. Their model would be further strengthened if a similar rescue can be shown with Hand>mblRNAi.

Minor points:

1. Fig. 1A,B: Ventral longitudinal muscles are covering the hearts on these images, so it's difficult to see the heart dimensions. This holds true for images throughout the manuscript. Where were the diameters measured (by the valves)? A better description and illustration would help the reader understand the situation.

2. Fig. 1 A',B': White line does not reflect the location where SOHA data are measured and should be horizontal for consistency. Where is ventral vs. dorsal?

3. Fig. 1D-F: Annotate 1 and 5 weeks in Figure, please. Also, why were 1 and 5 weeks tested? Is there an age-component in DM1 phenotype severity?

4. Fig. 3A: Transcriptional analysis was done at which stage of development?

5. Fig. 3: It is not clear, in which set the authors looked for miR-1 bindings sites (144 genes or the whole set)? Not well annotated. What is meant by 'heart-targeted'?

6. Fig. 4C,D: It looks like they are not shown in the same dorsal-ventral orientation. Also, it looks Mp is overexpressed in the VLM, but Hand-Gal4 only drives in the cardiomyocytes and pericardial cells? How was quantification done?

7. Fig. 4I: Why are some myofibers indicated in red in the model?

8. Fig. 5 D-E: Genotypes need to be better indicated in the graphs. Did the authors control for multiple UAS sites? Is UPRT a UAS control?

In the first paragraph of Result 3, the last sentence seems unfinished. "We identified a set of candidate genes, of which Multiplexin (Mp)"

In Method, the in silico screening for miR-1 target should be explained in more detail.

Significance

The presented data is a significant advance our knowledge of our understanding of the molecular mechanisms involved in DM1. I expect that scientists in the muscular disease field and beyond will find this work of high interest.

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Reply to the reviewers

_Reply to the reviewers _

Note: the three reviewers who provided comments were identified as Reviewers 2-4

Reviewer #2

1) I could not open any of the movies (while those associated with the BioRXiv preprint were fine). Some of the movies could be combined to minimize download/open clicking sequences.

• The movies were uploaded as .avi files, as per Review Commons instructions, and we tested our ability to view them on several computers at our institution before submission. We are relieved the reviewer was able to access the .mp4 formatted movies via BioRXiv. We will ask the Review Commons Managing Editor to make sure there are no problems with the videos uploaded with the revised manuscript.*

2) I really dislike reviewing papers without line numbers

• Line numbers have been added to the revised version.*

3) The manuscript could be made more relevant to malaria researchers by briefly discussing red cell invasion by merozoites (a single constriction and force against the cell cortex), migration of ookinetes (multiple constrictions during mosquito gut penetration) and sporozoites (long distance migration), but this is not a must.

• Constrictions during ookinete migration are now mentioned on lines 265-269, and the discussion of the constriction at the moving junction has been broadened to include other apicomplexan parasites lines 270-278.*

4) I would limit reporting of numbers to two digits, e.g. instead of 46.3% make it 46%; 2.56 +/- 0.38 to 2,6 +/- 0,4 etc

> We have adjusted all numbers in the text and figures to the appropriate number of significant figures based on measurement precision.

5) Millions of deaths, please rewrite, more like around 1 million from malaria and cryptosporidium; use citation (WHO)

> Done (line 40)

6) Motility: please don't mention flagella, which are used for swimming, in the same sentence / phrase / logic connection as lamellipodia, which are used for substrate based migration

> The sentence has been rewritten to make clear that cilia and flagella are not organelles involved in the substrate-dependent motility of other eukaryotic cells (lines 47-49).

7) In Figure 1B, I can see one microsphere and it's not clear if it moves completely back to the original position. In the movie it looks like it goes completely back, maybe exchange the last panel of the figure with a last frame from the movie? Or maybe better: replace with frames from movie 2, which is more striking and shows many beads being displaced?

> As suggested, Figure 1B now shows frames from the other movie (former Video 2), where bead movement is more obvious.

8) Please add the entire figure S1 to Figure 1. This is important for readers to understand and 'deserves' full figure status. Same for Figure S2.

*> We have moved most of former Figure S1 into a new main Figure 2, as suggested. We left the two graphs as Supplemental data (new Figure S1), since these graphs simply show that parasite motility in fibrin is similar to the previously described motility of parasites in Matrigel. *

*> Figure S2 has been moved to the main text, as suggested (in new Figures 3 and 6). *

9) I would encourage the authors to elaborate more on the data on Figure S2. It appears that motile parasites did mostly not exert forces above the level for non-motile parasites; for how much motility did they observe forces? The meaning of the x-axis does not become clear. Are those individual parasites per time point or time points of one parasite or of the analyzed matrix volumes over several parasites? How many parasites where observed? This is stated more clearly later but needs to be done already here.

> We have moved the data in former Suppl. Figure S2 into the main figures, broken it into two parts (Figures 3 and 6B-E) and included a new 3D volume view and additional explanatory detail in the figure legends and text to clarify these points of confusion (lines 100-116, 500-507, 564-570).

10) Please change 0.042 um into 42 nm etc

*> Done, lines 113-116. *

11) Please move some of the data in Figure S8 to the main figures e.g. Figure 4, where it would make a nice contrast / comparison to the mic2 mutant. Please also put a WT for comparison.

> Done; see revised Figure 6.

12) I wonder if the defect in directional migration of the mic2 mutant is also partly due to the parasite not being able to squeeze through narrow matrix pores and hence is deflected more often. While I understand (and agree) with the authors observation (interpretation) of the wt parasites not squeezing but pulling, it's hard to think that such squeezing would not still play a part.

*> The idea that the parasite needs to squeeze its way through pores in the matrix is intuitively appealing (and, in fact, what we had expected to see) but there is currently no data to support it. If squeezing were occurring, we should see an outward deformation of the matrix as the parasite pushes on the matrix fibers, but this is something we have never observed. We therefore think it is unlikely that the loss of directional migration is due to an inability to squeeze through pores in order to “stay on track”. *

13) Hueschen et al is now on BioRXiv

> The BioRXiv citation has been added (lines 293, 320).

14) The shaving off of antibodies could be brought into context to the work on sporozoites by Aliprandini Nat Micro 2018 and on trypanosomes by Enstler Cell 2007 (but not a must)

*> The two studies mentioned are intriguing and may be related to the well-documented anterior to posterior flux and shedding of GPI-anchored proteins from the surface of gliding Toxoplasma tachyzoites. What we are showing here is slightly different: the fluorescent antibodies on the cell surface seem to be “shaved” backwards at the constriction, much like surface bound antibodies are shaved backwards at the moving junction during invasion (Dubremetz 1985). In other words, there is a discontinuity in the density of surface staining at the constriction/junction. All of these processes may be related, but this is only speculation at this point and since the shaving of antibody at the constriction is a minor point of the paper (meant only to illustrate another similarity between 3D motility and invasion), we would prefer not to try to tie it to these other observations which may or may not be related. *

15) Anterior-posterior flux: best experimental evidence for this is Quadt et al. ACS Nano 2016 for Plasmodium and Stadler MBoC 2017 for Toxoplasma. The common observations and differences could be discussed as they pertain to the current study

> These two papers are now cited in our discussion of the linear motor model along with our speculation that the constriction reflects the motility-relevant zone of engagement of this rearward flux with ligands in the matrix (lines 319-322).

16) The loss of mic2 could lead to the loss of the capability to form discrete adhesion sites that reveal themselves as the observed rings in 3D. I suggest to be careful to hypothesize that the absence of this and MyoA reveals a completely different motility mechanism. To me it seems more likely that the absence of the proteins means that the existing mechanism doesn't work perfectly any more, ie the highly tuned migration machinery misses a key part and malfunctions.

*> The paragraph in question offered possible explanations for how parasites lacking the constriction could in fact move at normal speeds, not that motility was negatively affected. We have tried to make this more clear in the revision (lines 352-354), before describing the 3 possible explanations. *

17) Maybe reflect on whether 'search strategy' might be a better word than 'guidance system'

*> We have replaced the term “guidance system” in the title (lines 1-2), abstract (lines 33-36) and introduction (line 75) with more conservative references to the ability of the parasite to move directionally. The only place the term “guidance system” remains is in the final paragraph of the discussion, which is more speculative in nature, and where we now suggest it to be “part of” a guidance system. *

Reviewer #3

1) Extracellular matrix choice. The authors track the parasite movement first on Matrigel and next on fibrin. The authors exemplify the fibrin matrix on an image on Suppl. Fig 1 that shows a relatively quite large pore size, similar or greater than parasite size. Was the analysis done on parasites touching the fibers?

*> Previous Suppl Figure 1A showed a confocal image at only one z-plane which did indeed give the impression that the pores are relatively large. We have changed this image to a more informative maximum intensity projection (New Figure 2A) and included a video showing the entire imaging volume (new Video 4), which makes clear that the matrix contains many small fibers and that the pores are smaller than the previous single z-plane suggested, so the parasite is likely to be near to or in contact with fibers of the matrix at all times. In Suppl Figure 1D we purposely used a less dense matrix in order to make the matrix deformation more obvious to the eye. The density of the matrix in Fig. 1D has been added to the legend. *

2) Lack of movement of parasites. In many figures of the articles it is revealed that the majority of parasites in fibrin remain immobile (Suppl Fig 1, Fig 2, Video 5, Suppl Fig 2, Suppl Fig 8). The number of immobile parasites in Matrigel seem to be lower than in fibrin (Suppl Fig 1B) although no quantification is shown. How does the movement in fibrin and Matrigel compare? How does this compares with movement in stiff substrates in 2D? Could the lack of movement be caused by the large pore site in fibrin?.

> We have added a panel to Suppl. Figure S1 showing that the proportions of parasites moving in fibrin vs Matrigel are not significantly different. In fact, none of our measured motility parameters are different between fibrin and Matrigel. Not all parasites move during the 80s of capture used for these matrix comparisons; some of the parasites are likely dead, but others may have simply not initiated motility during this time window. We typically see between 30-50% movement in 3D motility assays of this duration and similar numbers in 2D trail assays although we have not explored the effect of 2D substrate stiffness.

3) Considering parasite movement: The authors consider that 3SD is a cutoff for considering parasite displacement. However, several timepoints fall behind this cutoff in the control without parasites and the knockouts with restricted movement.

> We chose three standard deviations from the mean as our cutoff, in order to eliminate 99.7% of the noise. Since we calculate 16807 vectors per comparison, this leaves us with ~50 vectors above the cutoff even in samples with no moving parasites. Not surprisingly, these vectors are found at random locations in the volume. New Figures 3 and 6B-E and the associated text (lines 100-116, 500-507, 564-570) hopefully clarify this point adequately; it is quite obvious in Figure 3C which vectors correspond to parasite-induced displacements and which correspond to random noise.

4) Imaging: Although the authors show a very detailed an illustrative table of the imaging acquisition conditions in table 1, it is unclear which microscope the authors used, as two microscopes are described in the methods section, a Nikon Eclipse TE300 widefield microscope and a Nikon AIR-ER confocal microscope. Which images were taken in each system? For the location of Table1 in the manuscript it seems that most images were taken with the Nikon Eclipse. Although this microscope has control over z, the images are quite noisy. How does the lack of confocallity might interfere with the analysis?

> The high temporal resolution needed for 3D force mapping of cells that move several microns per second meant that all these experiments were done using a widefield microscope equipped with a piezo-driven z-stage. The fastest confocal we tested was not as fast as the widefield. However, spatial resolution suffered as a result of having to use widefield, particularly in z,* and this did indeed make our data more noisy as suggested by the reviewer. This may be why we were unable to detect fibrin deformation in the knockout parasites. The only data collected on the confocal microscope were those shown in new Figure 2A; we have clarified this on lines 421-427. Future studies will explore other imaging modalities such as light sheet microscopy in an attempt to achieve better spatial resolution while maintaining the high frame rates required for force mapping. *

5) Nuclear constriction. The authors did not show any image or video exemplifying this.

The images in Suppl. Figure 6 have been replaced with data that show the nuclear shape more clearly.

6) Knockouts: The authors did not explain how did they generated the knockouts in the methods or did now show the efficacy of the knockout in any figure. If these knockout strains were a gift (I did not find it on the manuscript), the authors should indicate this more explicitly and reference the manuscript where they were described for the first time.

> Both of the stable knockout lines used were generous gifts from Dr. Markus Meissner. We cited the original papers describing these lines in the text and thanked Dr. Meissner for providing them in the Acknowledgements section. We have now included an additional citation at the first mention of each of the knockouts (lines 174, 188) to make it even clearer where they came from.

7) Discussion: Although the experimental methodology is sound the authors seem to make many assumptions and speculations on the discussion as how the appearance of this ring/constriction on the parasite translates into the helical movement of the parasite or the coupling of the ring with the cytoskeleton. Live imaging of actin dynamics or mathematical modelling could be used to support their claims.

> We imaged parasites expressing the actin chromobody but were unable to visualize a ring of actin at the constriction. However, due to the speed of the parasites and the need for a fast frame rate (~15 ms per image) to reconstruct the 3D image volumes, the actin chromobody signal could be under our threshold of detection. We need to develop new, more sensitive ways to visualize proteins at the constriction, and this will be a major focus of our work going forward.

*> We fully concur that mathematical modeling such as the work recently done by Hueschen et al on actin flow during motility and by Pavlou et al on the role of parasite twist during invasion has much to offer our understanding of these processes. Similar approaches may provide support to the speculations (not claims!) we offer in the discussion and, although beyond the scope of the current study, are a direction we intend to take this work in the future – particularly if we are able to improve the signal-to-noise in our force mapping. *

8) Quantification of experiments missing: Overall, the main figures lack quantification that sometimes can be found in the supplemental information and sometimes is missing. I would suggest including quantifications next to the events described in the main figures). Likewise, some of the supplemental figures lack quantification (Suppl Fig 7, how many parasites showed this protein trail?)… Overall, the authors should indicate how many parasites were quantified in each figure. As they usually refer to number of constrictions. This is overall a problem in main figures 3 and 5. Or for example in Suppl Fig 5: How many parasites were quantified in this figure? The authors only show number of constrictions, and as the authors described, a parasite might have more than one constriction.

> We have added further detail on the number of events/parasites quantified to both the figure legends and text throughout the manuscript, including the specific examples noted by the reviewer.

9) Videos: The videos lack scale of time. Although this that can be found in main figures, it would be helpful to have the annotation in the videos. Likewise, some references for positions in videos, such as the cross found on Fig1 would be helpful for parasites that present little movement.

> Time stamps have been added to all videos as suggested, and crosshairs have been applied to new Figure 1B and Suppl. Figures 7 and 8 to make the movement of the parasites more obvious. *

*

Reviewer #4

1) I am not sure about the premise that the "linear model" of gliding motility predicts uniformly forward direction. Previous videos of 2D gliding show sporadic motility, changes in direction, or even reversal of direction are not infrequent. However, the current model could explain these behaviors if one or more of the following conditions occur: 1) myosin motors might be coordinating activated to initiate motility, followed by relaxation, 2) actin fibers might be transiently arrayed in clusters that change density and polarity over time, or 3) adhesins, necessary to generate traction, might vary in density and spatial orientation across the surface of the parasite. Changes in these properties would be expected result in zones that promote or disfavor local forces needed for motility - and reversal of direction could occur when forward forces relax and external elastic forces predominate.

> The potential explanations offered by the reviewer for the frequent changes in direction of zoite motility are intriguing and worth exploring experimentally. The ability of actin fibers to periodically reverse polarity, or the presence of counteracting elastic forces are not components of the “standard” linear motor model of motility but, if they occur, could explain the patch gliding phenomenon and help refine our understanding of motility. Since the data in this manuscript do not in the end either strongly support or disprove the linear motor model – this may ultimately require higher resolution force mapping methods that can detect the forces responsible for forward motion – we have de-emphasized potential problems with the model in the introduction and deleted specific discussion of patch gliding as one of these problems (lines 61-64).

2) The model favored here: "we propose that force is generated, at least in part, by the rearward translocation of the subset of actin filaments that are coupled to adhesins at the circular ring of attachment" does not seem fundamentally different from the current model - other than it focuses the forces at a critical junction that the parasite migrates through. It seems to me that this is a refinement of the current model and not a replacement. As such, the authors might focus on how their data improve the model rather than pointing out prior deficiencies (although I get that editors like this style).

> We agree with the reviewer and have modified the text to be more circumspect on this issue* (lines 319-331). *

3) The finding that the absence of MIC2 affects the constriction formed by inward pull on the matrix is quite convincing and interesting. However, mutants that cannot form the constriction, still move at similar speeds. This suggest that the inward force is different from the motor itself and affects its ability to impart direction, rather than the ability to move per see. The interpretation of the MyoA defect is complicated since motility is certain to be disrupted, the potential role of an independent inward force may no longer be detectable.

> We agree with the reviewer on this point as well: the forces we have observed to date cannot explain forward motion. We stated this previously and have now emphasized the point further *(lines 322-324, 352-357). Because the parasite is moving forward, the forces responsible must be there but are likely below our threshold of detection. In order to visualize these forces, we are going to need new imaging modalities that can achieve better signal-to-noise than our current setup at the high frame rates required for force mapping. That said, we new data we have added to the manuscript are at least consistent with the narrow diameter ring of the constriction making a contribution to the parasite’s forward motion (new Suppl. Figure 10 and lines 347-351) *

4) Although I agree with the authors that there are striking parallels between motility in 3D and cell invasion, I am not certain about their conclusion that the construction seen during cell entry is due to the parasite pulling inwardly. When entering the host cell, the parasite must also navigate the dense subcortical actin network, which likely also aids in forming the constriction that is observed. It would be interesting to record this pattern under conditions where host cell actin is destabilized while parasite motility is intact- for example using cytochalasin D to treat wild type host cells during invasion by resistant parasites.

*> We do not conclude that the constriction during invasion is due to the parasites pulling inwardly, but we do propose that this possibility needs to be considered based on the noted similarities between invasion and motility and our clear (and somewhat surprising) demonstration that the moving parasite pulls on the matrix at the constriction during motility. During invasion, the parasite may indeed have to squeeze through the dense subcortical network – or it may use secreted proteins to loosen up the network so that no squeezing is required. We just don’t know, and our purpose here was simply to put this alternative possibility on the table because we believe it is a viable possibility that follows from the data presented. *

> We thank the reviewer for the suggestion of testing what happens when cytoD resistant parasites invade in the presence of cytoD; this is a clever idea that we will likely pursue in future work.

5) Not all of the color patterns shown in Figure 1A are consistent with the model. For example, GAP40 (yellow) does not appear in the model, there are two MLC boxes, but they are different shades, and ELC1/2 does not appear in the model.

> We thank the reviewer for catching this error; it has now been fixed.

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Referee #3

Evidence, reproducibility and clarity

The study provides new insight into the gliding motility of Toxoplasma gondii through the use of time lapse video microscopy combined with 3D traction force mapping. Substantial new insight is provided through the discovery that the parasite pulls inward on the matrix, creating a moving junction that it glides through during forward motility. This process of migration in 3D thus closely resembles the invasion of host cells. These data carefully documented and improve our understanding of how gliding motility operates. There are a few issues surrounding how previous data are presented and the relationship between this new inward force and the motor complex that require better explanation.

Major points

1. I am not sure about the premise that the "linear model" of gliding motility predicts uniformly forward direction. Previous videos of 2D gliding show sporadic motility, changes in direction, or even reversal of direction are not infrequent. However, the current model could explain these behaviors if one or more of the following conditions occur: 1) myosin motors might be coordinating activated to initiate motility, followed by relaxation, 2) actin fibers might be transiently arrayed in clusters that change density and polarity over time, or 3) adhesins, necessary to generate traction, might vary in density and spatial orientation across the surface of the parasite. Changes in these properties would be expected result in zones that promote or disfavor local forces needed for motility - and reversal of direction could occur when forward forces relax and external elastic forces predominate.
2. The model favored here: "we propose that force is generated, at least in part, by the rearward translocation of the subset of actin filaments that are coupled to adhesins at the circular ring of attachment" does not seem fundamentally different from the current model - other than it focuses the forces at a critical junction that the parasite migrates through. It seems to me that this is a refinement of the current model and not a replacement. As such, the authors might focus on how their data improve the model rather than pointing out prior deficiencies (although I get that editors like this style).
3. The finding that the absence of MIC2 affects the constriction formed by inward pull on the matrix is quite convincing and interesting. However, mutants that cannot form the constriction, still move at similar speeds. This suggest that the inward force is different from the motor itself and affects its ability to impart direction, rather than the ability to move per see. The interpretation of the MyoA defect is complicated since motility is certain to be disrupted, the potential role of an independent inward force may no longer be detectable.
4. Although I agree with the authors that there are striking parallels between motility in 3D and cell invasion, I am not certain about their conclusion that the construction seen during cell entry is due to the parasite pulling inwardly. When entering the host cell, the parasite must also navigate the dense subcortical actin network, which likely also aids in forming the constriction that is observed. It would be interesting to record this pattern under conditions where host cell actin is destabilized while parasite motility is intact- for example using cytochalasin D to treat wild type host cells during invasion by resistant parasites.

Minor points

Not all of the color patterns shown in Figure 1A are consistent with the model. For example, GAP40 (yellow) does not appear in the model, there are two MLC boxes, but they are different shades, and ELC1/2 does not appear in the model.

Significance

The study provides a conceptual advance that improves our understanding of gliding motility in apicomplexan parasites. It will spur future research in the area to better define the process, although it does not yet offer a new mechanistic foundation.

The work will be of interest to those working on motility in general and parasite systems in specific.

I have worked on cell motility and invasion in this group of organisms for many years, although we currently focus on other questions.

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, Stadler et al., characterize the biophysics behind Toxoplasma gondii locomotion on extracellular matrix. The authors describe the generation of a circular/ring structure that pull extracellular matrix in a highly localized way, creating a constriction on the parasite accompanied by a forward movement. In addition, they characterize the movement in two knockouts of parasite myosin and adhesins. The characterization of the biophysical forces necessary for parasites to complete their life cycles is timely and necessary and relevant to improve our understanding and to design new antiparasitic strategies. I would like to praise the authors for this effort However, a few major and minor corrections and clarifications are necessary before the publication of the article.

Major Comments:

Extracellular matrix choice. The authors track the parasite movement first on Matrigel and next on fibrin. The authors exemplify the fibrin matrix on an image on Suppl. Fig 1 that shows a relatively quite large pore size, similar or greater than parasite size. Was the analysis done on parasites touching the fibers? Lack of movement of parasites. In many figures of the articles it is revealed that the majority of parasites in fibrin remain immobile (Suppl Fig 1, Fig 2, Video 5, Suppl Fig 2, Suppl Fig 8). The number of immobile parasites in Matrigel seem to be lower than in fibrin (Suppl Fig 1B) although no quantification is shown. How does the movement in fibrin and Matrigel compare? How does this compares with movement in stiff substrates in 2D? Could the lack of movement be caused by the large pore site in fibrin?. Considering parasite movement: The authors consider that 3SD is a cutoff for considering parasite displacement. However, several timepoints fall behind this cutoff in the control without parasites and the knockouts with restricted movement.

Imaging: Although the authors show a very detailed an illustrative table of the imaging acquisition conditions in table 1, it is unclear which microscope the authors used, as two microscopes are described in the methods section, a Nikon Eclipse TE300 widefield microscope and a Nikon AIR-ER confocal microscope. Which images were taken in each system? For the location of Table1 in the manuscript it seems that most images were taken with the Nikon Eclipse. Although this microscope has control over z, the images are quite noisy. How does the lack of confocallity might interfere with the analysis? Nuclear constriction. The authors did not show any image or video exemplifying this. Knockouts: The authors did not explain how did they generated the knockouts in the methods or did now show the efficacy of the knockout in any figure. If these knockout strains were a gift (I did not find it on the manuscript), the authors should indicate this more explicitly and reference the manuscript where they were described for the first time.

Discussion: Although the experimental methodology is sound the authors seem to make many assumptions and speculations on the discussion as how the appearance of this ring/constriction on the parasite translates into the helical movement of the parasite or the coupling of the ring with the cytoskeleton. Live imaging of actin dynamics or mathematical modelling could be used to support their claims.

Minor comments:

Quantification of experiments missing: Overall, the main figures lack quantification that sometimes can be found in the supplemental information and sometimes is missing. I would suggest including quantifications next to the events described in the main figures). Likewise, some of the supplemental figures lack quantification (Suppl Fig 7, how many parasites showed this protein trail?).

Overall, the authors should indicate how many parasites were quantified in each figure. As they usually refer to number of constrictions. This is overall a problem in main figures 3 and 5. Or for example in Suppl Fig 5: How many parasites were quantified in this figure? The authors only show number of constrictions, and as the authors described, a parasite might have more than one constriction.

Videos: The videos lack scale of time. Although this that can be found in main figures, it would be helpful to have the anotation in the videos. Likewise, some references for positions in videos, such as the cross found on Fig1 would be helpful for parasites that present little movement.

Significance

Host-parasite interactions are driven by a combinations of biochemical and mechanical factors, but most research focuses on the molecular side. This article aims to better define the mechanical properties behind Toxoplasma movement. This is important, because understanding the biophysical determinants behind parasite movement is essential and has been historically ignored. To my knowledge, this manuscript is among the few that aim to define the physical cues driving toxoplasma movement.

Although the article is focused on the mechanobiology of Toxoplasma interactions with the extracellular matrix, the article is easy to read and accessible to molecular and cellular parasitologists/biologists.

My background covers host-parasite interactions in 3D bioengineered models. This review has been done together with an expert in mechanobiology. Most of the article falls behind our expertise except for computational modelling of single cell displacements.