!- limitations : human - Chomsky points out something very simple but profound - It is the same thing taught by Nagarjuna - A thing or process once named or positively defined by observable properties, is also negatively defined - once we have one ability, it also rules out countless other abilities

Reviewer #3 (Public Review):

The manuscript presents a theory of generalization performance in deterministic population codes, that applies to the case of small numbers of training examples. The main technical result, as far as I understand, is that generalization performance (the expected classification or regression error) of a population code depends exclusively on the 'kernel', i.e. a measure of the pairwise similarity between population activity patterns corresponding to different inputs. The main conceptual results are that, using this theory, one can understand the inductive biases of the code just from analyzing the kernel, particularly the top eigenfunctions; and that sample-efficient learning (low generalization performance with few samples) depends on whether the task is aligned with the population's inductive bias, that is, whether the target function (i.e. the true map from inputs to outputs) is aligned with the top eigenfunctions of the kernel. For instance, in mouse V1 data, they show that the top eigenfunctions correspond to low frequency functions of visual orientation (i.e. functions that map a broad range of similar orientations to similar output value), and that consistent with the theory, the generalization performance for small sample sizes is better for tasks defined by low frequency target functions. In my opinion, perhaps the most significant finding from a neuroscience perspective, is that the conditions for good generalization at low samples are markedly different from those in the large-sample asymptotic regime studies in Stringer et al. 2018 Nature: rather than a trade-off between high-dimensionality and differentiability proposed by Stringer et al, this manuscript shows that in the low-sample regime such codes can be disadvantageous for small sample sizes, that differentiability is not required, that the top eigenvalues matter more than the tail of the spectrum, and what matters is the alignment between the task and the top eigenfunctions. The authors propose sample-efficient learning/generalization as a new principle of neural coding, replacing or complementing efficient coding.

Overall, in my opinion this is a remarkable manuscript, presenting truly innovative theory with somewhat limited but convincing application to neural data. My main concern is that this is highly technical, dense, and long; the mathematical proofs for the theory are buried in the supplement and require knowledge of disparate techniques from statistical physics. Although some of that material on the theory of generalization is covered in previous publications by the authors, it was not clear to me if that is true for all of the technical results or only some.

Fixed population code, learnable linear readout: the authors acknowledge in the very last sentences of the manuscript that this is a limitation, given that neural tuning curves (the population neural code) are adaptable. I imagine extending the theory to both learnable codes and learnable readouts is hard and I understand it's beyond the scope of this paper. But perhaps the authors could motivate and discuss this choice, not just because of its mathematical convenience but also in relation to actual neural systems: when are these assumptions expected to be a good approximation of the real system?

The analysis of V1 data, showing a bias for low-frequency functions of orientation is convincing. But it could help if the authors provided some considerations on the kind of ethological behavioral context where this is relevant, or at least the design of an experimental behavioral task to probe it. Also related, it would be useful to construct and show a counter-example, a synthetic code for which the high-frequency task is easier.<br /> Line 519, data preprocessing: related to the above, is it possible that binning together the V1 responses to gratings with different orientations (a range of 3.6 deg per bin, if I understood correctly) influences the finding of a low-frequency bias?

I found the study of invariances interesting, where the theory provides a normative prediction for the proportion of simple and complex cells. However, I would suggest the authors attempt to bring this analysis a step closer to the actual data: there are no pure simple and complex cells, usually the classification is based on responses to gratings phases (F1/F0) and real neurons take a continuum of values. Could the theory qualitatively predict that distribution?

terrible analogy is terrible—and I say this as someone who doesn't even fall in line with the general anti-copyright sentiment that is prevalent on sites like HN and Reddit

This is the kind if definition of optimism I have in mind when thinking about how I try to approach the world. Combined with a (hopefully!) well balanced sense of Humour, a bit of stubbornness and the kind of “naivetë” [[Mihaly Csikszentmihalyi]] described in his opus magnum “Flow” I am convinced it’s, in the long run, a quite unstoppable combination and quality that can, in fact, be trained and developed.

Involve people in change, and allow them to make choices.

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Reply to the reviewers

1. General Statements

We thank the reviewers for their critical analysis of our manuscript. We have addressed all reviewer concerns and questions in our revised version. Along with other improvements requested by the reviewers, we added an MTT assay to validate our flow cytometry assays, normalized binding to surface area to better compare toxin binding between Leishmania and HeLa cells, and revised the discussion. We believe the revised contribution provides important novel insights into membrane integrity in a non-standard organism that will appeal to a broad audience.

Reviewer comments below are in italics.

Point-by-point description of the revisions

Reviewer 1

*Major Comments. The experimental work has been carried out carefully, including multiple biological replicates, convincing statistical analysis. Data presentation is extensive, including 6 supplementary figures. It is likely that the experiments could be reproduced by others, as the approaches do not seem to be especially difficult, and the methods are well documented. *

We thank the reviewer for this assessment.

*My major comment regarding revision is that this paper is quite long and extensive given the relatively restricted body of experiments and discrete conclusions. The principal discovery is that sphingolipids protect Leishmania parasites against somewhat artificial treatment with bacterial sterol-binding pore forming toxins, but they do not do so by obstructing toxin binding to sterols. A similar effect is seen for the antileishmanial drug amphotericin B, the most important agent studied. No further mechanistic insights are provided regarding the process whereby sphingolipids blunt toxicity of either the CDCs or amphotericin B. In addition, the experimental approach relies largely upon one methodology, dose-response curves. A report with such highly focused scope should be presentable with considerably more economy. In particular, the Discussion is long and diffuse, obscuring the presentation of the major conclusions. It could probably be cut in half and would in the process present the major deliverables of the paper with higher impact. *

We have condensed the discussion as requested, and to address Reviewer 2’s concerns, we provided a summary articulating the significance.

Significance

*The most notable advance is the observation that sphingolipids protect Leishmania parasites from the cytotoxic activity of the first line antileishmanial drug amphotericin B that binds to the major sterol in the parasite plasma membrane, ergosterol, and induces pore formation. This discovery suggests that parallel treatments with agents that selectively reduce sphingolipid levels in the parasite might act synergistically with amphotericin B, potentially allowing treatment with lower doses of this inherently toxic drug. This work will likely be of most interest to those with a focus on pharmacology and drug development for this and related parasites, but it will also be of some interest to those working on the basic biochemistry of these organisms. The senior authors are major workers in sphingolipid biochemistry in Leishmania parasites and thus are well positioned to address the relevant background in the field, much of which has come out of their laboratories.

The major limitation of this study is its relatively circumscribed scope, resulting in one principal conclusion: Leishmania sphingolipids blunt the potency of toxins or drugs that target sterols for pore formation, but they do not do so by impairing binding of these agents to sterols, as they do in mammalian cells. The work would be of higher impact if it addressed mechanistically how sphingolipids do decrease toxicity, e.g., do they prevent these agents from oligomerizing or from intercalating into the membrane to form pores. Such studies would require the application of an expanded repertoire of experimental methodologies going beyond the measurement of dose-response curves with various mutants and drugs.*

We agree with the reviewer that next steps include determining if Leishmania sphingolipids interfere with oligomerization or pore-insertion. One challenge is that these tools need to first be validated in Leishmania.

To address the reviewer concern about the limited range of experimental methodologies, we added an MTT assay (Supplementary Fig S2E) as validation of our flow cytometry assays. We have better summarized the significance and broad impact of our work in lines 466-476.

Reviewer 2

*In the abstract the authors describe that the pore-forming toxins engage with ceramide and other lipids and while it's clear that the levels of sphingolipids are important for the effect of these toxins there is limited evidence to show they physically interact as the word engage suggests. *

We agree with the reviewer that we do not show physical interaction. In the abstract, we are careful to only use the word “engage” in association with our proposed model. Our proposed model both explains our data, and uses those data to open new horizons by making falsifiable predictions that can be tested in the future. Direct engagement of toxins with lipids is one such prediction. For these reasons, we prefer to retain the word “engage” in the abstract.

*The authors conclude that the ergosterol on the Leishmania cell membrane is less accessible to the CDCs as it does not bind as much CDCs as a HeLa cell. What is the relative abundance of sterols in the HeLa membrane in comparison to a Leishmania cell. A HeLa cell is much bigger than a Leishmania cell and will therefore be able to bind a lot more CDC, was the MFI normalised for cell size? This would be important to know as the difference in intensity may be purely related to the difference in cell size. *

We thank the reviewer for this insight. We had not normalized MFI by cell surface area. We added MFI normalized to cell size (described on lines 573-577) and found that when area was accounted for, the promastigotes bound more toxin than HeLa cells. These data are now included as Supplementary Fig S1A, and discussed on lines 187-189.

*The authors are keen to prosecute that ceramide is important for differences between PFO and SLO action as the inhibitor has a much greater effect on the PFO treatment of ipcs- cells than SLO, as ceramide will accumulate in these cells. But for the SLO analysis they stated that the treatment of spt2- with myriocin had no change on the LC50 as the target of myriocin was spt2 while they noted was there a drop in the LC50 with PFO. Based on this I think the importance of ceramide is being overstated here, as spt2- cells have little ceramide in them. Moreover the authors also suggest that changes to the lipid environment rather than a single species might be important. Are there alternative targets the myriocin might inhibit when there is no spt2-, it is intriguing that there is a decrease in LC50 for PFO on spt2- myriocin treated cells. *

Clearly, IPC is very important for determining the cytotoxicity for the CDCs in Leishmania but I think the evidence for the role of ceramide and the sensing of it is less clear cut and the strength of the conclusions about this should be modified. In the results the authors conclude that the L3 loop is sensing ceramide and the data shows that the L3 loop is important but in the discussion they are more circumspect about the moieties L3 can detect. The authors should qualify these conclusions in the results a bit more.

As requested by the reviewer, we have qualified our statements in the results, lines 282, 297, 315.

*Minor comments *

*It would be helpful for the review process to include line and page numbers to highlight areas that I have concerns about. *

We agree with the reviewer and have added line numbers.

*In the first paragraph of the results is there a reference for the spt2- cell line that was used here. *

We have added the Zhang 2003 reference to the first paragraph of the results, line 161.

*In the second paragraph there is a disconnect between the statements about the phenotype of the ipcs- cells and the reference/evidence for it. *

We have added the reference to the earlier mention of the ipcs cells, and in the introduction, lines 118-120 and 167-169.

*On many of the graphs the letters a, b, c are alongside many of the symbols but it was unclear what they represented. *

The letters represent statistically distinct groups. These are used instead of stars and bars to reduce clutter on the figure. We have now explained the difference in the first figure legend in which they are used, lines 818-823.

*The colour scheme for figure 4 was confusing - yellow diamonds in A/B are spt2-/+spt2 but in C/D are iscl-, this makes it hard to compare between them. *

We have changed the color and symbols for the iscl- mutant in Fig 4 and Fig S6.

*The methodology states that various tests were used to define whether differences were significant but it was not clear from the figures when these were being applied only a few graphs had '*' associated with them. *

We have clarified this in the figure legends.

*There is no overall conclusion to the study at the end of the discussion just a series of limitations of the study, which is good to acknowledge but feels an odd way to finish the manuscript. *

We have revised the discussion in response to Reviewer 1, and included a summary to tie everything together, lines 466-476.

*Significance: *

Overall this is a strong manuscript with a set of experiments that have a clear strategy and purpose that was well written. This paper outlines the importance of the lipid composition for the cytotoxicity of both sterol specific toxins and amphotericin B in Leishmania, which will have significant implications for their study for other pathogens but also for the development of combination therapies to enhance the potency of amphotericin B, as such I think this will be of interest to both researchers interested in drug discovery and those interested in lipid metabolism.

We thank the reviewer for this assessment.

Reviewer 3

Major comments: 1) The idea that sphingolipids do not block toxin access relies on the work of CDC-based probes binding the accessible pool of cholesterol in mammalian membranes. The authors make the observation that ergosterol is not shielded by sphingolipids because the presence of them does not prevent CDC binding. Is it possible to show that Leishmania sphingolipids are able to actually sequester ergosterol or would it all be considered free and available to toxin binding?

Our interpretation of the binding data is that the Leishmania sphingolipids fail to sequester ergosterol from toxins, so ergosterol accessibility is independent of sphingolipids. Similar to mammalian cells, there could be an “essential” pool of ergosterol bound to other proteins/lipids that is inaccessible to toxins. However, detecting that pool is technically challenging.

We have revised the manuscript to clarify this, lines 454-456.

* 2) The statistical analysis applied to each experiment, while defined in the figure legends, are presented mostly using uncommon methods of presentation, making it difficult to determine if the correct analysis was applied.*

We have clarified the statistics and use of letters. The letters represent statistically distinct groups. These are used instead of stars and bars to reduce clutter on the figure. We have now explained the difference in the first figure legend in which they are used, lines 818-823.

* 3) The binding of these toxins to Leishmania cells appears to be independent of their lipid composition, but Figure 1A-D suggests that these toxins do not bind very well to Leishmania; a ~65 fold increase in toxin added only results in a maximal 3 fold change in amount of toxin bound. Therefore, the authors need to demonstrate that this increase in binding is not simply the result of adding more ug of each CDC. *

Leishmania are smaller than HeLa cells, which accounts for the apparent reduced binding. We added Supplementary Fig S1A, which normalized MFI to estimated surface area. When normalized to surface area, Leishmania bound to toxin better than HeLa cells. We further note that the dose-dependent increase in cytotoxicity argues against non-specificity of increased toxin.

* 4) The authors use HeLa cells to compare the ability of these toxins to bind to sterol containing membranes, but it is unclear how a mammalian cell line, which lacks ergosterol, can inform upon the differences in binding to Leishmania membranes when their data shows almost no cholesterol is found in the Leishmania membrane. The use of HeLa cells to compare the toxicity of these CDCs is simply a control experiment for the lytic activity of these proteins, and should not be used as a direct comparison of their LC50s, as a mammalian plasma membrane lipid composition is significantly different from that of Leishmania. If the authors want to use HeLa cells as a direct comparison to show that sphingolipids in mammalian cells also protect them from CDC pore formation, they must demonstrate the HeLa cells which have genetic defects in sphingolipid biology or which have been treated with sphingomyelinases are more sensitive to these CDCs. *

We agree with the reviewer that to argue sphingolipids in mammalian cells are protective would require additional data beyond the scope of this manuscript. We are not making any statements about the role of sphingolipids in mammalian cells, which have a controversial role in CDC damage and membrane repair (see e.g. Schoenauer et al 2019. PMID: 29979630). Since the head group of sphingomyelin interacts with cholesterol (Endapally et al 2019), but the IPC head group is not expected to interact similarly with ergosterol, we choose to remain focused on Leishmania sphingolipids.

Given our focus on Leishmania, why include HeLa cells at all? We think including HeLa cells provides an important and relevant point of reference because there are situations where both human cells and Leishmania promastigotes could encounter pore-forming toxins. This comparison provides insight to the following question: “In a mix of promastigotes and human cells (for example during a blood meal), which cells would die first from the bacterial PFT?” Comparing cytotoxicity to HeLa cells provides a point of reference in judging how cytotoxic CDCs are to Leishmania promastigotes, and how sensitive the spt- promastigotes become.

We have rephrased the manuscript (lines 208-209) to better clarify that HeLa cells are a reference point so readers can evaluate the relative sensitivity of sphingolipid-deficient promastigotes.

* 5) The authors need to demonstrate that the mutant cholesterol recognition motif (CRM) and the glycan binding mutant proteins can still bind to both Leishmania and Hela cell membranes to serve as controls for their lack of lytic activities. Without this, they cannot conclude that "Leishmania membranes engage the same binding determinants used by CDCs to target mammalian cells". *

The glycan binding and ΔCRM mutants are unable to bind to HeLa cells. These toxin mutations were previously characterized (Mozola & Caparon, 2015 and Farrand et al 2010), showing that their defect lies in binding to cells, but not oligomerization or pore-formation. Since their defect lies solely in binding, if these toxins were able to bind to spt2- cells, they would kill the spt2- cells. This enables us to use these toxin mutants to ask if the CRM or glycan-binding is essential for toxin binding to Leishmania. Since the only defect in these mutant toxins is binding (either to glycans or cholesterol), the failure of these mutants to kill allows us to conclude that both of these binding surfaces on the toxin are essential for cytotoxicity in L. major.

We have clarified the manuscript, lines 236-240. *

Minor comments: 6) Multiple figures lack adequately defined axes. Examples include, but are not limited to: Figure 1A-D where the X-axis is plotted as logarithmic based 2 but this is not defined. Figure 2 the Y axis is plotted as logarithmic based 10 but is not defined. *

We have updated the figure legends to indicate where log axes are used.

7) The authors state that "Promastigotes with inactivated de novo sphingomyelin synthesis has a significant increase in total sterols" in reference to Figure 1E. Not only is there no significance indicated for the spt2-/-, the authors only indicate a significance point for the Myr (not yet defined) + WT sample in "Other sterols".

We have rephrased this to indicate a trend, line 181.

8) The authors use increases in membrane permeability as a read out for specific lysis using PI uptake, however, they then refer to this read out as killing of Leishmania, without measuring the viability of these cells. Therefore, the authors should provide additional experiments that demonstrate the death of the different Leishmania strains treated with the cytolysins.

As requested, we have now provided an additional experiment to validate Leishmania death. We have now added MTT assay as Fig S2E, and discussed in the results, lines 202-205.

9) It is not clear how the authors calculated their LC50 values in Figure 2. According to the figure legends, the authors used HU/ml ranges that would be sub lethal or not completely lysed within this range to most of the Leishmania strains tested. The data presented in Figure are not clear that the correct LC50 calculations were used as none of the Specific Lysis curves do not reach saturation with the concentrations presented, and one does not even reach 50% Lysis.

We thank the reviewer for catching this discrepancy. The legend in Fig 2 did not include the correct ranges of toxin dose used for PFO. We have corrected the legend to indicate the toxin range used. To calculate LC50, we used linear regression on the linear portion of the death curve to determine the concentration at 50% lysis. This gives us a way to determine LC50 even without the use of very large (and costly) amounts of toxin to get extensive saturation on the kill curve.

* 10) Figure 4 and Figure S6 are very difficult to interpret. Figure S6 would benefit by breaking up each graph into multiple graphs that would allow the reader to see more of the curves individually. Additionally, there are multiple conditions were it appears that a different number of experiments (2-4 totals) were preformed but statistical analysis was applied to these data. *

We updated the labels on Fig 4 for improved readability. We broke Fig S6 up into multiple graphs. We have removed unpaired data (eg the n of 4 noted by the reviewer), and re-checked our stats. This change did not alter our conclusions. The apparent n of 2 was overlap of data points due to poor jittering of the datapoints. We have increased the jitter on the data points to make all three reps more distinct.

* 11) The authors state "In contrast to myriocin-treated ipcs- L. major, which contain low levels of ceramide, myriocin treated iscl- L. major contain low levels of IPC" but do not provide a reference or point to data to support this claim. *

We have qualified these statements to say ‘are expected to’ on lines 306-307.

* 12) Figure 5 E would benefit in presentation by being broken up into 4 separate graphs based on the toxin used, as it is difficult to determine which data points are being compared. *

We compare by toxin used in Fig 5A-D. The purpose of Fig 5E is to compare between toxins. We included all of the data points (including resistant control strains) for completeness. The main focus is the spt2- and ipcs- parts of Fig 5E.

* 13) The authors state that "myriocin did not inhibit growth more than 25% promastigotes at 10 μM" but this data is not presented. *

We have now added these data as Fig 6A.

14) Multiple graphs lack legends or have axis that are not defined.

In order to improve readability and avoid cluttering the figures, where the legends and axes are the same across multiple graphs, they are included only once for a given row and/or column.*

Significance:

Overall, the experiments presented were conducted to analyze each question, but many of the results are observational, without considering the impact of altered lipid species on the findings. The data suggests an existence of a protective mechanism for the parasite from CDCs, but it unclear how these finding inform upon the CDC or Leishmania fields. CDCs have been known to target sterols within membranes and that altered local membrane environments can have substantial impacts on CDC binding. This work suggests that the altered lipid species of Leishmania membranes, compared to a mammalian membrane, could dramatically effect the sequestering power of sphingolipids or other lipids, and thus change how CDCs bind to them. This work advances is likely to have specialized audience of Leishmania researchers looking at the dynamics of their membranes.*

We believe this work will be valuable to a broad audience because it will be of interest to researchers studying membranes in general, pathogenic eukaryotes and pore-forming toxins. Most membrane biology work is done either in opisthokonts or in model liposomes, so there are few studies on biomembranes in other taxonomic groups, including many different human pathogens. We provide a blueprint for examining the membranes of non-standard organisms, establish L. major as a pathogenically relevant model system, and report on key differences in sterol sequestration compared to mammalian cells. These findings provide important perspectives for the generalization of biomembranes, especially when compared to prior work in opisthokonts.

We have clarified our significance in lines 466-476.

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Referee #2

Evidence, reproducibility and clarity

Summary

One of the major treatments of the parasitic disease Leishmaniasis is the drug amphotericin B, which targets ergosterol and a route to increasing its potency would be to increase the accessibility ergosterol on the surface of the parasite. With that in mind the authors investigated sterol-binding ability of two cytotoxic toxins PFO and SLO. The authors clearly show that these toxins are readily able to bind to the surface of the parasite regardless of the levels of sphingolipids present, yet the presence of inositol phosphorylceramide and to a lesser extent ceramide affect overall cytotoxicity. The L3 loop of the toxin was shown to be important for the sensitivity of the different toxins to the lipid composition of the membrane.

Major comments

In the abstract the authors describe that the pore-forming toxins engage with ceramide and other lipids and while it's clear that the levels of sphingolipids are important for the effect of these toxins there is limited evidence to show they physically interact as the word engage suggests.

The authors conclude that the ergosterol on the Leishmania cell membrane is less accessible to the CDCs as it does not bind as much CDCs as a HeLa cell. What is the relative abundance of sterols in the HeLa membrane in comparison to a Leishmania cell. A HeLa cell is much bigger than a Leishmania cell and will therefore be able to bind a lot more CDC, was the MFI normalised for cell size? This would be important to know as the difference in intensity may be purely related to the difference in cell size.

The authors are keen to prosecute that ceramide is important for differences between PFO and SLO action as the inhibitor has a much greater effect on the PFO treatment of ipcs- cells than SLO, as ceramide will accumulate in these cells. But for the SLO analysis they stated that the treatment of spt2- with myriocin had no change on the LC50 as the target of myriocin was spt2 while they noted was there a drop in the LC50 with PFO. Based on this I think the importance of ceramide is being overstated here, as spt2- cells have little ceramide in them. Moreover the authors also suggest that changes to the lipid environment rather than a single species might be important. Are there alternative targets the myriocin might inhibit when there is no spt2-, it is intriguing that there is a decrease in LC50 for PFO on spt2- myriocin treated cells.

Clearly, IPC is very important for determining the cytotoxicity for the CDCs in Leishmania but I think the evidence for the role of ceramide and the sensing of it is less clear cut and the strength of the conclusions about this should be modified. In the results the authors conclude that the L3 loop is sensing ceramide and the data shows that the L3 loop is important but in the discussion they are more circumspect about the moieties L3 can detect. The authors should qualify these conclusions in the results a bit more.

Minor comments

It would be helpful for the review process to include line and page numbers to highlight areas that I have concerns about.

In the first paragraph of the results is there a reference for the spt2- cell line that was used here.

In the second paragraph there is a disconnect between the statements about the phenotype of the ipcs- cells and the reference/evidence for it.

On many of the graphs the letters a, b, c are alongside many of the symbols but it was unclear what they represented.

The colour scheme for figure 4 was confusing - yellow diamonds in A/B are spt2-/+spt2 but in C/D are iscl-, this makes it hard to compare between them.

The methodology states that various tests were used to define whether differences were significant but it was not clear from the figures when these were being applied only a few graphs had '*' associated with them.

There is no overall conclusion to the study at the end of the discussion just a series of limitations of the study, which is good to acknowledge but feels an odd way to finish the manuscript.

Significance

Overall this is a strong manuscript with a set of experiments that have a clear strategy and purpose that was well written. This paper outlines the importance of the lipid composition for the cytotoxicity of both sterol specific toxins and amphotericin B in Leishmania, which will have significant implications for their study for other pathogens but also for the development of combination therapies to enhance the potency of amphotericin B, as such I think this will be of interest to both researchers interested in drug discovery and those interested in lipid metabolism.

Expertise in the molecular cell biology of trypanosomes and leishmania.

this mechanism can be defeated by paraphrasing the output with another model

I think the emphasis is wrong here. The regulation is secondary. The long tail of weird events is the more important thing.

the stochastic parrots argument could be defeated as models get bigger and more complex

Author Response

Reviewer #1 (Public Review):

Sayin et al. sought to determine if bacterial drug resistance has impact on drug efficacy. They focused on gemcitabine, a drug used for pancreatic cancer that is metabolized by E. coli. Using an innovative combination of genetic screens, experimental evolution, and cancer cell co-cultures to reveal that E. coli can evolve resistance to gemcitabine through loss-of-function mutations in nupC, with potential downstream consequences for drug efficacy.

Major strengths include:

• Paired use of genetic screens and experimental evolution

• The spheroid model is a creative approach to modeling the tumor microbiome that I hadn't seen before

• Rigorous microbiology, including accounting for mutation rate in both selective and non-selective conditions

• Timely research question

Major weaknesses of the methods and results include the following:

1) Limited scope of the current work. Just a single drug-bacterial pair is evaluated and there are no experiments with microbial communities, animal models, or attempts to test the translational relevance of these findings using human microbiome datasets.

We agree with the reviewer that uncovering evidence from human microbiome datasets will be very exciting and complementary to our study. However, since gemcitabine is administered intravenously it’s unclear whether it will impose a considerable selective pressure on the gut microbiome. Therefore, it also remains unclear if adaptive mutations, as those we identified, are expected to be found in datasets for the gut microbiome. While metagenomics datasets that are bacterial-centric of infected pancreatic tumors will be ideal for addressing the reviewer’s suggestion, they do not exist to the best of our knowledge. It should be noted however, that our work generated hypotheses that can be tested in pancreatic tumor tissues infected with gammaproteobacteria and can be tested in the future by targeted sequencing for the specific genes of interest (e.g, nupC and cytR).

2) No direct validation of the primary genetic screen. The authors use a very strict cutoff (16-fold-change) without any rationale for why this was necessary. More importantly, a secondary screen is necessary to evaluate the reproducibility of the results, either by testing each KO in isolation or by testing a subset of the library again.

We used a strict cutoff to allow the reader to focus on a manageable list of gene names in the main figure (2E). To partly address this limitation in scope, we also included results from pathway enrichment analysis in the same figure (2F). This analysis utilizes all enrichment values and is therefore independent from any choice of cutoff value. We also now refer the reader to explore more of the hit genes in the supplementary information (line 152).

As the reviewer suggested we evaluated the reproducibility of the results by performing two validation screens. The first validation screen was performed as a biological replicate of the original screen and relied on the original collection of knockouts strains. The second validation screen was performed with a knockout strain collection that was cloned independently from the strains used in our original screen. The results from these two completely independent biological replicates are presented on supp. figure 1D. The results (resistance/sensitivity) from the two screens are highly correlated. We refer to this comparison in the main text (lines 142-147).

3) Some methodological concerns about the spheroid system. As I understood it, these cells are growing aerobically, which may not be the best model for the microbiome. Furthermore, bacterial auxotrophs are used and only added for 4 hours, which will really limit their impact. It also was unclear if the spheroids are truly sterile. Finally, the data lacks statistical analysis, making it unclear which KOs are meaningful. Delta-cdd looks clearly distinct by eye, but the other two genes are more subtle.

The 4 hour time interval chosen to address two opposing requirements of the co-culture system – mitigate overgrowth of the bacterial cultures (which hinders spheroid growth irrespective of the drug) while still allowing enough incubation time to allow for drug degradation. As the reviewer notes, removal after 4 hours may limit the bacteria impact. However, such a limitation will only result in underestimation the bacterial impact (but will have no impact on how we evaluate how strains compare to one-another). We now comment on this in the methods section (lines 699-705).

We do not expect the spheroid to remain infected after bacterial removal since we treat spheroids with antibiotics. We didn’t not detect any bacterial growth in the 7 days post infection in the microscope and we did not observe influence on spheroid growth when compared to spheroid that were not infected. Growth of spheroid before infection was performed w/o antibiotics and we did not detect any evidence of bacterial growth prior to introducing the bacteria intentionally (the cell-line itself was also tested for animal pathogens and bacterial contamination prior to the experiments).

We repeated the spheroid experiments and observed similar shifts in the EC50 fronts. We now include these replicates as supplementary figure 7. We comment on these replicates in the main text (lines 273-274).

Maybe this is just a regional thing, but something I really began to notice several years ago is that (a) what Jamie is saying is true, but (b) it's evident that people actually love the unfalsifiability aspect of non-concrete claims—it's safe refuge that they actively seek out.

Two opposing examples from real life that particularly stuck out: * "Slow down. [You're going too fast.]" * "[...] since you always take so long"

(These were two different instances/contexts with, I think, a year+ in between them; it wasn't the contrast between them that made me notice the phenomenon. Rather, I recognized each one at the time as being part of this phenomenon. They just serve as good examples because of how easily they could be made concrete—and therefore falsified in light of the facts: "without defining exactly what 'too fast' means, what is an acceptable speed?", "without defining what it means to take too long, what is an acceptable amount of time to take?"—both arising from wholly disingenuous complaints that were forms of externalized gut reactions rather than anything that would hold up under scrutiny...)

I'm trying to give some context here. I'm setting the scene trying to emphasize that it was just a normal day. Details, such as the "basement of my dorm" and the "2 long hours" are meant to help the reader understand that it was just a regular day of homework. I tried to be descriptive in the first half of the paragraph so the reader could put themself in my shoes. Most people can relate to grinding out homework (or work) and just going through the motions.

This piece is an essay that I wrote for the Stacks exploration essay. In this piece, we were tasked to provide specific detail on our journey through the library. I'm proud of the concrete specific detail shown in this essay and the clear narrative arc that shows my journey around the library

The creativity I was talking about previously stems from these next few sentences. I like that I decided to embody William and his positioning when saying, “It’s almost as if he’s been looking towards the past instead of the opportunities in front of him. Maybe much like I have.” I liked that I could tie the previous concrete details of him sitting away from my window to something larger, like my college transition. I thought that was pretty creative. In my previous writing history, creative writing was always put on the back burner. I have probably written 100 of the same cookie-cutter literary analysis essays with the same boring tone. Before this essay, however, I had perhaps written five creative works for a class. Honestly, I was just surprised that I had these creative thoughts within me, but I was proud that I did. It was a validating moment for me.

Who is the first scholar? Or is this just an inadvertent duplication of the same fact?

Thus far, Scheper has only been making the argument to do as I say because I know best. He's not laying out the reasons or affordances that justify this approach.

In this second principle it's ostensibly just "the possibility of linking", but this was broadly done previously using subject headings or keywords along with indices and page numbers which are logically equivalent to these note card addresses.

How was this not obvious from the jump? Was the author of the essay so distracted by shiny object syndrome they failed to see the obvious?

It's like taking notes in a language class... the goal is to read and write with fluency, so you practice these things regularly and gain fluency over time. Taking notes about the grammar and syntax of a language is highly unlikely to get you to fluency. Note taking is the wrong tool for a number of processes and the user should very quickly get a gut feeling for what's useful and what is not.

This author obviously missed the boat here.

Author Response

Reviewer #1 (Public Review):

1) This study performs an interesting analysis of evolutionary variation and integration in forelimb/hand bone shapes in relation to functional and developmental variation along the proximo-distal axis. They found expected patterns of evolutionary shape variation along the proximo-distal axis but less expected patterns of shape integration. This study provides a strong follow-up to previous studies on mammal forelimb variation, adding and testing interesting hypotheses with an impressive dataset. However, this study could better highlight the relevance of this work beyond mammalian forelimbs. The study primarily cites and discusses mammalian limb studies, despite the relevance of the suggested findings beyond mammals and forelimbs. Furthermore, relevant work exists in other tetrapod clades and structures related to later-developing traits and proximo-distal variation. Finally, variations in bone size and shape along the proximo-distal axis could be affecting evolutionary patterns found here and it would be great to make sure they are not influencing the analysis/results.

We appreciate the reviewer’s comments, and we acknowledge the importance of including examples of non-mammalian lineages in our study. We attended to the recommendation and included more examples of other tetrapod taxa in our text and in our references, providing a more inclusive discussion of limb bone diversity beyond mammals. We also explain below why the results obtained are not inflated by variation of bigger versus smaller sizes of bones.

Reviewer #2 (Public Review):

10) Congratulations on producing a very nice study. Your study aims to examine the morphological diversity of different mammalian limb elements, with the ultimate goal seemingly to test expectations based on the different timing of development of the limb bones. There's a lot to like: the sample size is impressive, the methods seem appropriate and sound, the results are interesting, the figures are clear, and the paper is very well written. You find greater diversity and integration in distal limb segments compared to proximal elements, and this may be due to the developmental timing and/or functional specialization of the limb segments. These are interesting results and conclusions that will be of interest to a broad readership. And the large dataset will likely be valuable to future researchers who are interested in mammalian limb morphology and evolution. I have one major concern with how you frame your discussion and conclusions, which I explain below. But I think you can address this issue with some text edits.

We sincerely thank the reviewer for his constructive recommendations and for his appreciation of our work. We addressed the issue raised as detailed below.

11) Major concern - is developmental timing the best hypothesis?

You discuss two potential drivers for the relatively greater diversity in distal elements: 1) later development and 2) greater functional specialization. Your data doesn't allow you to fully test these two hypotheses (e.g. you don't have detailed evo-devo data to infer developmental constraints), and I think you realize this - you use phrases like "consistent with the hypothesis that ...". You seem to compromise and conclude that both factors (development + function) are likely driving greater autopod diversity (e.g. Lines 302-306). Being unable to fully test these hypotheses weakens the impact of your conclusions, making them a bit more speculative, but otherwise, it isn't a critical issue.

But my concern is that you seem to favor developmental factors over functional factors as the primary drivers of your results, and that seems backwards to me. For instance, early in the Abstract (Line 32) and early in the Discussion (Line 201) you mention that your results are consistent with the developmental timing hypothesis, but it's not until later in the Abstract or Discussion that you mention the role of functional diversity/specialization/selection. The problem with favoring the development hypothesis is that your integration results seem to contradict that hypothesis, at least based on your prediction in the Introduction (Line 126; although you spend some of the Discussion trying to make them compatible). Later in the paper, you acknowledge that functional specialization (rather than developmental factors) might be a better explanation for the integration results (Lines 282-284, 345-347), but, again, this is only after discussions about developmental factors.

When you first start discussing functional diversity, you say, "high integration in the phalanx and metacarpus, possibly favoured the evolution of functionally specialized autopod structures, contributing to the high variation observed in mammalian hand bones." (Line 282). This implies that integration led to functional diversity in the autopod. But I'd flip that: I think the functional specialization of the hand led to greater integration. Integration does not result solely from genetic/developmental factors. It can also result from traits evolving together because they are linked to the same function. From Zelditch & Goswami (2021, Evol. & Dev.): "Within individuals, integration is customarily ascribed to developmental and/or functional interdependencies among traits (Bissell & Diggle, 2010; Cheverud, 1982; Wagner, 1996) and modularity is thus due to their developmental and/or functional independence."

In sum, I think your results capture evidence of greater functional specialization in hands relative to other segments. You're seeing greater 1) disparity and 2) integration in hands, and both of those are expected outcomes of greater functional specialization. In contrast, I think it's harder to fit your results to the developmental timing hypothesis. Thus, I recommend that throughout the paper (Abstract, Intro, Discussion) you flip your discussion of the two hypotheses and start with a discussion on how functional specialization is likely driving your results, and then you can also note that some results are consistent with the development hypothesis. You could maintain most of your current text, but I'd simply rearrange it, and maybe add more discussion on functional diversity to the Intro.

Or, if you disagree and think that there's more support for the development hypothesis, then you need to make a better case for it in the paper. Right now, it feels like you're trying to force a conclusion about development without much evidence to back it up.

We thank the reviewer for his thoughtful and thorough comment. We agree that the results provided, particularly those of integration, support the hypothesis that functional specialization contributes to the uneven diversity of limb bones. We addressed the concerns by substantially changing our discussion, particularly moderating (and removing) sections on the developmental constraints and adding new arguments for other possible drivers for the diversity of limb bones, such as function. However, the goal of the paper was to test whether the data corroborate - or not - the predictions derived from the developmental hypothesis, and they largely do. Therefore, we decided to keep the developmental hypothesis presented first in the introduction and in the discussion section, as we believe this sequence provides more coherence considering the hypothesis tested (we believe that detailing the role of functional specialization particularly in the introduction would mislead the reader to think that we directly tested for these parameters). Following the discussion of the integration results, we then go on to discuss the possible role of functional specialization on the results obtained (lines 262-285, see also lines 216-234). Yet, these are not tested in this paper and remain to be tested in a future analysis focusing specifically on the role of ecology and function in driving variation in the mammalian limb.

12) Limitations of the dataset

Using linear measurements is fine, but they mainly just capture simple aspects of the elements (lengths and widths). You should acknowledge in your paper the limitations of that type of data. For example, the deltoid tuberosity of the humerus can vary considerably in size and shape among mammals, but you don’t measure that structure. The autopod elements don’t have a comparable process, meaning that if you were to measure the deltoid tuberosity then you’d likely see a relative increase in humerus disparity (although my guess is that it’d still be well below that of the autopod). And you omit the ulna from your study, and its olecranon process varies considerably among taxa and its length is a very strong correlate of locomotor mode. In other words, your finding of the greatest disparity in the hand might be due in part to your choice of measurements and the omission of measurements of specific processes/elements. I recommend that you add to your paper a brief discussion of the limitations of using linear measurements and how you might expect the results to change if you were to include more detailed measurements and/or more elements.

We followed the recommendation and included a discussion about the dataset limitations, acknowledging for the possible impact of the measurements and the bones chosen in the results obtained (Lines 235-260).

Reviewer #3 (Public Review):

32) This paper uses a large (638 species representing 598 genera in 138 families) extant sample of osteologically adult mammals to address the question of proximodistal patterns of cross-taxonomic diversity in forelimb bony elements. The paper concludes, based on a solid phylogenetically controlled multivariate analysis of liner measurements, that proximal forelimb elements are less morphologically diverse and evolutionarily flexible than distal forelimb elements, which the paper concludes is consistent with a developmental constraint axis tied to limb bud growth and development. This paper is of interest to researchers working on macroevolutionary patterns and sources of morphological diversity.

Methodological review Strengths:

The taxonomic dataset is very comprehensive for this sort of study and the authors have given consideration to how to identify bony elements present in all mammalian taxa (no small task with this level of taxonomic breadth). Multivariate approaches as used in this study are the gold standard for addressing questions of morphological variations.

The authors give consideration to two significant confounders of analyses operating at this scale: phylogeny and body size. The methods they use to address these are appropriate, although as I note below body size itself may merit more consideration.

We sincerely thank the reviewer for his appreciation of our study. We addressed the main concerns pointed out below.

Weaknesses:

33) The authors assume a lot of knowledge on the part of the reader regarding their methods. Given that one of their key metrics (stationary variance) is largely a property as I understand it of OU models, more explanation on the authors' biological interpretation of stationary variance would help assess the strength of their conclusions, especially as OU models are not as straightforward as they first appear in their biological interpretation (Cooper et al., 2016).

We acknowledge that this may not be straightforward and now include a more extensive explanation of the approach and the metrics used. We detailed the explanation about the stationary variances in the methods, contextualizing the biological meaning (lines 456-469).

34) It is unclear what the authors mean when they say they "simulated the trait evolution under OU processes on 100 datasets". Are the 100 datasets 100 different tree topologies (as seems to be the case later "we replicated the body mass linear regressions with 100 trees from Upham et al (2019)." If that is so, what is the rationale for choosing 100 topologies and what criteria were used to select the 100 topologies?

We understand the explanation may have been confusing. Globally, we used a parametric bootstrap approach to assess the uncertainty around point estimates for morphological diversity and integration. That is, we first simulated 100 datasets on the maximum clade credibility tree (MCC tree, that summarizes 10,000 trees from Upham et al. 2019) – using the best fit model on our original data (i.e., an OU process) with parameters estimates from this model fit. The model (an OU process) was then fit to these 100 simulated traits, and the distribution of parameters estimates obtained was used to assess the variability around the point estimate (for the determinant, the trace, and the measure of integration) obtained on empirical data. We did not used the simulated dataset to estimate the significance of the stationary variances. We fitted the empirical datasets with 100 trees randomly sampled from the credible set of 10,00 trees of Upham et al (2019) – instead of using the MCC – to further assess the variability due to the tree topology and branching times uncertainties. We included this expanded explanation in the methods in lines 421-428 and 471.

35) The way the authors approach body mass and allometry, while mathematically correct, ignores the potential contribution of body mass to the questions the authors are interested in. Jenkins (1974) for example argued that small mammals would converge on similar body posture and functional morphology because, at small sizes, all mammals are scansorial if they are not volant. Similarly, Biewener (1989) argued that many traits we view as cursorial adaptations are actually necessary for stability at large body sizes. Thus size may actually be important in determining patterns of variation in limb bone morphology.

We agree with the observation. We believe that categorizing the groups according to size would provide a meaningful overview on the effect of size on the diversity and evolution of limb bones. Although insightful and worthy of investigation, we were particularly interested in understanding whether developmental timing corresponds to bone diversification more broadly across Mammalia and thus considered only the size residual values. This issue will be addressed in our future works. We discussed in the lines 329-341 the potential contribution of body size to limb segment diversification and the importance of considering this aspect in future studies.

36) Review of interpretation.

The authors conclude that their result, in showing a proximo-distal gradient of increasing disparity and stationary variance in forelimb bone morphology, supports the idea that proximo-distal patterning of limb bone development constrains the range of morphological diversity of the proximal limb elements. However, this correlation ignores two important considerations. The first is that the stylopod connects to the pectoral girdle and the axial skeleton, and so is feasibly more constrained functionally, not developmentally in its morphological evolution. The second, related, issue arises from the authors' study itself, which shows that the lowest morphological integration is found in the stylopod and zeugopod, whereas the autopod elements are highly integrated. This suggests a greater tendency towards modularity in the stylopod and zeugopod, which is itself a measure of evolutionary lability (Klingenberg, 2008). And indeed the mammalian stylopod is developmentally comprised of multiple elements (the epiphyses and diaphysis) that are responding to very different developmental and biomechanical signals. Thus, for example, the functional signal in stylopod (Gould, 2016) and zeugopod (MacLeod and Rose, 1993) articular surface specifically is very high. What is missing to fully resolve the question posed by the authors is developmental data indicating whether or not the degree of morphological disparity in the hard tissues of the forelimb change over the course of ontogeny throughout the mammalian tree, and whether changing functional constraints over ontogeny (as is the case in marsupials) affect these patterns.

We thank the reviewer for sharing such an interesting reinterpretation of the results. Combined to the recommendations from the other two reviewers, we substantially changed our discussion, specially modifying the interpretation of results concerning trait integration. We discussed the possible role of the functional variation at the articulations on element integration in lines 263-285.

The fact of the matter is, since it's not possible to know what the law is until a judge or jury has determined it as a matter of fact in a court of law, it's impossible to justly enforce this provision. If a court determines something to be illegal, they'll let Reddit know. There's no need for a "consensus" of self-appointed non-lawyer reddit police to decide for people. Also, since when do the enforcers get to ultimately decide what's right or wrong without any oversight? Oh, that's right, on Reddit which was designed to be tyrannical and fascist. 🙄

This is so stupid. I have a relevant question/comment to add to the thread, but someone has decided that no more value can come of this thread - yet it's in search results, so new people are seeing it all the time.

If people don't want notifications on an old thread, they should mute notifications on it - not declare it dead because they bore easily.

One could start a new thread talking about it, but that just daisy chains the topic across multiple threads.

Reddit is dumb for having this "feature" and it originated to censor people, which is abhorrent.

Example of Brian Eno using ITunes as a digital music zettelkasten. He's got 2,800 pieces of unreleased music which he plays on random shuffle for serendipity, memory, and potential creativity. The experience seems to be a musical one which parallels Luhmann's ideas of serendipity and discovery with the ghost in the machine or the conversation partner he describes in his zettelkasten practice.

!- Futures Thinking: The value of Coal, Oil and Gas in our current industrial society - If we do away with coal, we cannot manufacture - How do we find a solution to this? - Efficacy - can we get rid of / redesign infrastructure so that we can eliminate unnecessary use of coal / oil / gas? - ie. relocalize to eliminate need for energy intensive transportation, locally produced bio-fertiilzed food production to get rid of fossil fuel fertilizers, replace 24/7 refrigerators in every home with fruit and veg underground cold cellars and only very small fridge or freezer with ultra insulation for very low energy consumption

!- Futures Thinking : summary - our generation has the most wisdom to deal with the problem, even though it is an unprecedented problem - We need diversity of opinions and perspectives. Like in evolution, that diversity will emerge an optimal solution - To consciously culturally evolve, we need to put all ideas on the table and discuss openly - An open, interpersonal, people-centered knowledge ecosystem such as Indyweb is suitable for such a process

!- Prototypes : Cosmolocal between Finland, Hawaii and Iceland - Michaux is helping a group in Hawaii learn from Finland's experiences and then both of those can be used to demonstrate to Iceland - knowledge transfer between different communities of practice - this could benefit from an interpersonal, open, cosmolocal knowledge network such as Indyweb

!- Futures Thinking : Maslow's Hierarchy framing for Manufacturing - global supply chains are very fragile and not resilient - such systems will begin to fragment as different parts become more scarce, more expensive, it affects anything downstream of the value chain - cars and computers will be produced less if microchips or the minerals that make them up become more scarce - more primitive, available, less energy dense minerals and technologies available within short distance (ie. 500 km) will come to dominate

!- Futures Thinking : Maslow's Hierarchy framing for Minerals - need frank discussion about what we need for which futures trajectory, how much actually exists - from that, the truth will emerge that our current plans are unrealistic and we will have to change trajectories to adapt

!- Futures Thinking : Maslow's Hierarchy framing for Minerals - There just isn't enough copper to meet the target of full electrification - We would need 6.2x the copper we've mined since 4000 BC. - At current mining extraction rates, it would take 180 years to mine all this material, if it existed in the first place!

!- Futures Thinking : Maslow's Hierarchy framing for Minerals - Effective recycling won't have impact until many years into the future because most of the non-fossil fuel systems have not yet been built. There will be a 10 year lag time before we have major amounts to recycle

!- Futures Thinking : Maslow's Hierarchy framing for Water - future may see us going to decentralized water systems due to energy intensity of operating current system of long networks of pipelines and pumps - sanitation and water closely linked, poor potable water leads to poor sanitation, and to increased disease burden

!- Futures Thinking : Maslow's Hierarchy framing of food - will have to greatly relocalize - autonomous of any destructive petrochemicals that result in soil sterilization/death - Green growth solution, exemplified by BASF is to use GMO technology that uses more petrochemicals, AI and robots - this is not sustainable in the long term, in fact disasterous -

! - summary: how we got here 3 reasons: cheap, abundant (until recently), high calorific value (fossil fuel) energy abundant (until recently) minerals cheap credit

All three are no longer true.

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys clarify ICE attorneys' professional incentives for pursuing cases where the non-citizen respondent is neither dangerous nor a flight risk. Specifically, courtroom experience is a key factor when assigning immigration judgeships. Therefore, ICE attorneys may choose to detain non-dangerous individuals in order to acquire trail experience necessary for career advancement.

Source Excerpt: * RI 7: I did think when Brito came down that [ICE] would perhaps release people. Because pre-Brito they would arrest somebody, you know, for a driving without a license charge. And it's still my burden to show that that guy should be let out. Even though that's all he has on his record for 20 years or whatever. I don't think [ICE released people] to the extent it really should have; they still try to put on their case even when their cases are weak. * RI 51: One of the nice things about the Boston immigration court, though, is that a lot of the trial attorneys are former district attorneys. And so they're used to like conferencing the case. [ICE attorneys] don't just come straight from law school to government. They typically have experience in a forum where they're used to meeting to communicate with the other side. * RI 69: And it is easier to get into a judgeship if you were previously in law enforcement because you've already gone through the background checks. And more court experience is one heavy factor when weighing of the immigration judgeship. And for immigration judgeships, prosecutors are always going to have more court experience than the people representing the non-citizens.

Link to Data Source: * Research Interview 7: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/PV33MN * Research Interview 51: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/2OP5YN * Research Interview 69: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/ZFK3WF

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys illuminate how they provide important value to respondents, especially those held in custody, unfamiliar with U.S. laws, and/or non-English speaking. Competent lawyers can acknowledge and effectively rebut the government's claims; without them, the government would likely ride roughshod over a respondent's argument (RI #7, #52). Capable attorneys can review the government's case and prepare appropriate responses, including composing legal strategy, piecing together a compelling story on why and how the respondent is not dangerous, and collecting evidence supporting the respondent's counterarguments (RI #23).

Source Excerpt: * RI 7: Now, post Brito, the government presents the case and talks about how terrible my client is, and all these crimes they've committed, or whatever it is they want to say. And then my response rubuts that. I would say, "Nope. That's not true. Here's what happened." Or [I would say], "Yes, I acknowledge that. But look at all the wonderful ways he's changed since then." So, you're acknowledging their argument and rebutting it. * RI 23: In a practical sense, it means that we are actually getting paperwork from the government ahead of our bond hearings, which is just magical. \(\dots\) I think my stress levels going into a hearing changed a little bit because I knew what the government had and what they were likely to say. And I knew that if they thought someone was secretly a gang member, that would be at the top of their submission rather than after I've put together my 20-minute presentation. But, you know, if I think they have anything, then I can't tell my client, "Well, let's just hang back, see what the government comes up with." I need to have proof that my client is not dangerous. I had a client who was convicted of embezzlement. It was not great sounding. Okay. And he and another family member were co-defendants and the other family member had, to my mind, done a great deal more. More bad guy stuff than my client. And so I'm not going to let the government say, "Look, he's got a federal conviction." I have to think through what motivates my client has to stay here. I have to think through my argument that my guy has relief. How am I going to map out for the judge that that relief is not a pipe dream? [I have to explain,] "No, your honor. It's gonna be like this, he's gonna do A, he's gonna do B, he's going to do C. And you're gonna see that he's eligible for D." So I have to have that tight. The burden might be on the government, but if I can't say he's eligible for anything, I don't think any judge can ignore that. And the burden isn't really on the government to say what he's eligible for, especially when I have to do the fact-finding. * RI 52: I think that the burden-shifting made it much easier for detained immigrants to be able to get bond because instead of this ridiculous proving a negative of my client is not a flight risk. My client is not a danger to society, it was the government's job to actually demonstrate how they were a flight risk or a danger to the community.

Link to Data Source: * Research Interview #7: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/PV33MN * Research Interview #23: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/1K5HBD * Research Interview #52: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/RD0IPI

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys demonstrate that an IJ's ignorance of Brito should not be an excuse. First, as RI #71 explains, courts are interactive and allow both attorneys to professionally remind IJs of legal developments and IJs to clarify how a recent legal development affects the case before the court. Second, as RI #69 explains, courts can revisit past decisions and correct due process failures.

Source Excerpt: * RI #71: I assume the IJ doesn't know anything about this. And just put it on the record. And sometimes, I don't advise this specifically. But I know my friends, and I know that they say things like, as I'm sure your honor is where he, you know, in Brito, V blah, blah, blah, blah, blah, blah, blah. So if they're not aware, then they can ask further questions. \(\dots\) It also helps them immediate situation to sort of put the judge on notice, like, remember, we're in Massachusetts, remember, you know, because you're doing video calls all over the country. Remember, here in Massachusetts, we have this. * RI #69: Certain judges were doing detainee cases, and others were not. [My client] got in front of this judge for a bond hearing. The IJ said Brito standards apply, but she forgot to consider alternatives to detention. Literally did not even consider them even though we have put them in our packet and mentioned them. We had argued about that when she gave her decision. Still, she said I find that he's a flight risk. Here's why. Period. And there was no consideration that a GPS ankle monitor can actually ensure that he'll come back. Nothing. And so we went, we went back to the district court and did a motion to enforce the judgment. To argue that she failed to consider alternatives to detention. And Brito requires that. And then we went back for a new bond hearing in May, and she did release with an ankle monitor. And she literally said, "I did not complete the analysis." And I remember thinking, it's because this alternative to detention thing is kind of new. And the last time she was deciding detention cases, this wasn't a thing. And so she didn't, like it wasn't of part of her mindset of what needed to be done. We ultimately got what we wanted out of that case, but it was just more circuitous than I would have liked.

Link to Data Source: * Research Interview #69: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/ZFK3WF * Research Interview #71: https://data.qdr.syr.edu/privateurl.xhtml?token=515b519b-f318-4142-9443-d972822d547c&file=doi:10.5064/F60LYDGR/C91RC1

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys demonstrate that an IJ's ignorance of Brito should not be an excuse. First, as RI #71 explains, courts are interactive and allow both attorneys to professionally remind IJs of legal developments and IJs to clarify how a recent legal development affects the case before the court. Second, as RI #69 explains, courts can revisit past decisions and correct due process failures.

Source Excerpt: * RI #71: I assume the IJ doesn't know anything about this. And just put it on the record. And sometimes, I don't advise this specifically. But I know my friends, and I know that they say things like, as I'm sure your honor is where he, you know, in Brito, V blah, blah, blah, blah, blah, blah, blah. So if they're not aware, then they can ask further questions. \(\dots\) It also helps them immediate situation to sort of put the judge on notice, like, remember, we're in Massachusetts, remember, you know, because you're doing video calls all over the country. Remember, here in Massachusetts, we have this. * RI #69: Certain judges were doing detainee cases, and others were not. [My client] got in front of this judge for a bond hearing. The IJ said Brito standards apply, but she forgot to consider alternatives to detention. Literally did not even consider them even though we have put them in our packet and mentioned them. We had argued about that when she gave her decision. Still, she said I find that he's a flight risk. Here's why. Period. And there was no consideration that a GPS ankle monitor can actually ensure that he'll come back. Nothing. And so we went, we went back to the district court and did a motion to enforce the judgment. To argue that she failed to consider alternatives to detention. And Brito requires that. And then we went back for a new bond hearing in May, and she did release with an ankle monitor. And she literally said, "I did not complete the analysis." And I remember thinking, it's because this alternative to detention thing is kind of new. And the last time she was deciding detention cases, this wasn't a thing. And so she didn't, like it wasn't of part of her mindset of what needed to be done. We ultimately got what we wanted out of that case, but it was just more circuitous than I would have liked.

Link to Data Source: * Research Interview #69: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/ZFK3WF * Research Interview #71: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/C91RC1

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys clarify ICE attorneys' professional incentives for pursuing cases where the non-citizen respondent is neither dangerous nor a flight risk. Specifically, courtroom experience is a key factor when assigning immigration judgeships. Therefore, ICE attorneys may choose to detain non-dangerous individuals in order to acquire trail experience necessary for career advancement.

Source Excerpt: * RI 7: I did think when Brito came down that [ICE] would perhaps release people. Because pre-Brito they would arrest somebody, you know, for a driving without a license charge. And it's still my burden to show that that guy should be let out. Even though that's all he has on his record for 20 years or whatever. I don't think [ICE released people] to the extent it really should have; they still try to put on their case even when their cases are weak. * RI 51: One of the nice things about the Boston immigration court, though, is that a lot of the trial attorneys are former district attorneys. And so they're used to like conferencing the case. [ICE attorneys] don't just come straight from law school to government. They typically have experience in a forum where they're used to meeting to communicate with the other side. * RI 69: And it is easier to get into a judgeship if you were previously in law enforcement because you've already gone through the background checks. And more court experience is one heavy factor when weighing of the immigration judgeship. And for immigration judgeships, prosecutors are always going to have more court experience than the people representing the non-citizens.

Link to Data Source: * Research Interview 7: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/PV33MN * Research Interview 51: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/2OP5YN * Research Interview 69: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/ZFK3WF

<br>Analytic Note: Excerpts from research interviews with private immigration attorneys illuminate how they provide important value to respondents, especially those held in custody, unfamiliar with U.S. laws, and/or non-English speaking. Competent lawyers can acknowledge and effectively rebut the government's claims; without them, the government would likely ride roughshod over a respondent's argument (RI #7, #52). Capable attorneys can review the government's case and prepare appropriate responses, including composing legal strategy, piecing together a compelling story on why and how the respondent is not dangerous, and collecting evidence supporting the respondent's counterarguments (RI #23).

Source Excerpt: * RI 7: Now, post Brito, the government presents the case and talks about how terrible my client is, and all these crimes they've committed, or whatever it is they want to say. And then my response rubuts that. I would say, "Nope. That's not true. Here's what happened." Or [I would say], "Yes, I acknowledge that. But look at all the wonderful ways he's changed since then." So, you're acknowledging their argument and rebutting it. * RI 23: In a practical sense, it means that we are actually getting paperwork from the government ahead of our bond hearings, which is just magical. \(\dots\) I think my stress levels going into a hearing changed a little bit because I knew what the government had and what they were likely to say. And I knew that if they thought someone was secretly a gang member, that would be at the top of their submission rather than after I've put together my 20-minute presentation. But, you know, if I think they have anything, then I can't tell my client, "Well, let's just hang back, see what the government comes up with." I need to have proof that my client is not dangerous. I had a client who was convicted of embezzlement. It was not great sounding. Okay. And he and another family member were co-defendants and the other family member had, to my mind, done a great deal more. More bad guy stuff than my client. And so I'm not going to let the government say, "Look, he's got a federal conviction." I have to think through what motivates my client has to stay here. I have to think through my argument that my guy has relief. How am I going to map out for the judge that that relief is not a pipe dream? [I have to explain,] "No, your honor. It's gonna be like this, he's gonna do A, he's gonna do B, he's going to do C. And you're gonna see that he's eligible for D." So I have to have that tight. The burden might be on the government, but if I can't say he's eligible for anything, I don't think any judge can ignore that. And the burden isn't really on the government to say what he's eligible for, especially when I have to do the fact-finding. * RI 52: I think that the burden-shifting made it much easier for detained immigrants to be able to get bond because instead of this ridiculous proving a negative of my client is not a flight risk. My client is not a danger to society, it was the government's job to actually demonstrate how they were a flight risk or a danger to the community.

Link to Data Source: * Research Interview #7: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/PV33MN * Research Interview #23: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/1K5HBD * Research Interview #52: https://data.qdr.syr.edu/file.xhtml?persistentId=doi:10.5064/F60LYDGR/RD0IPI

it's not just the tools, it's the time, meaningful connections, and PD for teachers

I tried to introduce the economic principle of decision-making, and I used the example of choosing between apples and bananas to explain this theory. This is because I often see in scientific articles that they use simple and daily instances to help readers understand a scientific theory, so I wanted to do the same. It wasn't clear in the first draft how one makes decisions between apples and bananas. However, after revising this paragraph and using more direct language such as listing that one might compare their tastes or nutrients subconsciously, I made the paragraph clearer. This is an example of one successful revision i did.

What I have gathered from this is that language makes us the people we are today and in this context that is correct because language is the one thing that we as people or social beings can’t be without it it connects us to not just people but places and beings around us and it transcends across the globe and it’s very interesting to think about language like this.

Hughes is just as much a representation of this country and it's history as the 'good parts' that people pay attention to. For the company to face him is for them to face their ignorance and racism. Hughes refuses to hide for the comfort of white people who want to forget the past.

Maybe a bit extreme? Why not keep it but just come to terms with it? Find something better that makes it faint a bit. Or is this what is meant?

Is this just redundant w/ Fig 2 BC? I see that the values are a lot higher in Fig 2 BC and those were 6-day old, instead of the 5-day old plants shown here. Sorry if it's an ignorant question.

That's a you-problem. The pieces are there—the language is stable, and there's a ludicrously backwards compatible World Wide Wruntime that you can trust to be around—it's on you if it fails.

When taking a risk some people are just more comfortable with taking a bigger risk and others because they see that the outcome of this can be bigger and better. Others it takes a while because they see what would be the outcome but they are not sure if they want to take the risk.

What homework could look like in a ChatGPT world

Critical editing becomes a more important skill than summation. When the summation synthesis comes for free, students distinguish themselves by understanding what is correct and correcting what is not. Sounds a little bit like "information literacy".

Alan Berg, recognized as the first widely distributed political talk radio voice

Hosting a liberal talk radio show out of Denver, Colorado. He is murdered by someone who called him on the air and who Berg called a Nazi.

Enforcement of the FCC Fairness Doctrine

The case was decided at the D.C. Circuit court level with the future Justice Warren Berger writing the opinion. The opinion forced action at the FCC.

Office of Commun., United Ch., Christ v. FCC. 425 F.2d 543 (D.C. Cir. 1969). (see https://casetext.com/case/office-of-commun-united-ch-christ-v-fcc)

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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

Wade believes it is his responsibility, with a platform and a magnitude of followers, to speak out for those whose voices won't be heard and those who won't/can't/don't have the platform and following he has.

This is for me the crucial issue. Creators of the pre-TwitterMigration Mastodon argue that they oppose scaling / growth as a goal. but they need to face the reality, that their network just grew by around 50%

General comments: 1-This is a really important work because in this day and age, transforming DNA into an organism or a cell is an essential tool for any molecular biologist

2-And we still don’t understand how to transform the vast majority of organisms on Earth!

3-I applaud this effort for developing robust and accessible transformation tools for the amoeba Acanthamoeba castellanii. I believe this is an important organism to study but equally important are the general trends/approaches about what works to transform an organism

4-accumulating more and more of this knowledge on transformation across different organisms is essential if we want to access the biology of many more organisms

5-Also kudos for the detailed and meticulous transfection optimization! I really enjoyed your use of the N/P ratio and the combinatorial approach over a range of DNA and PEI concentrations. This is solid science!

Specific comments: 1-Figure 2 → Is it possible to include cell counts, in addition to the RFU signal? This is not a major comment. It’s just that there are cell counts in your other figures so it might be good to include for this figure as well. No need to repeat this experiment if you don’t have cell counts though!

2-Figure 4 → Would it be possible to include arrows or something to indicate which parts of the figure you would like the reader to focus on? It’s great having all of the data included but it may help your narrative if you point in the figure itself to certain key differences or features of the data. Or you might consider including a table to summarize all the data from the figure? May be the table could contain standard deviation around the average for each treatment (or something to show the distribution of the signal but with a single number)?

3-Figure 6 → Would it be possible to include indication of statistical significance?

Questions: 1-How long are plasmids maintained in the transformed cells?

2-Is there a robust selection that could enable you to produce stably transformed lines?

3-And related to Q2, is it possible to produce stable mutant lines (gene deletions or gene introductions), perhaps by transiently transforming CRISPR genes?

4-Does Acanthamoeba castellanii easily undergo transfection in nature? Is there any evidence for that based on its genome?

5-Related to Q4, are there known viruses that infect Acanthamoeba castellanii? Knowledge about these viruses may inform alternative methods of gene delivery and also serve as evidence for the transfection rate in nature.

6-I was wondering if you have tried transfecting Acanthamoeba castellanii with one big plasmid containing two fluorescent genes? Does that work? If yes, is the gene expression worse or better compared to having the genes on two plasmids and transfecting the plasmids together?

I agree with this statement. I think that some of the keys ways that I have learned/observed myself learning in this course are through reading, synthesizing, note taking, making connections, discussing, and reflecting. All of these methods require specific tools or collaborators in order to be put into use, but it's the act of doing these things that makes you think critically about what you are doing, and why. I think that once we ask ourselves these questions, we start to understand how important our work is, and how the information that we have impacts the rest of our learning process, and that of others. It's very easy for instructors to use tools to teach, but unless students are also actively using them, they won't remember anything that they learned later.

how to configure celery?

To configure Celery, you need to create a Celery instance and define the broker and backend to use for managing and storing tasks and results. You can do this by creating a celery.py file and adding the following code:

Copy code from celery import Celery

app = Celery()

app.config['CELERY_BROKER_URL'] = '<your-broker-url-here>' app.config['CELERY_RESULT_BACKEND'] = '<your-backend-here>' The CELERY_BROKER_URL is the URL of the message broker that Celery will use to manage and queue tasks. The CELERY_RESULT_BACKEND is the backend that Celery will use to store the results of completed tasks.

Next, you need to define your Celery tasks. This is simply a matter of creating functions and decorating them with the @app.task decorator. For example:

Copy code @app.task def add(x, y): return x + y You can then run the Celery worker using the celery command and passing the name of your Celery app:

Copy code celery -A celery worker -l info This will start a Celery worker process that will run in the background and listen for tasks to process.

For more information and additional options, you can check out the Celery documentation on configuration.

explain the above process in more detail.

To configure Celery, you need to create a Celery instance and define the broker and backend to use for managing and storing tasks and results. You can do this by creating a celery.py file and adding the following code:

Copy code from celery import Celery

Create a new Celery instance

app = Celery()

Configure the Celery instance with the broker and backend

app.config['CELERY_BROKER_URL'] = '<your-broker-url-here>' app.config['CELERY_RESULT_BACKEND'] = '<your-backend-here>' The CELERY_BROKER_URL is the URL of the message broker that Celery will use to manage and queue tasks. This can be a URL to a message queue service like RabbitMQ or Redis, or it can be a database like SQLite or MongoDB. The CELERY_RESULT_BACKEND is the backend that Celery will use to store the results of completed tasks. This can be the same as the broker, or it can be a separate service.

Next, you need to define your Celery tasks. This is simply a matter of creating functions and decorating them with the @app.task decorator. For example:

Copy code @app.task def add(x, y): return x + y This defines a task named add that takes two arguments, x and y, and returns their sum.

Once you have defined your tasks, you can start the Celery worker process by running the celery command and passing the name of your Celery app:

Copy code celery -A celery worker -l info This will start a Celery worker process that will run in the background and listen for tasks to process. You can then submit tasks to the worker by calling the task function and passing the arguments you want to use:

Copy code result = add.delay(10, 20) This will submit the add task to the Celery worker with the arguments 10 and 20. The delay method returns a AsyncResult object that you can use to check the status of the task and retrieve the result when it's finished. For example:

Copy code result.ready() # Returns True if the task has completed result.result() # Returns the result of the task You can also use the apply_async method to submit tasks with additional options, such as a countdown or a scheduled time to run the task:

Copy code result = add.apply_async((10, 20), countdown=60) This will submit the add task with the arguments 10 and 20, and the task will be scheduled to run in 60 seconds.

For more information and additional options, you can check out the Celery documentation on configuration and task execution.

Simple explanation - Imagine you want to make a cake, but it takes a long time to bake. You don't want to just stand in the kitchen and wait for it to finish, so you put the cake in the oven and set a timer. Then, you can go do other things and come back when the timer goes off. That's like a background task.

In the same way, a website might have a task that takes a long time to finish, like uploading a big file or sending a lot of emails. Instead of making the person using the website wait, the website can use something called Celery to put the task in the "oven" (or run it in the background) and send a response to the person using the website right away. Then, Celery can let the website know when the task is done.

So, Celery is like a helper that makes it easy to run long tasks in the background and get notified when they're finished. It's like a timer for websites.

Another helpful verified purchase Amazon review on some mice. I found this while looking for a mouse that was (1) ergonomic, (2) rechargeable, and (3) wireless.

Reviewer gives some thoughts on the different ways ppl use mice and how that effects they need (or lack thereof) for a wrist brace.

A helpful Amazon review on 4 ergonomic wireless mice. I found this while looking for a mouse that was (1) ergonomic, (2) rechargeable, and (3) wireless. The viewer covered 4 different mice: - Anker B2C - 2 of the Jellycomb - MV045 - MV09D - iClever TM209G

This page is for the iClever, but the reviewer claims that it's just like the Jelly Comb MV045.

If you're simply reading to inform yourself about a topic, it may be worth reading a couple of book reviews, and listening to an interview or two, rather than invest the significant amount of time necessary to really engage with the book.

A few hours of skimming and reviews/interviews may get you to 25% understanding and retention, which in many cases may be more than enough for your needs of being basically informed on the topic. Compared to the 50 - 100 hours necessary for a deep, analytical engagement with the text, that would only get you to 50% understanding and retention.

I like your summary of the article but disagree with the author. If one is measuring the "understanding percentage" of a book, I think he's actually looking for short-form information or answers (like gathered from a book review or blog post) rather than a book. For me, the value of the best books comes from the inspiration and impulse for original thought it gives. Not everything in a book is valuable for everyone, nor should it be.

For textbooks, of course cutting the detail gives you an overview more quickly, but it's just an overview. And this entire point doesn't apply to fiction books.

Maybe it's a question of what type of understanding we want from each book. Sometimes that's answers, sometimes that's understanding, sometimes diversion or inspiration -- they're not all the same. Consulting reviews but not the book is an awesome shortcut to get answers, but then you're not reading the book, but the reviews.

I think the gap between thoughtful, slow reading and quick reading is much larger. Assuming of course that slow reading means thinking about the sentences, probably highlighting passages and scribbling a few notes.

Doesn't apply to every book of course, and depends on the aspect of understanding you want (your own thoughts or what the author meant).

Griner was not using marijuana to just get high for fun but because it has helped her with her pain and inflammation that she encountered over her career

this is a problem that has been affecting the people in the community as well, not only cops and such

All about her demeanor not actually the things she did.

Thesis: not polarization but white-supremacy-filled judicial tyranny

It isn’t clear to me that the judiciary is filled with white suprematists, but the judiciary is increasingly swinging conservative appointed by far right ideologues fueled by white suprematism.

Reviewer #3 (Public Review):

This manuscript provides a helpful and transparent guide on the application of granger-causality (GC) to calcium datasets. This is a useful entry point toward understanding the suitability and limitations of GC to neural data. However, it is not entirely convincing that the variations of GC analysis provided in this manuscript can be effectively applied to large-scale calcium datasets without prior knowledge of the underlying circuit, especially when such networks are likely to contain redundancy and recurrent links.

I would like to acknowledge that, at the outset, I held an unfavorable prior belief toward GC, for reasons that are well addressed in this manuscript, including the dangers of applying spectral GC to nonlinear networks, as well as a variety of pathologies that can undermine naive GC.

The manuscript has been helpful, both for its effective presentation of both bivariate GC and its multivariate extension, as well as the practical considerations that are essential to applying it to real-life data. It was particularly helpful to see a treatment of the challenges and their possible resolutions. I commend the authors for their transparency - they should certainly be rewarded rather than punished for their transparency.

Major<br /> 1. Redundant signals: throughout the brain, it's expected that a population of neurons can encode the same information. It's unclear how GC (both the original and the modified versions) can handle this redundancy. Given how pervasive redundant signals are in the brain, this should be addressed in both simulation and experimental data. For example, in one of the manuscript's simulated networks, replace one neuron with 10 copies of it, each with identical inputs and outputs but with the weights scaled by 1/10. Such a network is functionally equivalent to the original but may pose some challenges for the various versions of GC. I believe this issue also accounts for the MVGC results in the hindbrain dataset. It might be more appropriate to apply GC to groups of neurons (as indeed the authors cited), instead of applying it at the single-cell level with redundant signals.<br /> 2. Similarly, there is recurrent connectivity throughout the brain. The current manuscript appears to assume feedforward networks. Is the idea that GC cannot be applied to recurrent networks? If so, this needs to be clearly stated. If the authors believe that GC can recover casual links even in the presence of recurrent connectivity, this needs to be demonstrated.<br /> 3. Both BVGC and MVGC appear to be extremely sensitive to any outlier signals. The most worrying aspect is that the authors developed their corrections and pipelines with the benefit of knowing the structure of the underlying system, whereas in the case where GC would be most useful, the user would be unable to rely on prior knowledge of the underlying structure. For instance, the motion artifact in Fig 3a-c was a helpful example of a vulnerability of naive GC, but one could easily imagine scenarios involving an unmeasured disturbance (e.g. the table is bumped) causing a similar artifact, but if the experimenter is unaware of such unmeasured disturbances then they will not be included in Z, and hence can result in the detection of widespread spurious links.<br /> There is a circularity here that's concerning. It seems that one already needs to have the answer (e.g. circuit connectivity) in order to clean up the data sufficiently for BVGC or MVGC to work effectively. Perhaps the authors would be interested in incorporating ideas from the systems identification literature, which can include the estimation of unmeasured disturbances, perhaps in conjunction with L1 regularization on the GC links. This is certainly out of scope for the present work, but it would be worth acknowledging the difficulties of unmeasured disturbances and deferring a general solution to future work. Similar considerations apply to a common unmeasured neuronal input (e.g. from a brain region not included in the field of view of the imaging).<br /> 4. Interpretation - would it be correct to state that BVGC identifies plausible causal links, while MVGC identifies a plausible system-level model? I think these interpretations, carefully stated, might provide a helpful way of thinking about the two GC approaches. Taking the results of the paper together, neither BVGC nor MVGC is definitive - BVGC may overestimate the true number of causal links but MVGC is prone to a winner-take-all phenomenon that may represent just one of many plausible system-level models that can account for the observed data. This should be more clearly stated in the manuscript.<br /> 5. "correlation completely misses the structure" - links are signed, so they should be shown with "bwr" colormap, with zero mapped to white (i.e. v_min is blue, 0 is white, v_max is red, |v_min| = |v_max|, this is natively supported in PyPlot and can be trivially implemented or downloaded in MATLAB). It is misleading that correlation appears to miss certain links marked in black, until one realizes that these links are inhibitory. It would substantially aid clarity and consistency if all panels followed this signed "bwr" convention. I think the emphasis for the GC panels is on whether links are detected, rather than the weight of the link, so I would suggest indicating detected inhibitory links as -1 (blue) and detected excitatory links as +1 (red), and link not detected as 0 (white).

see this right here is important because I disagree with it. Redon doesn't really seem to fit the mold of Symbolist according to Larson, and it seems pretty clear to me that his work purposefully evades definition and overall narratives. I think that many scholars make it their job to understand and interpret the noirs into something tangible when they are very purposefully not. These works represent something that's just out of reach, that we can't quite put our finger on but somehow still recognize. It's supposed to be ineffable, because it was when Redon imagined it.

Here's a snapshot of an aspect of the society in which R. resides (or at least his perception of an aspect of the society in which he resides). One manifestation of a sort of state of moral decay. It sounds like this sort of thing could reasonably be assumed to have happened just as R. surmises.

I'm not sure you need to call this out separately from the user flow. It's one of the main purposes of doing a user flow. So maybe you can just indicate that the user flow helped you identify the pages you needed to design.

It's interesting how sports go hand and hand with politics. It seems it has been like that since the start of sports, and it will continue to be like that till the end.

ngrok

Issues: When someone gets cancelled, it's not just their reputation that is harmed, but their livelihood as well.

don't own it

problems with platforms

This is something I've started noticing more as I got into product design. For the most part, I think these changes are welcome since they allow for a better user experience. However, it's also with just some further changes they can be used somewhat unethically. One example is how Facebook conducts experiments with these type of design changes.

http://scripting.com/2022/12/03.html#a152558

Anecdotal evidence for the slow death of Twitter and higher engagement on Mastodon.

Calling someone a creature shows that they don't see them as equal, it is very dehumanizing. It's hard to understand why they wouldn't want to give a child a second chance, you never know why someone would do something they might be in a hard spot and just need a little help.

!- for : innovation biases - crative cliff illusion

Author Response

Reviewer #1 (Public Review):

In this paper, Quiniou and colleagues show via orthogonal methods that human thymopoiesis releases a large population of CD8+ T cells harboring a/b paired TCRs that (i) have high generation probabilities, (ii) have a preferential usage of some V and J genes, (iii) are shared between individuals and (iv) can each recognize and be activated by multiple unrelated viral peptides, notably from EBV, CMV and influenza.

Major strengths of the paper

Quiniou et al. generated single-cell sequencing datasets of the earliest stages of TCR beta chain gene recombination. And then showed that a subset of them is highly clustered also having high generation probability.

They show that these T cells can bind multiple antigens, both via the use of public antigen-specific datasets as well as corroborating experimental TCR expression and binding essays.

Minor weaknesses

To what extent is TCR clustering and high Pgen and cross-individual sharing correlated? What is the Pgen of the sequences clustered with the high Pgen cells? Can you comment on the correlation between these three phenomena?

Indeed, there is a significant positive correlation between the Pgen and the number of connections among the clustered TCRs, as was reported in Fig.1F of the original manuscript. Furthermore, this correlation is true for both private and public TCRs, as was reported in figure 2B of the original manuscript.

To show the link between the three phenomena, we now have added two supplementary figures showing a high positive correlation between Pgen and the number of connections, and between cross-individual sharing and the number of connections, and to a lesser extent between Pgen and cross-individual sharing (Figure 2-figure supplement 4C and D in the manuscript supplementary information).

However, we would like to emphasize that the difference in the mean Pgen of the clustered and dispersed TCRs is of about 20-fold. This is a high difference for a biological process (and highly statistically significant), but a small one compared to the 10-log10 span of the Pgens of the two populations. Factually, what we observed is not that clustered sequences have a high Pgen, but that they have a higher Pgen than the non-clustered sequences. Yet, many CDR3s with high Pgen do not cluster, and vice versa, indicating that a high Pgen is not the only (nor most important) driver of clustering. We have now added these as Figure 1-figure supplement 3E-F of our revised manuscript.

In other words, to what extent is this surprising to see that highly clustered TCRs have higher Pgen and are more shared?

That for a given CDR3 there is a correlation between having a high Pgen and being public is not surprising as both suggest a positive selection during evolution. What is more surprising is that there are CDR3s forming large clusters that occupy over 20% of the repertoire and that co-cluster between individuals with different HLA, “indicating a convergence of specificities between individuals’ clustered repertoires”. This suggests a surprising selection process that could depend less on HLA than the “classical” selection.

These points are now better emphasized in the revised manuscript.

Potential Impact of the paper

This work highlights an intrinsic property of the adaptive immune response: to generate TCRs with high generation probability that can efficiently bind multiple antigens. This finding has, therefore important impact on drug discovery and vaccine design.

We thank the reviewer for his appreciation.

Reviewer #2 (Public Review):

This study analyses the T cell receptor (TCR) repertoire of double positive human thymocytes, and compares this to mature single positive CD8 cells. The first major finding is that the repertoire post-selection is enriched for groups of TCRs with high generation probabilitites, similar sequences, and for TCRs previously annotated for viral specificity. This data is clearly presented and convincing. The extent of analysis of the human thymocyte repertoire is still very limited, and the paper adds significantly to this important question.

We thank the reviewer for his appreciation.

The second major finding is much more controversial. The authors first investigate the publicly available databases and show that there is a substantial proportion of TCRs which have been annotated to multiple viral specificities, a fact which is well-known to the specialists in the field, but not previously addressed.

Indeed, we are not aware of reports disclosing “a substantial proportion of TCRs which have been annotated to multiple viral specificities”. Actually, one could wonder why “a fact which is well-known to the specialists in the field” is not mentioned and discussed in published articles? To us, it reveals that this point has been overlooked by immunologists as recently in Zhang et al, 2021 where authors aiming at identifying highly specific T cell clones with a new modelling approach, excluded all clones binding more than 1 peptide. Thus, it makes it important to report it, as we do. Furthermore, we would also like to emphasize that we do more than just reporting that some TCR have “been annotated to multiple viral specificities”. We show from a manual curation of public databases that (i) some TCR have been reported to bind to tetramers presenting peptides from unrelated viruses; (ii) that such TCRs co-cluster using Levenshtein distance or GLIPH2 based clustering method; and (iii) that some of these TCRs indeed recognize different, unrelated peptides without significant sequence homology upon re-expression in carrier T cells.

The authors acknowledge that this in silico analysis is mostly based on unpaired alpha/beta sequence data, and that the chain pairing may influence specificity. They, therefore, perform a number of functional assays, demonstrating examples of T cells which respond by interferon gamma production to more than one peptide.

We thank the reviewer for pointing to the fact that, beyond tetramer binding, we performed cumbersome functional studies to document polyreactivity.

The paper is mostly very clearly written and presented and provides some fascinating novel perspectives on T cell cross-reactivity.

We thank the reviewer for his appreciation

The findings will surely be of interest to a broad readership - indeed anyone interested in how adaptive immunity works.

The link between the different sections of the paper is the weakest aspect. The relationship between thymic selection and polyspecificity, and also the real relationship between in silico "cross-reactivity" as evidenced by multiple annotations and the functional polyspecific T cells remains unclear.

Our flow of reasoning/analyzing was as follow. As we were studying the thymic selection of TCR repertoires, (1) we discovered a massive clustering within these repertoires. As for thymocytes this cannot be accounted for by a history of immune responses, this triggered our attention and led us to analyze the properties of these TCRs. This led us (2) to discover in these thymic repertoires “TCRs which have been annotated to multiple viral specificities”, that we were not aware of. We were so much intrigued by these observations that we wanted to substantiate them using datasets of paired  TCRs. As (3) we could confirm these observations in such datasets, this led us (4) to investigate these TCRs in functional studies. This is the link for the 1-to-4 sections.

To make this link clearer, we have reworked the titles of the different Results’ sections such as to emphasize the switch from thymocyte bulk sequencing studies to that of single peripheral cell sequencing studies.

The mechanistic molecular details underlying polyspecificity also remain unclear.

Indeed, we believe that solving the structure of polyreactive TCRs interacting with different peptides will be needed for a molecular understanding of polyreactivity, but that it falls beyond the present work.

But overall, lots of interesting new data, and some very intriguing hypotheses for the community to follow up on.

We thank the reviewer for his overall comment

Reviewer #3 (Public Review):

In this manuscript, the authors propose that there is a special, previously unrecognized, high-frequency population of a/b TCRs that are shared between people, have high generation probabilities, and react to many unrelated viral epitopes. Here is the main flow of the results, with comments on the strengths of the conclusions:

"Thymopoiesis selects a large and diverse set of clustered CDR3s with high generation probabilities" -- this seems correct and has been noted in earlier work by Mora and Walczak and others.

So far, Mora and Walczak selection models in humans are based on studying PBMCs (our ref n° 27 in the revised version), not thymic DP and SP sorted cells, even in the mouse derived models for which they used the total thymic cells (our ref n° 27).

Selection leads to a focusing of the CDR3 length which likely increases the degree of clustering and increases Pgen.

To address this question, we compared the CDR3 length distribution between DP CD3+ cells and CD8 SP cells from our thymic dataset. We did not observe major changes. The distribution and the mean of CDR3 length for the two cell populations remained identical. We only observed a small shifting in the CDR3 length distribution towards shorter sequences post-selection. This is now reported in the new Figure 1-figure supplement 3C in the revised manuscript.

"Clustered CDR3s are enriched for publicness " This also seems correct and again it makes sense: publicness is equivalent to having been independently rearranged (and sequenced) in another individual, which is determined by Pgen, and clustering is also determined to a large extent by Pgen (the factors that contribute to Pgen, shorter CDR3s for example, are largely shared between neighbor TCRs).

We agree that theory could have indeed predicted that. In any case, to our knowledge, this is the first report of large clusters of just selected thymocytes’ CDR3s that moreover co-cluster between individuals with different HLA.

"Clustered public CDR3s are enriched in viral specificities" -- This claim is not justified by the data, which comes from sequence matching against literature-derived databases. Rather, what is true is that "Clustered public CDR3s are enriched in public viral specificities".

We changed “CDR3s are enriched in viral specificities” for “clustered public CDR3s are enriched in public viral specificities".

But this might be a simple consequence of the previous observation, that "clustered CDR3s are enriched for publicness". One would need experimental specificity data on the very same datasets to make a conclusion about viral specificities in general.

We based our interpretation on experimental data.

Indeed, we manually curated databases to identify CDR3s that bind specific tetramers/dextramers. This type of “experimental specificity data” is for immunologists a paradigmatic and yet unchallenged mean to define specificity.

We make the observation that there are more CDR3s from a TCR that does bind tetramers/dextramers presenting viral peptides in clustered than in dispersed CDR3s. This is a highly statistically significant fact, that we now report as a fact that we leave open to discussion/challenge by our community.

"Identification of polyspecific TCRs" -- In this section, the authors report that some of the CDR3 clusters contain CDR3 sequences from literature-derived TCRs with multiple specificities. They conclude that these must represent polyspecific TCRs. The problem with this conclusion is that even having the same CDR3beta, let alone similar CDR3beta sequences, does not imply the same specificity. One can see the problem if one imagines a very deeply sequenced dataset, and focuses on a short CDR3 length with high frequency. With sufficient sampling, one will be able to navigate from nearly any single CDR3beta to any other CDR3beta of the same or similar length by jumping between single-mismatch variants. But this doesn't imply that all the TCRs from which these CDR3s were sampled, which likely have many different Vbeta genes and completely different TCRalpha sequences, must all bind the same thing.

We will first point to the fact that we did not analyze “a very deeply sequenced dataset”, but only the 18 000 most abundant sequences per sample. Singletons were excluded. In addition, we did not mean to say that all the connected TCRs have the same specificities, regardless of their position in the cluster. Clustering algorithms, whether LV distance of GLIPH2 for example, are now commonly used to infer specificity of clusters and it is admitted that the closer the TCR sequences are, the more they share their specificities.

That said, it is precisely because we acknowledge the limitation of bulk sequencing for inferring specificities that we turned to also analyze single-cell datasets.

We made this more apparent by the new sections of the results that more clearly indicate the shift from unpaired bulk thymocyte sequencing and paired single peripheral cell sequencing.

"Binding properties of polyspecific TCRs" -- Here the authors look to validate these results with paired TCR sequences. They analyze a public dataset made available by 10X genomics, featuring single-cell gene expression, TCR sequencing, and dextramer UMI counts for ~150,000 T cells. This is an amazing dataset with lots of interesting features, but, like any large high-throughput dataset, it needs to be analyzed with care.

We can assure the reviewer that we were always very careful. Actually, we even started by carefully reviewing the 10X proposed methodology, in which we identified major biases. This led us to explore this dataset cautiously and without preconceived ideas.

The authors claim to see evidence for large-scale cross-reactivity. This comes mainly from a set of dextramers for A03 and A11-restricted peptides. But these dextramers appear to be binding in a uniquely non-specific manner (by comparison with the other dextramers) and non-TCR-dependent manner in this experiment. One can see this, for example, by comparing the consistency of binding within expanded clonotypes: for a specific dextramer like A*02-GIL(Flu), positive binding for one cell in a clonotype greatly increases the likelihood of binding for other cells in the clonotype, suggesting that the binding is mediated by the TCR.

This is not true for the A03 and A11 dextramers (except for a few expanded clonotypes in an A*11 donor). TCR sequence doesn't appear to be the determining factor for binding to these dextramers; rather it may be expression of KIR genes or other surface proteins that can interact with MHC.

These are indeed striking binding patterns that are remarkably similar for a single CDR3 beta associated with more than 40 different CDR3s alpha (and moreover from two donors). The first attitude of immunologists would indeed be of discarding this observation for non-fitting the paradigms. We would like to rather propose an agnostic view at these results.

These results show that a series of five A03 and A11 dextramers loaded with various peptides bind to cells that express a given CDR3 beta associated with a multitude of CDR3alpha. If it would be an MHC to KIR binding, then such dextramers should bind to most cells, independently of their TCRs. We have added two supplementary figures (Figure 4-figure supplement 8B-C) to show that this is not the case, and that further show very different binding patterns.

If it would be a binding to “other surface proteins”, it would likely be the same.

We identified a CDR3 from donor 3 which binds preferentially to A03 and A11 dextramers. However, it binds to only 4 out of 5 of these. If the binding is non-specific and non-TCR-dependent, a binding for the A0301 RIAAWMATY BCL2L1 dextramer should also have been observed. Moreover, we identified this same CDR3beta in two other cells from donor 1 and 4, and that were associated with a different CDR3alpha. Except for only one binding, these TCRs didn’t show binding to the A03 and A11 dextramers.

Moreover, we identify another CDR3 from donor 1 that is associated with a strong binding to one A1101 dextramer presenting an EBV peptide when associated to many different CDR3alpha. The binding to the other A03 and A011 dextramer is weaker and seem to depend more on the CD3alpha.

If the binding of A03 and A011 dextramers is non-specific and non-TCR-dependent, why is there such a difference between the binding of A1101 IVTDFSVIK and A1101 AVFDRSDAK dextramers?

"Polyspecific T cells are activated in vitro by multiple viral peptides" Here the authors explore polyspecificity experimentally. First they report that polyclonal populations of T cells, sorted for binding to one dextramer, can also produce IFN gamma upon stimulation with a distinct peptide, albeit more weakly than for the cognate peptide.

This is indeed true for CMV+ sorted cells that respond better to CMV peptides than to EBV ones, but not true for EBV+ sorted cells that also respond better to CMV peptides than to EBV ones.

But it's not clear that the concentrations of the peptides are appropriate for stringently detecting cross-reactivity.

We wonder what does mean “stringently”? It is possible that stringently mainly means defining the conditions that eliminates what does not fit the current paradigm?

More factually, the peptide concentration used for these experiments, presented in Fig. 5A-B, was 1 µg/mL, i.e. ~1 µM for a 9-10 aa-long peptide. This is clearly a physiological concentration for viral peptides, routinely used in in-vitro recall assays. We can thus rule out that the observed cross-reactivity is simply due to an excess peptide stimulation.

Then the authors actually synthesize and characterize individual TCRs. Here what is seen is consistent with expectation and does not seem to support the idea of substantial fuzzy cross-reactivity: binding to the cognate peptide is 3-4 orders of magnitude stronger than to the alternative peptides.

We respectfully disagree. First, as shown in Fig. 5C TCR#35-13 (cognate peptide HLA-A2-restricted Flu MP 58-66) indeed recognizes the alternative HLA-A2-restricted CMV IE1 184-192 peptide with a 3-4 higher log EC50; yet, the EC50 of this TCR is approx. 10e-6 M, i.e. 1 µM, which remains a physiological concentration. Second, this is not the case for TCR#36-150 (same cognate peptide HLA-A2-restricted Flu MP 58-66), which actually recognizes the alternative HLA-A2-restricted EBV BMLF1 280-288 peptide with a 4-fold lower EC50.

The only exception is the GAD 114-122 TCR, where the different peptides appear to be closer in binding strength. But in this case, the authors state that they "analyzed their response to a set of peptides comprising their cognate peptide and peptides with no significant structural commonalities, selected by testing combinatorial peptide libraries". If the competitor peptides came from peptide library screening then the observation of strong binding to alternative peptides does not seem as surprising as a TCR that binds well to a Flu peptide, say, and also a CMV peptide, selected from a smallish set of possibilities.

As explained above, this TCR does not stand as an exception compared to Flu-reactive TCRs. Moreover, it should be noted that this GAD 114-122 TCR recognizes its cognate peptide in a similar or even lower concentration range compared to the Flu-reactive TCR #36-150. It should also be pointed out that, contrary to the Flu-reactive TCRs, here we did not have any reference dextramer binding data to guide our peptide selection, which is why we resorted to combinatorial peptide libraries. Thus, although different strategies were used, peptide selection was “guided” in both instances.

It is pretty well established that TCRs are cross-reactive, both for nearby peptides and also for sequence-dissimilar peptides.

We agree and had notably quoted the landmark paper by Don Mason estimating that each TCR may respond to over 106 different peptides from an estimated repertoire of > 1010 peptides. Based on the Don Mason estimate of cross reactivity, the chance to find a cross reactive peptide at random would be around 10-4.

Here, we just tested a few peptides from different viruses. If Don Mason’s estimates are correct, for a given TCR, the chance to find even just 1 cross-reactive peptide among these few peptides would be at most of 10-3, the chance to find 2 cross reactive peptides would be of 10-6 and that to find 3 or more cross reactive peptides would have be infinitesimal.

Thus, if the polyreactivity that we described is part of this general cross reactivity, our results are at least highlight a major previously unreported bias in the selection of these cells.

The question is whether widespread, functionally relevant (not just dextramer binding at some concentration) poly-reactivity to diverse viral peptides is a defining feature of a large fraction of the TCR repertoire. The paper does not appear to present sufficiently strong evidence to support this claim.

We agree with the reviewer that more work is needed to “fully” appreciate the role of polyreactive cells!

Author Response

Reviewer #1 (Public Review):

Pan et al. examined the role of oligodendroglial exocytosis, and specifically the role of L-type prostaglandin D synthase (LPGDS), in modulating oligodendrocyte differentiation and myelination. The topic of autocrine and paracrine signaling within the oligodendrocyte lineage is under-studied and the authors use a novel approach for oligodendrocyte precursor-specific inhibition of VAMP-mediated exocytosis using inducible expression of botulinum toxin with the PDGRFa-CreER transgenic mouse line (PD:ibot). Using a combination of in vitro culture systems and immunohistological analysis in vivo, the authors find ibot expression in OPCs leads to reduced oligodendrogenesis and myelination, leading to a behavioral deficit in rotarod performance. Additional transcriptomic analysis in PD:ibot mice revealed Ptgds, the gene encoding LPGDS, was significantly overexpressed in both mature oligodendrocytes and OPCs. Further pharmacological experiments with cultured OPCs showed direct LPGDS inhibition led to a similar inhibition of oligodendrogenesis as PD:ibot mice. Together, this study reveals VAMP-mediated exocytosis in OPCs is required for normal oligodendrogenesis and identifies LPGDS as a new chemical regulator of oligodendrocyte myelination. These findings are strengthened by careful characterization of the PD:ibot mouse line and effective use of culture systems and pharmacology to uncover a cellular mechanism. Quantification is performed at several levels of resolution using immunohistochemistry, electron micrography, and protein/transcriptomic analyses and control experiments were largely carefully considered.

We thank the reviewer for recognizing the strength of our study.

Despite these strengths, there are some points that need to be further addressed. The interpretation of autocrine/paracrine signaling relies on a critical culture experiment in which PD:ibot OPCs were cultured in the presence of PD:ibot or control OPC well inserts. However, these results had a marginal effect size, raising questions as to the extent to which VAMP inhibition specifically had effects through the blockade of exocytosis (resulting in an autocrine/paracrine signaling deficit) or inhibited oligodendrogenesis in a cell-intrinsic mechanism (e.g. VAMP-dependent trafficking of critical myelination components, such as PLP (Feldmann et al., 2011)).

We agree with the reviewer that both cell autonomous and cell non-autonomous effects may contribute to the defect associated with VAMP inhibition. We performed additional experiments to investigate the contribution of cell non-autonomous mechanisms. We took advantage of the fact that all OPCs purified from PD:ibot mice were not botulinum-GFP-expressing (efficiency ~65% Figure 6B, page 24). The GFP- cells in PD:ibot OPC cultures did not express botulinum toxin and were competent in exocytosis. We compared the development of GFP- control cells in cultures generated from PD:ibot mice vs. control cells in cultures generated from control mice. Interestingly, we found that the percentages and sizes of lamellar cells in control cells in PD:ibot cultures were smaller than in control cells in control cultures (Figure 6C, D text page 25). Although both groups of cells were competent in exocytosis, they were surrounded by exocytosis-deficient vs. exocytosis-competent neighbor cells. The differences in the growth capacity of control cells in the presence of different neighbor cells reveal cell non-autonomous contributions of botulinum-expressing cells in oligodendrocyte development.

As described above under Essential Revisions 4), we performed additional experiments on the role of the secreted protein L-PGDS in oligodendrocyte development. We found that adding a protein that inactivates PGD2, HPGD extracellularly to oligodendrocyte cultures inhibited their development (Figure 7F, G, page 33). Adding L-PGDS protein extracellularly to PD:ibot oligodendrocyte cultures rescued their development defect (Figure 9A, B, page 33). Moreover, overexpressing Ptgds in PD:ibot mice partially rescued the myelination defect (Figure 9E-H, page 36). These observations further strengthened our conclusion that cell non-autonomous mechanisms contribute to the effect of botulinum toxin on oligodendrocyte and myelin development.

Nevertheless, these results do not rule out the cell autonomous effect of botulinum on oligodendrocyte development and, therefore, we included the potential contribution of both cell autonomous and cell non-autonomous mechanisms in the text.

Additionally, the authors claim the reduced number of oligodendrocytes in PD:ibot mice in vivo is not due to oligodendrocyte apoptosis and provide evidence by cleaved caspase-3 immunostaining of the cerebral cortex. While statistically not significant, the data is highly variable.

We thank the reviewer for pointing out the variability of the caspase-3 results. We performed a more thorough analysis of activated caspase-3 at multiple developmental stages. Again, we did not find any statistically significant difference in apoptosis between PD:ibot and control oligodendrocytes, OPCs, or cells of other lineages (Figure 3-figure supplement 1, text page 13).

If true, this would suggest oligodendrocyte differentiation is inhibited, which would coincide with a reduction of OPC proliferation. A complementary experiment comparing the rates of OPC proliferation between control and PD:ibot mice in vivo would provide further clarity on how oligodendrocyte density is being reduced.

We analyzed OPC proliferation in vivo by staining and quantifying Ki67+PDGFRa+ cells. Intriguingly, we found a modest increase in OPC proliferation in PD:ibot mice (Figure 3-figure supplement 3, text page 14).

The relevance of these myelination deficits is assessed with a rotarod assay, however, the mice used for these experiments are several times older (2-5 months) than those used for all other histological quantification (P8-P30). The large variance in results could be due to age-related differences in myelination, and it is unclear whether the deficits at early timepoints show a linear progression with age.

We thank the reviewer for the insightful comment. We have separately labeled data points from 2 months old and 5 months old mice (Figure 3Q-S, text page 17). With the data we have so far (n=20-27 per genotype), there isn’t a striking progression of phenotype with age. Future analysis at multiple time points may resolve any age-dependent changes in the phenotype.

Reviewer #3 (Public Review):

The authors pose an important question of whether oligodendrocyte lineage cells have an autocrine/paracrine signaling loop that contributes to their differentiation and myelination. While prior studies have demonstrated oligodendrocyte lineage cells have cell-intrinsic pathways that impact differentiation and myelination, there isn't a strong precedent for oligodendrocytes to promote their own differentiation via autocrine/paracrine mechanisms. The notion that oligodendrocyte lineage cells promote their own differentiation in an autocrine/paracrine manner is an intriguing one that adds a new layer to our understanding of how oligodendrocyte maturation is controlled. I anticipate this paper will prompt a new direction of future investigations to uncover the extent of oligodendrocyte autocrine/paracrine signaling.

To test the possible role of oligodendrocyte-secreted molecules on oligodendrocyte development, Pan et al. utilized a mouse model where the release of a subset of secretory vesicles (specifically VAMP1/2/3-dependent vesicles) is blocked. Blocking this vesicular release prevented or delayed the differentiation of oligodendrocytes in vivo and in vitro. Further, the authors identified changes to the mRNA and secreted protein levels of prostaglandin D2 synthase (L-PGDS). Prior RNA sequencing and snRNA sequencing datasets of the oligodendrocyte lineage have identified Ptgds as a highly abundant mRNA transcript in oligodendrocyte lineage cells, particularly mature oligodendrocytes. Ptgds encodes L-PGDS, which has an unknown role in oligodendrocyte function. L-PGDS has been shown to regulate Schwann cell myelin formation in the peripheral nervous system, prompting the question of whether this protein acts similarly in the central nervous system. The paper has a clear set of well-rounded experiments, with a few remaining points that would strengthen the conclusions:

We thank the reviewer for the positive comments on our study.

One of the foundational conclusions of the study is that VAMP1/2/3-dependent exocytosis is critical to oligodendrocyte maturation, by using a PDGFRa-CreER mouse line combined with iBot mice that express botulinum toxin in Cre-expressing cells (abbreviated as PD:iBot). Prior work has demonstrated in vitro that oligodendrocyte morphological maturation, myelin gene expression and myelin protein transport can all be impacted by the loss of VAMPs, including VAMP3. This paper establishes the importance of these SNARE proteins in the oligodendrocyte lineage in vivo: the number of mature (CC1+) oligodendrocytes and myelin basic protein staining is substantially reduced in PD:iBot mice.

1) The data in Figure 3M suggests that PD:iBot oligodendrocytes (GFP+) are lacking MBP+ sheaths and that any myelin formed is by the smaller percent of oligodendrocytes that do not express botulinum (GFP- cells). Furthermore, the efficiency of iBot expression (as evaluated by GFP+ cells) shows that 80% of OPCs and just 60% of oligodendrocyte lineage cells express GFP at P8 and supplementary data shows just 30% of oligodendrocyte lineage cells express GFP at P30. This raises the question of whether PD:iBot cells are unable to differentiate and die. While the authors show no change in caspase-dependent apoptosis in PD:iBot cells in vivo and in vitro, the data still suggests that blocking VAMP-dependent exocytosis itself slows or prevents the progression to a fully myelinating oligodendrocyte in vivo rather than the putative autocrine/paracrine signals are required for OPC differentiation. Confirming whether botulinum-expressing cells also contribute to the population of surviving, differentiated oligodendrocytes in vivo to strengthen the conclusions that autocrine/paracrine secreted molecules contribute to the oligodendrocyte maturation in vivo.

We thank the reviewers for raising a key point in characterizing the consequence of botulinum toxin expression in oligodendrocyte-lineage cells. We analyzed the overlap between GFP+ botulinum-expressing cells and the population of differentiated oligodendrocytes (Olig2+PDGFRa-CC1+ cells) and found that botulinum-expressing cells can survive and become differentiated oligodendrocytes (Figure 3-figure supplement 2, text page 14). Additionally, we performed a more thorough analysis of activated caspase-3+ apoptotic cells than was included in first submission and did not detect statistically significant differences between PD:ibot and control mice (Figure 3-figure supplement 1, text page 13).

2) The paper has complementary in vitro data to pinpoint a mechanism that results in hindered oligodendrocyte maturation. The authors conduct a well-designed set of in vitro co-culture experiments in Fig4 K-M that led them to conclude oligodendrocyte morphology is impacted by secreted molecules from other oligodendrocytes.

2a) The key experiment is the transwell co-culture experiment with control and iBot cells, which suggests that blocking secretion itself has the predominant impact on cell morphology: by eye, both group3 and 4 show the largest reduction in lamellar area and the difference between group 3 and 4 is slight. At day 3 of culture (Fig 4E), the authors show the clearest effect as a reduction in cells with lamellar morphology. The quantification of the lamellar cell area is less obvious than the % of cells with arborized vs lamellar shape, as seen in Figures E & F. I would recommend that the authors show representative images of these observations and quantification of morphologies for the transwell experiments. The impact of secreted factors may be clearer with this measure.

We added representative images (Figure 6G). We quantified both the % and size of lamellar cells. The size of lamellar cells is significantly higher in group 4 than in group 3. Although the % of lamellar cells is numerically higher in group 4 than in group 3, the difference is not statistically significant. To further assess whether cell non-autonomous mechanisms contribute to the oligodendrocyte development defect in PD:ibot mice, we performed additional analysis in culture. We took advantage of the fact that all OPCs purified from PD:ibot mice were not botulinum-GFP-expressing (efficiency ~65% Figure 6B). The GFP- cells in PD:ibot OPC cultures did not express botulinum toxin and were competent in exocytosis. We compared the development of GFP- control cells in cultures generated from PD:ibot mice vs. control cells in cultures generated from control mice. Interestingly, we found that the percentages and sizes of lamellar cells in control cells in PD:ibot cultures is smaller than in control cells in control cultures (Figure 6C, D, text page 25). Although both groups of cells were competent in exocytosis, they were surrounded by exocytosis-deficient vs. exocytosis-competent neighbor cells. The differences in the growth capacity of control cells in the presence of different neighbor cells reveal cell non-autonomous contributions of botulinum-expressing cells in oligodendrocyte development.

2b) On a related note, the cell morphology data is dependent on MBP staining. The authors show that MBP protein is reduced in cells from iBot mice. Since MBP+ cell area/arborized or lamellar structure is being quantified, there remains the possibility that the cells could display a more complex morphology (lamellar) that may be missed by only staining for MBP. The authors use a CellMask dye to show cellular morphology, which is a great idea. The authors state that it labels the plasma membrane; however, the methods (and images) indicate that a cytoplasmic CellMask was used (cat.no. H32720 labels nuclei and cytoplasm, not membranes). These conclusions about cell morphology vs simply MBP expression would be strengthened by an alternative membrane label (e.g., a CellMask plasma membrane dye).

We thank the reviewers for the insightful suggestion. We used the membrane version of CellMask and repeated the transwell co-culture experiment. The new results are consistent with the results based on MBP (Figure 6-figure supplement 1, text page 23). In addition, we used the membrane version of CellMask for all the new cell culture experiments (L-PGDS rescue, HPGD etc.)

3) The authors sought to identify what secreted factors may be affected by blocking VAMP1/2/3-dependent exocytosis. Pan et al. opted for a strategy of examining transcriptional changes, asserting that important genes may be upregulated in response to compensate for blocked secretion. While this is an indirect way to identify secreted candidates, the authors found a fortuitous result that Ptgds was substantially increased in the PD:iBot oligodendrocyte cells. To confirm that L-PGDS secretion is reduced from iBot cells, the authors show Western blots. By eye the change in L-PGDS is variable, however, the authors conduct several experiments with an inhibitor and product of L-PGDS that nonetheless indicate L-PGDS activity can contribute to the morphological maturation of oligodendrocytes. A caveat is that the AT-56 inhibitor reduces MBP+ cells, and the quantification of morphology is dependent on MBP staining (again, see my note in 2b about the CellMask dye). A report on differentiation (% MBP+ cells) may be a more accurate reflection of the result.

We repeated the AT-56 experiment using the membrane version of CellMask and again found that AT-56 inhibits oligodendrocyte maturation (Figure 7-figure supplement 2, text page 33).

The key, compelling experiment demonstrating the role of prostaglandin D2 is the authors' rescue experiment in Fig 4G.

As described above under Essential Revisions 4), we performed additional rescue experiments on the role of L-PGDS in oligodendrocyte development. We found that adding L-PGDS protein extracellularly to PD:ibot oligodendrocyte cultures rescued their development defect (Figure 9A, B, page 34). Moreover, overexpressing Ptgds in PD:ibot mice partially rescued the myelination defect (Figure 9E-H, page 36).

4) Although it's not a direct demonstration that L-PDGS secretion from oligodendrocytes is the key factor, the global L-PDGS knockout mice phenocopy many of the observations of the PD:iBot mice. This is a nice set of observations consistent with the author's hypothesis that L-PDGS impacts oligodendrocyte maturation. Future work should pinpoint whether oligodendrocyte-derived L-PDGS is critical.

We agree with the reviewer that pinpointing whether oligodendrocyte-derived L-PGDS promotes oligodendrocyte development and myelination is an interesting direction to pursue in future work. We are breeding L-PGDS conditional knockout mice to address this question and may report the results in a separate paper in the future.

Minor points:

1) The authors demonstrate that PD:iBot expresses botulinum and loses VAMP2 protein levels in oligodendrocyte lineage cells, but there is no demonstration of whether VAMP3 is expressed or similarly affected. Prior work has demonstrated in vitro that oligodendrocytes express both VAMP2 and VAMP3 (VAMP1 not detected). This would more clearly demonstrate which VAMP-mediated vesicular transport is blocked for the effects observed.

We agree with the reviewer and examined VAMP3 levels with Western blot. We found diminished levels of VAMP3 in oligodendrocyte-lineage cells from PD:ibot mice (Figure 1 J, M, text page 10).

2) It is satisfying to observe a behavioral effect in the PD:iBot mice. I would advise caution in interpreting any direct link between oligodendrocytes maturation and the rotarod behavioral difference at this time. Blocking secretion from PDGFRa-Cre expressing cells may have many indirect effects (beyond myelination) in both the CNS and other cell types that can express PDGFRa and VAMPs1/2/3. I was pleased that the authors did not conclude any direct links at this time.

We agree with the reviewer.

Overall, the authors had a well-rounded manuscript with clearly described and thoughtful experiments. The data support the conclusion that VAMP-mediated exocytosis is critical for oligodendrocyte maturation. The evidence that reduced L-PDGS secretion from the oligodendrocytes can explain the effects of the iBot mice is not as clear cut, but their data does demonstrate that L-PDGS is an important molecule for the differentiation of oligodendrocytes. This work will lead a new direction for future studies to investigate autocrine/paracrine signaling in oligodendrocyte maturation.

We thank the reviewer for the positive comments on our manuscript. As detailed in Essential Revisions 4), we now provide additional evidence on the potential contribution of L-PGDS in the oligodendrocyte development defect in PD:ibot mice.

Narrative of it's your job...you've got to do your job.

This provides credibility in the article and background information for the story. It's interesting to note that Serena Williams was the chosen person to compare in this story –– she's in a similar position to Osaka and understands the mental health aspects of being a professional athlete.

Laughing is a form of criticism that isn't as serious but just as neccesary.

That could be a problem, if it actually send to the permanent failure hook in this case. Then you would have to hit their bounces API to check whether it's actually a permanent failure / hard bounce for the recipient as opposed to just for this message.

I wish we would have been taking more responsibility with taking care of Earth. For a lot longer than just now. It's as if we are procrastinating that responsibility a bit.

Author Response

Reviewer #1 (Public Review):

They established a "behavioral transcriptomics" platform as they cultured mouse primary cell explant on an apparatus, imaged the cells over time, and analyzed cells with differential physiological status by scRNA-seq. They showed evidence that the system recapitulated physiological features of airway cells, including chemical-induced damage response. They further utilized the system to isolate cells of different cellular features and analyzed gene expression through scRNA-seq. The study demonstrates an interesting establishment and application of an in vitro system mimicking in vivo.

However, several major concerns need to be resolved.

First, whereas the overall study seems to focus on the establishment of airway epithelial cell explant apparatus and its application, take home messages that are delivered by the authors seem to emphasize the transcriptome analysis part. The authors introduced "spatial transcriptomics" and"behavioral transcriptomics" in the abstract but it is hard to appreciate that the study resolves spatial transcriptomics. This causes unnecessary confusion. Second, probably related to the first question, it is hard to find the novelty of the study. Third, probably the last and most important part of the manuscript is to analyze the cells by Smart-seq. But the analysis was performed on the SO2 injured animal only and lacked experiment on wildtype mice. If the authors tried to prove the feasibility of the technique rather than resolving physiological mechanism here, then I would recommend explaining why wild type experiment was not performed.

The method described in the manuscript consists of two components: a novel tissue imaging platform, and characterization of a cellular behavior. Both steps can be generalized to different tissue contexts and different cellular behaviors, respectively. We have revised the title and abstract to specify the scope of this study and have also revised the text accordingly.

Live imaging allows us to observe cell behaviors in intact tissues but does not provide information on cell type. By profiling cells that are observed by live imaging to share a behavior at single-cell resolution rather than bulk, we can separate out sources of transcriptional variation, like cell type identity, in order to identify the transcriptional signatures that reflect cell behaviors.

Single-cell sequencing (via Smart-seq) has been previously performed in wild-type mouse trachea (Montoro et al., 2018), and identified underlying cellular heterogeneity. However, the steady state tracheal epithelium is largely quiescent, characterized by slow turnover and a lack of visible cell motility. We performed daily imaging of trachea explants from uninjured mice over 4 days and did not observe any significant displacement of epithelial cells. Furthermore, we also imaged an uninjured explanted tracheal epithelium every 40 minutes for over 19 hours with no significant directional movement (new Movie 3). We added the following text to the manuscript: “Imaging of trachea explant controls from uninjured mice over 19 hours revealed no cellular displacement in the airway epithelium (Movie 3).”

In contrast, regeneration activates cell motility followed by cell proliferation. Therefore, we chose tissue regeneration as the more suitable biological context for this study to examine cellular dynamics. We leveraged the gene signatures derived from the previous wild-type study (Montoro et al., 2018) to identify different cell types and make like-for-like comparisons. We used an independent regeneration dataset in the same tissue but with a different injury model (Plasschaert et al., 2018) to test whether the molecular signatures derived in our study that differentiate moving and non-moving cells are generalizable to other contexts.

Reviewer #2 (Public Review):

Kwok et al. devise a method that uses a transgenic mouse line to make the link between cell behaviour in intact living tissue and subsequent dissociation into distinct groups forsingle cell sequencing. Specifically, they set up a mouse airway culture system in which it is possible to maintain live cells for multiple days and then preserve the same tissue. The analysed tissue section can be fixed and known cell types identified via classical staining protocols. In this system they imaged a number of tissue phenotypes such as ciliary beating, mucociliary clearing and airway regeneration. With respect to airway regeneration they observe that there was cellular heterogeneity between cells with the capacity to move and so-called non-movers, which the authors were able to quantitively track.To make the link with single cell sequencing, they use the Kaede transgenic mouse lines,which contains a green fluorescent reporter gene, that can be converted into a red fluorescent reported gene by illuminating a defined tissue section, in this case regions enriched for movers or non-movers. After dissociation of the tissue, cells were FACSsorted using the reporter protein. Subsequent single cell RNAseq revealed distinct gene signatures that were associated with the mover versus the non-mover phenotype. These phenotypes could also be detected in previously published data sets.

The conclusions of the paper are supported by the data that is presented, but the comparison to existing mouse injury data could be improved. A weakness of the paper is the implication that the technique can be used for any of the phenotypes that they have examined. However, in order to be assessed by this method,there need to be a reasonably large number of cells that show similar behaviour in a region that can be photoconverted. If it is indeed possible to do the photoconversion at the single cell level, the authors should demonstrate that such resolution is possible, or otherwise clearly state this limitation of the technique they have developed.

We recognize that the approach in this study does not involve photoconversion at single-cell resolution. While single-cell photoconversion and subsequent intermittent live imaging has been demonstrated in other systems such as zebrafish (Green and Smith, 2018) and mouse skin (Park et al., 2017), the throughput of doing downstream single-cell analysis would be limited, especially in a cell type-specific manner. Having observed a relatively homogeneous behavior of cells within a small region (~200 μm diameter, Movie 1 and Movie 2) of the airway epithelium, we photoconverted a small area with several hundred cells. Subsequent single cell sequencing allowed us to compare differences in gene expression between basal cells of slow/non-moving regions to basal cells of fast/moving regions.

Reviewer #3 (Public Review):

In this manuscript, the authors identify a pressing need to couple visualized in situ cell behaviour with deep molecular profiling of visualized cells, aiming to move beyond inferences made from time-lapse tissue sampling approaches or the analysis of transcriptional kinetics to identify the molecular pathways that drive cellular behaviour in situ. The authors identify live cell imaging combined with deep molecular profiling of theimaged cells as one possible solution. To this end, the authors establish a novel platform for live cell imaging of tracheal epithelial cells using explants of mouse trachea that allows long-term visualization of cell behaviour, and try to couple live-cell imaging to the transcriptional cell states.

Combining single-cell RNA-seq analyses with live cell imaging offers the unique opportunity to link transcriptional and anatomic, morphological or movement phenotypes of individual cells. To be able to do this in intact tissues at baseline and in response to injury would allow a far more detailed and integral analysis of cellular behaviour in their physiological context. As such, the approach of the authors is interesting and clearly focused on achieving this goal. The only data that can support a claim of successfully achieving this ambitious goal are presented in figure 3, where an advanced mouse model(the Kaede-Green mouse) is used that allows labelling individual cells by photo-conversion, followed by isolation of individual cells by flow cytometry and plate-basedsc RNA-seq analysis of sorted cells. By taking this approach, the authors are able to identify transcriptional differences at the group level between tracheal epithelial cell subsets that differ in their movement after injury.

While this in itself is a remarkable accomplishment, and an interesting observation, the relationship between the 'behaviour' of the cells observed with live cell imaging (the movement after injury) versus the transcriptional phenotype remains rather elusive. One explanation could be that active movement of cells depends on a specific transcriptional program, that is lacking from the non-moving cells. Another explanation could be that the tracheal epithelial cells are inherently heterogeneous, and one subset has the capacity to move whereas others do not, and the transcriptional profile merely identifies these heterogeneous populations. The observation that non-mover cell populations contain both basal and club cells, whereas mover regions only have basal cells seems to support this notion to some extent. However, the authors then claim to use basal-cell derived signatures (excluding the club cells) from mover and non-mover regions and compare this to literature data from another injury model to show that these signatures also identify distinct subsets in a mouse model of polidocanol-induced injury. How the distinction basal vs club cells in the non-mover regions is made remains unclear, and would seem challenging from the number of cells analyzed (as presented in figure 3).

The identification of two behavioural phenotypes of basal cells (mover vs non-mover) in this manuscript is based on group-level phenotypes: the cells belong to a region of moversor a region of non-movers. This is relevant for figures 2 (including supplemental) and 3. In figure 2 supplemental 2C, it seems evident that within one region (or focussing only on all moving regions?), the behaviour of all cells within that region/selection is quite uniform:the variation is really very limited, and all cells seem to speed up and slow down in a highly coordinated fashion within the selected regions shown. At the same time, in figure2D, the distribution of regions across speed categories at 26-36 hours pi (the peak of the movement in suppl 2C) seems almost bimodal, with regions belonging either to non-mover(range 0.5 - 2.5 uM/hr) or mover (range 3.0-7.0 uM/hr) phenotypes. However, all regions display an increased movement at 16h pi compared to the pre-injury movements (Figure2C), indicating that all cells will be induced to induce movement to some extent.

My main concern with this analysis is that the behavioural phenotype of the epithelial cells is assumed to be homogeneous within each region, allowing a contrast to be made in figure3 for the transcriptional phenotypes on the basis of moving phenotypes rather than on the basis of the main variation within the dataset.

For instance, from the t-SNE plot (3B) - for what it's worth of course - and the heatmap (3C) there seems to be at least one non-mover cell that transcriptionally has a higher resemblance to the mover cells than to the other non-mover cells. Of course that can just be the variability present in the dataset, but it could also indicate that non-mover regions are not completely homogeneous, and even more so, that the moving vs non-moving associated transcriptional phenotype is a gradual transition rather than 2 clearly separate sub-phenotypes.

All-in-all, this manuscript describes an interesting technical advance and shows some of the applications thereof. However, the approach also has its limitations: The requirement to mark cells with specific behavioural features for follow-up transcriptomic analysis (such as by photoconversion) necessitates the division of the epithelial cells into major categories on the basis of certain cellular phenotypes (such as movement) that can be visualized by live cell imaging. This limits the analysis opportunities to group-based contrasts in cellular behaviour as also used here by the authors.

Also, the use of explanted tissue is of course less ideal than in vivo imaging, but most likely the only technically feasible approach at this moment. At the same time, the capacity to combine image-based features with single-cell transcriptomic data is an important advance, even when initially only possible in explanted tissue from mouse models carrying all kinds of fluorescent reporters. To strengthen the manuscript, it would therefore be important to discuss the limitations of the approach, as well as to provide a more comprehensive overview of the possible applications that the authors foresee.

We thank the reviewer for the feedback. Our data demonstrates that the movement behavior is an injury-induced phenotype. 24 hours after injury (hpi), the “mover” transcriptional program is transiently enriched, while the “non-mover” transcriptional program is also transiently decreased, consistent with a cell state that is induced by injury (see Figure 4A, 24-hpi).

SO2 removes nearly all the luminal cells (Rock et al., 2009) so we removed the club cells to compare injury response in basal cells. Distinguishing basal vs club cells is done by hierarchical clustering and comparison to established cell type signatures (Montoro et al., 2018). We apologize that the initial presentation did not make this clear. In the revised manuscript, we have provided an additional figure supplement demonstrating the hierarchical clustering (Figure 3 - figure supplement 1A), and the disjoint expression of canonical markers Krt5 (basal) and Scgb1a1 (club), which enabled us to assign unambiguous cell-type identities to discovered clusters (Figure 3 - figure supplement 1B).

We agree with the reviewer that all cells, including cells that we classified as “mover” and “nonmover” are induced to move compared to pre-injury as suggested by Figure 2c. However, “mover” and “non-mover” cells differ dramatically in the amplitude and collective directionality of movement. We investigated the movement phenotypes in detail, including high-resolution imaging at shorter time intervals (10 min). We found that the slow “non-movers” had a large circular directionality variance (akin to oscillations), whereas the rapid “movers” moved directionally across the field of view. We quantified this with particle image velocimetry in Figure 2 – figure supplement 3C-D, and we revised the text to provide additional details about this result.

The reviewer also raises concern about whether the movement is homogeneous enough to account for the variation in the datasets. We used our imaging data to determine the time points in which the mover and non-mover phenotypes varied the most (around 40 hrs post injury) between different regions (Figure 2 - figure supplement 2A, C) but we have also demonstrated that the movement within each region is indeed relatively homogeneous (~200 μm diameter, Movie 1 and Movie 2).

We acknowledge that the presented data did not eliminate the possibility of another main variation within the dataset. We now perform PCA on the dataset, which confirmed that while the first principal component (PC) is associated with a solitary pulmonary neuroendocrine cell, the second PC is strongly associated with the difference between moving and non-moving cells (p=0.003, Wald test). When analyzing only the basal cells, we find that PC-1 provides a very clean separation and overlaps perfectly with the moving vs non-moving distinction (p<2 x 10-16, Wald test, Figure 3 - figure supplemental 2a). Taken together, with this additional analysis we can confirm that our focus on this behavioral phenotype reflects the main variation within the dataset.

We appreciate the reviewer’s nuanced question about the single outlier cell. While we do observe a transcriptional phenotype that is clearly distinct, as the reviewer points out, there is a very small degree of overlap between the two cell type clusters visible on the t-SNE plot in Figure 3B. Given that the physical process of movement is a matter of degree, it is possible that this particular cell is simply not moving as much, and thus activating movement-related transcriptional programs to a lower degree. To analyze this question further in response to this question, we analyzed the separability of these groups by training a machine learning (k-nearest neighbor) classifier to distinguish these clusters (new Figure 3 - figure supplement 2b). We found that the groups could be distinguished with a high accuracy of 98.7% (95% CI: 92.7-99.9) using 5 or more of the signature genes that we defined in Figure 3C. This additional analysis we continue to conclude that while the groups have a very small degree of overlap, the moving and non-moving phenotypes are strongly separable.

We acknowledge the limitations of this approach to groups of cells (see response to Reviewer 1) and both the limitations and advantages of using a tissue rather than cells, and we added these points to the discussion section.

Sports teams really grow when they have strong roots in the community, just like the church.

hmmmm, maybe everyplace should become the landfill then realize what that causes and then gain this goal and starts to work for it. Maybe that will help a lot. Other than that, it's really cool how they have that goal and are getting closer and closer to it.

Bono gets it!

This ties up his argument nicely. He hones in on the fact that chess was meant as a mimic to real life, and it's only fitting that you can adversely take examples in real life, and break them down using chess as a reference and as a point of comparison.

Some may say it is okay to be rude on social media because it is posted on a public platform but that is not always the case. Just because something is posted online does not make it acceptable to harass someone publicly in their comment section.

When people have nothing else to do, they look for excitement by publicly humiliating strangers online. It gets to the point where a user forgets the purpose of the cancellation that it's just constant harassment.

One example that exemplifies this the most was the TikTok in the link below about 'couch guy', a person who was stalked and harassed over his "lukewarm" reaction to the arrival of his long-distance girlfriend. He was essentially just a normal person who ended up in a viral TikTok and and then had his entire life investigated along with his body movements analyzed by random people on the internet. It seems a little scary that a large portion of public shaming just seems to occur by mob justice.

Authors' response (28 November 2022)

GENERAL ASSESSMENT

This interesting preprint by Suárez-Delgado et al. explores the mechanism by which activation of the Hv1 voltage-activated proton channel is dependent upon both the voltage and pH difference across the membrane. The authors are the first to incorporate the fluorescent unnatural amino acid, Anap, into the extracellular regions of the S4 helix of human Hv1 to monitor transitions of S4 upon changes in voltage or pH. The authors first checked that Anap is pH insensitive for practical use in Hv1, where changes in local pH are known to occur when the voltage sensor activates and the proton pore opens. Anap was incorporated at positions throughout the S3-S4 linker and the extracellular end of S4 (up to the 202nd residue) of hHv1 and some positions showed clear voltage-dependent changes in fluorescence intensity. The authors also obtained fluorescence spectra at different voltages and observed no spectral shifts, raising the possibility that voltage dependent changes in fluorescence intensity could primarily be due to fluorescence quenching. Upon mutation of F150, the Anap signal at the resting membrane voltage increased, suggesting dequenching upon removal of F150. The authors also discovered that the kinetics of Anap fluorescence upon membrane repolarization have two phases (rapid and slow) under certain pH conditions and that there is a pH-dependent negative shift of the conductance-voltage (G-V) relation compared with the fluorescence-voltage (F-V) relation in some mutants. The biphasic kinetics of the fluorescence decay upon repolarization were explained by modelling a slower transition of return from intermediate resting state to a resting state. The pH-dependent shift of the G-V relation from the F-V relation provides insight into mechanisms of ΔpH-dependent gating of Hv1, a longstanding enigma. Overall, the approaches are rigorous, the figures show important results, and this work paves the way for the use of Anap fluorescence to study Hv1 gating and modulation.

We thank the reviewers for the careful reading and assessment of our manuscript and for the constructive criticism. We have tried to respond to all the essential revisions, both by rewriting sections and performing some experiments or new analysis. Below we respond one by one to all the points raised. Please also note that we have added an author to the manuscript, who has carried out new experiments included in this revised version of the preprint.

RECOMMENDATIONS

Revisions essential for endorsement:

1) In its current form, the narrative of the preprint has two threads. One on the mechanisms of Anap fluorescence changes (mainly quenching) and another on a previously unappreciated transition of the voltage sensor, as revealed by Anap. Our impression is that the preprint suffers somewhat from this split focus, which could be resolved by explaining why Anap was used to explore voltage sensor activation in Hv1 in the introduction. Perhaps the authors could also explain the advantage of smaller sized fluorophores compared to other maleimide-based fluorophores earlier in the introduction, or the utility of being able to insert Anap into transmembrane segments. The authors should more clearly point out how they exploited the advantages of Anap as a tool in this study. It would furthermore be helpful to discuss previous studies using nongenetic tools for VCF and spell out how they have delineated key aspects of Hv1, which would help to emphasize how several positions studied here (for example, 201 and 202) could not be labelled with cysteine-based fluorophores.

We think that this is a very useful suggestion and we have expanded the introduction to more pointedly indicate the contributions of previous voltage-clamp fluorometry experiments in Hv1 channels and to clearly explain why we chose to pursue the use of a genetically-encoded small fluorophore such as Anap.

2) We think the authors should be cautious about understanding the physicochemical nature of Anap using prodan as a model. It would be helpful to discuss the possibility that undetected spectral shifts due to a nonquenching mechanism could be overlooked, even though major signal changes can be explained by fluorescence quenching in their data. Regarding the mechanisms of remaining voltage-dependent fluorescence changes of F150A-A197Anap, it would be helpful for the authors to suggest possible ideas about which residues might account for remaining signals.

The beautiful spectral data for Anap is impressive. However, the physicochemical basis of the fluorescence change of Anap cannot be understood by simple extension of findings for prodan, which shows structural similarity to Anap. Our understanding is that changes in Anap fluorescence can only reveal a change in the structural relationship between Anap and one of its neighbors because the physicochemical basis of Anap fluorescence is complicated. For example, fluorescence could also be affected by the electrostatic environment, stretch of peptide bond, etc. Previous studies, including those of TRP channels, showed that the kind of environmental changes that Anap faces in ion channels do not necessarily induce large spectral shifts, unlike in cell-free spectral analyses using distinct solvents. Further, only minor shifts in spectra occur upon local structural change, as seen in previous work including Xu et al. Nat. Commun. 2020 11:3790. Such minor shifts could be perhaps overlooked even when Anap is incorporated into S4 and exposed to environmental change. Therefore, it is not easy to decode the physicochemical basis of Anap fluorescence changes. F150A-A197Anap has increased fluorescence and no change in spectral pattern, leading the authors to conclude that F150 quenches Anap fluorescence of A197 position. However, a significant amount of fluorescence change still occurs upon changes in membrane potential after F150 is changed to alanine (Figure 4). It is very likely that quenching is not the only mechanism underlying the observed voltage induced change of Anap fluorescence of Hv1. The authors suggest that remaining voltage-dependent fluorescence change of F150A-A197Anap could be due to interaction with other aromatic residues, but this has not been tested.

Thank you for pointing out our oversimplified discussion of the mechanisms of Anap fluorescence changes in Hv1 channels. We have taken into account your comments and present a more nuanced and toned-down discussion of the possible mechanisms at play in our experimental system.

3) The current version of the preprint is missing important control experiments, ideally performed using western blots to measure protein expression or, if that is not possible, proton current and fluorescence measurements, to demonstrate that protein expression or functional channels are not seen for all mutants in the absence of ANAP (but in the presence of the tRNA and Rs construct). A similar control for imaging would be to use ANAP alone without encoding.

We thank the reviewers for this recommendation. We show that the number of cells showing mCherry fluorescence is greatly diminished in the absence of L-Anap, but in the presence of the tRNA and synthetase. As suggested, we have included results of control experiments in which we attempted to record currents from cells expressing the constructs: F150A-A197tag, Q191tag, A197tag and L201tag co-expressed with the tRNA and synthetase-coding plasmid (pANAP) and in the absence of L-Anap. We struggled to find red fluorescing cells and recorded currents from a relatively small number of these cells, most of which was leak current. We now include these data in Figure 1-Supplement 1B. These control experiments show that there is very little leakage of expression of channels that did not incorporate Anap.

4) Aromatics in the S4 segment were ruled out as potential quenchers on the assumption that they would move together with Anap during gating. It should be noted, however, that Hv1 is a dimer and therefore a fluorophore attached to S4 in one subunit could be quenched by S4 aromatics in the neighboring subunit if were close to the dimer interface. In Fujiwara et al. J. Gen. Physiol. 2014 143:377-386, for example, W207 does not appear very far from labeled positions in the adjacent S4. This possibility should be mentioned in the discussion.

We appreciate the reviewers' concern regarding the role of other aromatic residues near Anap incorporation sites, especially the ones close to the subunit interface given that Hv1 is a dimer. We now mention the possibility that other residues could be quenching groups, especially given the fact that some quenching remains in the double mutant F150A-A197Anap (line 272 in results and line 432 in discussion). We have also included a new analysis of the ratio of Anap/mCherry fluorescence (at resting membrane potential) for all insertion sites. This shows a decreased ratio as Anap gets inserted in residues closer to the c-terminus of S4, which is evidence of a quenching group located near the center of the transmembrane domains (Figure 4-Supplement 1).

5) It is not clear whether the Anap spectra purely represent Hv1 incorporated into the plasma membrane or perhaps include signals from the cytoplasm or channels in internal membranes (whether assembled or incompletely assembled). It would be helpful to provide a more complete presentation of the data obtained and to provide more information in the Methods Section. In the Methods section, it is stated "The spectra of both fluorophores (Anap and mCherry) were recorded by measuring line scans of the spectral image of the cell membrane, and the background fluorescence from a region of the image without cells was subtracted". How are signals from cell membranes specified in this method being discriminated from those associated with the cytoplasm and intracellular membranes? If spectral data include signals from free Anap in the cytoplasm or Hv1 in intracellular membranes, spectral shifts upon membrane potential changes will be difficult to detect, even when Anap is incorporated into Hv1 and senses environmental change by voltage-induced conformational change. In Figure 3E, wavelength spectra were shown as standardized signals for different voltages. Amplitude change would be demonstrated (spectrum at different voltages without standardization should be shown).

We appreciate the concern related to the origin of the fluorescence signals and we have improved both the presentation and the associated figures. Since this is also a concern for the experiments that determined the pH-dependence of Anap incorporated at position Q191, we have included a figure supplement 1 to Figure 2 in which we explain how the membrane was visualized. We use mCherry fluoresce as an indication of plasma membrane-associated channels, since its red fluorescence is easier to detect in the membrane than Anap fluorescence (even though cytoplasm dialysis in whole-cell should diminish the amount of free Anap, it is difficult to distinguish Anap fluorescence in the membrane by itself). Once the membrane associated mCherry fluorescence is detected, the measurement of the spectrum from a very small membrane area is insured because the spectrograph slit delimits light collection to a very small vertical area and the horizontal line scan further limits light measurement. These procedures are now made explicit in methods section and supplementary figure mentioned before. Moreover, we explain that they were also followed in experiments where the cell was under voltage-clamp. The spectral data in Figure 3E is now presented without normalization to show the voltage-dependent change in amplitude without changes in peak emission wavelength.

In Figure 4, spectra were compared between A197Anap and F150A-A197Anap, showing increases of fluorescence in F150A-A197Anap. Was this signal measured at resting membrane potential? How does the spectrum change when the membrane potential is changed?

As in the experiments of figure 1E, the spectra were obtained in non-patched cells. Thus, the signal was measured at the HEK cell resting potential (~ -30 mV) and a ΔpH ≈ 0.2. We have now incorporated that information in the methods section and the figure description. On the other hand, we did not perform experiments measuring the double mutant spectra at different voltage steps, so we cannot respond to the second question.

Rationales for the confirmation of signals originating from the cell surface for Hv1 Anap might include the observations that: a) some mutants showed slightly different spectral patterns (in particular, Q191Anap showed a small hump at longer wavelengths, which is proposed to represent FRET between mCherry and Anap) and b) signal intensity was voltage dependent (if signals originate from endomembranes, they should not be voltage dependent). Mentioning these two points earlier in the text might help to alleviate concerns about the location of the protein that contributes to the measured signals.

These are great suggestions and we have incorporated them to the text (lines 156, 190 and 216 Results section), along with a better explanation of procedures followed to measure mostly membrane-associated fluorescence (see new Figure 2-Supplement 1).

6) In Fig 5, the fluorescence kinetics do not really match the current activation kinetics for panels A, B, and C. Is there an explanation for this mismatch? It would be helpful to have the fitted data in the figure. A more thorough comparison of the kinetics of currents and fluorescence would be helpful throughout the study.

We believe that the kinetics of fluorescence and current does not match because the current activation rate is overestimated due to a small amount of proton depletion present in recordings from large currents. This is an unavoidable problem in proton current recordings, even with the high concentration of proton buffer used in our experiments and the long time-intervals between each voltage pulse. For this reason, we did not undertake a systematic exploration of kinetics. Nonetheless, the current and fluorescence rates are very close and have the same voltage dependence, indicating a close correlation between voltage-sensor movement and current activation. We now explain this limitation in the manuscript text (line 223 and 327, results section).

7) Which construct of hHv1 was used to obtain the data in Figure 6? Unless we missed it, this information is not provided in the text or figure legend. Is it for L201Anap? This figure also shows an intriguing finding that the G-V relationship is negatively shifted from the F-V relationship at pHo7-pHi7 but not at pHo5.5-pHi5.5. A shifted G-V relation with the same ΔpH contrasts with what has been reported in other papers. However, the authors did not really discuss this surprising finding in the light of previous references. Could the shift of the G-V relation between two pH conditions with the same ΔpH be due to any position-specific effect of Anap? If Figure 6 represents L201Anap mutant, the presence of Anap at L201 probably makes such shift of G-V curve in Figure 6C? The authors should openly discuss this finding in relation to what has been reported in the literature.

Yes, construct L201Anap was used in Figure 6. This is stated now in the figure legend and in the corresponding main text. We agree that the leftward shift of the GV with respect to the FV in pHi7-pHo7 is an intriguing finding, suggesting that coupling between S4 movement and proton permeation can be regulated by the absolute value of the pH. We discuss this in the results section. The DeCoursey group has shown evidence in W207 mutants of hHv1 that the absolute value of pH can modulate the voltage dependence of the conductance. Although we had mentioned these results, we now mention them more prominently and also discuss the possibility that this might be a unique feature of introducing Anap at L201.

8) The authors suggest that the small hump near 600 nm in Figure 1E represents FRET between Anap and mCherry. It is surprising that FRET can take place across the membrane. Can the authors point to another case of FRET taking place across a cell membrane? One possibility might be that misfolded proteins place mCherry and Anap close to each other. It is also curious that only A191Anap did not show such a FRET-like signal. Also, if there is FRET, why wouldn't this also contribute to the voltage-dependent changes in fluorescence?

We thank the reviewers for bringing up this point. Based on published data, we assumed that mCherry could not be excited by 405 nm radiation, thus our conclusion that the observed emission near 604 nm is FRET between Anap and mCherry. We have now measured the excitation of the Hv1-mCherry construct and observe that the 405 nm laser is capable of exciting mCherry and produced ~2 % emission (as compared to 514 nm excitation), which is almost the same as that observed for the Hv1Anap-mCherry channels. We now conclude that the second hump in the emission spectrum near 600 nm is due to direct excitation of mCherry.

On the other hand, FRET across the membrane has been demonstrated for the membrane-bound hydrophobic anion dipicrylamine and membrane-anchored GFP (Chanda, et al. A hybrid approach to measuring electrical activity in genetically specified neurons. Nature neuroscience, 2005, vol. 8, no 11, p. 1619-1626.) and dipicrylamine and GFP in the c-terminus of CNG channels (Taraska & Zagotta, Structural dynamics in the gating ring of cyclic nucleotide–gated ion channels. Nature structural & molecular biology, 2007, vol. 14, no 9, p. 854). Finally, single-molecule FRET between dyes placed extracellularly and intracellularly in Hv1 channels has been demonstrated (Han et al. eLife 2022;11:e73093. DOI: https:// doi. org/ 10. 7554/ eLife. 73093).

A191Anap shows the hump at ~600 nm, but we think it's less evident because Anap at 191 is less quenched (see Figure 4-Supplement 1 and answer to point 4 above).

9) F150A-A197Anap shows a leftward shift of the F-V relation compared with the G-V relation only when ΔpH=1. Another unusual finding with F150A-A197Anap is the very small shift of the G-V relation between ΔpH=0 and ΔpH=1, when other reports in the literature suggest it should be 40 mV or more. Are these peculiar properties simply due to the absence of Phe at position 150, which might play a critical role in gating as one of the hydrophobic plugs of Hv1? To address this possibility, it would be ideal to compare different ΔpH values with and without F150 when Anap is incorporated at a different position (such as L201Anap). Regardless, it would be helpful to discuss this point.

We now discuss these changes in the discussion (lines 440-446).

10) In Figure 1E, I202Anap exhibits a blue shift in its spectrum suggesting the environment of Anap on I202 is more hydrophobic than the other sites. We presume these spectra were obtained at a negative membrane voltage, but the text or legend should clearly state how these were obtained. The authors should also explain whether the whole cell or edge was imaged. If these are at negative membrane voltages, might the Anap spectrum shift to higher wavelengths (i.e. more hydrophilic) when the membrane is depolarized? Did the authors find any spectral shift for I202Anap when doing a similar test as depicted in Figure 3E?

Yes, the spectrum of I202Anap was obtained at the resting potential (~ -30 mV), as were all spectra in Figure 1E. We now indicate this clearly in the methods section and in the figure legend. Fluorescence was measured from the membrane region as indicated by mCherry fluorescence and as illustrated in Figure 2-Supplement 1. We did not explore this mutant further and we cannot answer the question of whether a depolarizing potential might produce a red shift of the spectrum.

11) In Figure 3E, spectra are shown as normalized signals for different voltages, but an amplitude change should also be demonstrated by providing raw spectra at different voltages.

We have changed figure 3E to show non-normalized data that now show the increase in fluorescence intensity and no wavelength shift in the fluorescence spectrum of Anap (see also response to point 5).

12) In Figure 4, spectra are compared between A197Anap and F150A-A197Anap, showing increase of fluorescence in F150A-A197Anap. Were these obtained at a negative membrane voltage? How do these spectra change when membrane potential is changed?

See response to point 4 of "Revisions essential for endorsement" section.

Additional suggestions for the authors to consider:

1) The authors propose that Anap fluorescence tracks an S4 movement involved in the opening of the channel. They also argue that the existence of more than one open state could explain why the increase in florescence upon depolarization lags the proton current in most cases. While they convincingly show that Anap is not pH sensitive per se, when incorporated into the protein, the fluorescence efficiency of the fluorophore could still be affected by protonation of channel residues in the immediate environment when the channel opens, even after S4 has completed its movement. To address this alternative explanation, the authors could use Hv1 mutants with strongly reduced proton conductance. Channels bearing mutations corresponding to N214R or D112N were used successfully to isolate Hv1 gating currents from the much larger proton currents (De La Rosa & Ramsey, Biophys. J. 2018 114:2844-2854; Carmona et al. PNAS 2018 115:9240-9245; Carmona et al. PNAS 2021 118: e2025556118). Perhaps, they could be used with patch clamp fluorometry as well?

This is an interesting suggestion that could be explored in a follow up study.

2) The data showing that Hv1-197Anap is quenched by Phe at position 150 are very nice. Yet, it would be useful to show that the quenching is specific to F150 using a negative control. F149, for instance, is just next to F150 but points in a different direction, so its mutation to alanine should not affect Hv1-197Anap fluorescence.

This is an interesting suggestion, but, as suggested by reviewers, we think there is a possibility that other aromatic residues could contribute to quenching. Given the absence of a reliable structure for Hv1, prediction of the relative positions of any resides is very difficult and thus we did not attempt the suggested experiment.

3) A major finding of this work is the identification of a slow kinetic component that is highly sensitive to ΔpH. Earlier studies found that the ability of Hv1 to sense ΔpH is altered by some channel modifications, e.g., in the loop between TMH2 and TMH3 (Cherny et al. J. Gen. Physiol. 2018 150:851-862). Did the authors check whether any of these modifications alter the transition responsible for the slow kinetic component? For instance, a suppression of the transition resulting from a H168X mutation would help tighten the link to ΔpH sensing.

We did not carry out any of these experiments.

4) We understand that it is difficult to tightly control intracellular and extracellular pH when Hv1 is heterologously expressed in mammalian cells. The G-V relation is not always reliable because accumulation of protons or depletion of protons upon Hv channel activity will alter gating, as the authors have previously published (De La Rosa et al., J. Gen. Physiol. 2016 147:127-136). Could the kinetic analysis of Anap fluorescence be affected by similar alterations to proton concentration in the vicinity of Hv1? It would be helpful for the authors to comment on this specifically.

Thanks for this suggestion. Yes, we think that the kinetics, specially of ionic currents can be affected by even small changes in the pH gradient, for this reason we did not attempt a systematic kinetic analysis. We mention this in the text where we compare the voltage dependence of current and fluorescence activation for construct A197Anap (line 223).

5) Quenching of Anap by Phe could be verified in cell free conditions using a spectrophotometer with different concentrations of Phe, or citing the literature if it has already been reported.

We attempted this experiment but were unsuccessful in observing Anap quenching by phenylalanine at the concentrations of phenylalanine that can be attained in aqueous solution. We suspect that Phe quenching of Anap could happen by electron transfer or ground-state complex formation, in which case near proximity is necessary and higher concentrations of Phe would be required to detect quenching in solution. However, we measured the absorbance of Anap in the absence and presence of phenylalanine (Phe) (and tyrosine (Tyr)) at the concentrations that can be achieved in aqueous solution (8 mM and 1mM, respectively). Absorbance measurements can detect ground-state complex formation even at relatively low concentrations (J.R. Lakowicz, 1999, Principles of Fluorescence Spectroscopy). We observed that the absorbance of Anap is modified by the presence of Tyr or Phe, indicating that these amino acids indeed interact with Anap, possibly through ground-state complex formation. We include this data for the reviewers to inspect.

6) The authors did not cite any example of Anap incorporation into S4 helices, but there are several recent papers where Anap was utilized to probe motion of S4 in other channels. Examples include Dai et al., Nat. Commun. 2021 12:2802 and Mizutani et al. PNAS 2022 119:e2200364119.

Thanks for this observation, we have included these important results in the discussion.

7) In the Anap-free negative control (with only A197TAG plasmid transfection), the mCherry signal seems positive (Supplementary Figure 1, left row, second from the top). Is this due to unexpected skipping of the TAG codon to make mCherry-containing partial polypeptides? It would seem like an explanation is needed.

Thanks for bringing this up. We do not know the exact origin of these leak expression of red fluorescence. We think that, as suggested, there is a possibility that skipping of the Amber codon can lead to a methionine at the end of S4 acting as a second translation initiation site, giving rise to truncated channels that would express mCherry but not currents. This is consistent with the fact that we cannot detect currents in the absence of Anap but we see a small number of red cells.

8) The data of Figure 3E are shown as data with different membrane voltages. But there is no information about membrane voltage for Fig. 1E and Fig. 2A and Fig. 4B. Are these from unpatched cells? Please clarify.

See response to point 4 of "Revisions essential for endorsement" section.

9) G-V relations are shown for F150A-A197Anap, but current traces of F150A-A197Anap are missing.

We have modified the figure to include current and fluorescence traces.

10) On Page 11, Line 303 says "experimental F-V relationship is positively shifted by 10 mV with respect to the G-V curve". But looking at the data Fig5D, the shift at ΔpH=2 seems the opposite. Perhaps "positively" should be "negatively" in this sentence?

Thanks for pointing out this mistake. We have found that this misunderstanding was provoked because of a mistake with the image labeling of F-V and G-V curves for the ΔpH=2 data, we have now corrected the figure. The shift of F-V is indeed positive to G-V as stated before.

(This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

The writer might be constructing a narrative that points to the role of genders and how women are represented in sports. What are they supposed to/not supposed to do? How are they meant to act?

The story presents Serena Williams' perspective on the matter and how she sees it. This shows a credible source since Williams is said to be one of the greatest athletes in the world and has experience with performing on the biggest stages.

Noticing a theme of 'openness' in many of these tactics specifically being open about things that are often left unsaid particularly in "professional" academic contexts.

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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 #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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It's not just damaging to a student or just a person, but the persistent regurgitation of this belief ensues a greater bridge to cross in order to develop a counternarrative that can be healing as well as a means towards equality.

Literacy is how we make meaning of the world around us. It is communication at it's finest and the conversations that we have with the text in front of us. Just like it is hard for a non English speaker to read English, it is the same concept when we put irrelevant text in front of students who have never engaged with the topic.

Although not as strong an opinion today, it's crazy to think that during this time people truly felt that their faith in God is what gave them good fortune. Funny how the rich were able to "pay" for their way into heaven and said it was because they were better than the poor!

It's always interesting for a student to challenge their own thinking and questions the things that may be thought of as "absolute truths". Growth and understanding, empathy even, comes from these realizations.

Something I feel like that occurs so often within the church is that people expect a perfect preservation of tradition and philosophy. Realistically, traditions change. The interpretations and hermeneutics of the Bible differ from place to place and over time, tradition transforms. To be accepting of others, regardless of their gender, sex, identity, etc, is to love everyone just as you love yourself—something Jesus spent his life teaching, and what is essential to living under the image of God. In my opinion, there is no room for hate. It’s essential we embrace everyone, as we are all created by God.

Reviewer #1 (Public Review):

The authors tackle an interesting problem: how do ant colonies regulate foraging in response to their collective hunger? In previous work, the authors related the colony's response to individual ants sensing their own food levels and its temporal dynamics. Looking more carefully at the spatial dynamics of ants, the authors now find that foragers tend to move toward the depth of the nest when their food load is high and toward the nest exit when it is low. This is an elegant and computationally inexpensive set of rules that explains the spatiotemporal dynamics of the system.

Overall, the paper is written clearly, the methods are sound, and I agree with the interpretation of the results.

I do have a few comments and suggestions:

1) How exactly are the inward outward directions defined? Is it simply, away, or towards the entrance? It is not clear from the text, and since this system is not symmetric (cubic with entrance at one of the corners) the authors should clarify.

2) To analyze the biased random walk analysis of the ants, the authors "coarse-grained" the steps as being "inwards" "outwards" and "stay". It's not clear how this level of granulation is justified. Since the authors have access to the actual trajectories and all trophallaxis events, why not just calculate the actual turning angles between consecutive steps the ants take? This would give an actual assessment of both the bias and the noise imposed on the random walks, which the authors could then use directly in their models.

3) It would be important to better connect the author's previous mechanism (relating the colony's response to individual ants sensing their own food levels and its temporal dynamics) to the new mechanism (spatial-temporal dynamics). Are they mutually exclusive? It would be useful to elaborate on this in the Discussion.

4) It would be useful to add a few supplementary movies from the experiments, showing ants moving toward the entrance with low food loads, and moving away from the entrance with high food loads.

Reply to Andy https://forum.zettelkasten.de/discussion/comment/16865#Comment_16865

Andy, I'll take you at your word. You're right that none of it requires extraordinary genius--though many who seem to exhibit extraordinary genius do have variations of these practices in their lives, and the largest proportion of them either read about them or were explicitly taught them.

With these patterns and practices being so deeply rooted in our educational systems for so long (not to mention the heavy influences of our orality and evolved thinking apparatus even prior to literacy), it's a bit difficult for many to truly guarantee that they've done these things independently without heavy cultural and societal influence. As a result, it's not a far stretch for people to evolve their own practices to what works for them and then think that they've invented something new. The common person may not be aware of the old ideas of scala naturae or scholasticism, but they certainly feel them in their daily lives. Commonplacing is not much different.

By analogy, Elon Musk might say he created the Tesla, but it's a far bigger stretch for him to say that he invented a new means of transportation, or a car, or the wheel when we know he's swimming in a culture rife with these items. Humans are historically far better at imitation than innovation. If people truly independently developed systems like these so many times, then in the evolutionary record of these practices we should expect to see more diversity than we do in practice. We might expect to see more innovation than just the plain vanilla adjacent possible. Given Margolin's age, time period, educational background, and areas of expertise, there is statistically very little chance that he hadn't seen or talked about versions of this practice with several dozens of his peers through his lifetime after which he took that tacit knowledge and created his own explicit version which worked for him.

Historian Keith Thomas talks about some of these traditions which he absorbed himself without having read some of the common advice (see London Review of Books https://www.lrb.co.uk/the-paper/v32/n11/keith-thomas/diary). He also indicates that he slowly evolved to some of the often advised practices like writing only on one side of a slip, though, like many, he completely omits to state the reason why this is good advice. We can all ignore these rich histories, but we'll probably do so at our own peril and at the expense of wasting some of our time to re-evolve the benefits.

Why are so many here (and in other fora on these topics) showing up regularly to read and talk about their experiences? They're trying to glean some wisdom from the crowds of experimenters to make improvements. In addition to the slow wait for realtime results, I've "cheated" a lot and looked at a much richer historical record of wins and losses to gain more context of our shared intellectual history. I'm reminded of one of Goethe's aphorisms from Maxims and Reflections "Inexperienced people raise questions which were answered by the wise thousands of years ago."

this! it's easy to look at the wrongs but our kids come with so many advantages we just need to listen to them!

money as a commodity

nullifies it's utility

delusion

want money as a measuring stick

Usenet

rideshare Simpson episode toilet paper roll

unregulated

Connecting ideas across space and time when you don't know how they'll fully relate in advance is a tough design problem.

Outliner programs, first developed for computers in 1968, are great if you know the structure of a space in advance, but creating hierarchies by guessing about relationships in advance often turn out wrong or create other problems as one progresses.

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

_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.

Discussion, revision and decision

Decision

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

Dr. Bañuelos: Verified manuscript

Dr. Morris: Verified with reservations

Revision

Response to Reviewer 1 (Dr. Bañuelos)

1. Most importantly, I would like to see an introduction that explains the authors’ general arguments about grading changes – including the trajectory of these changes at Dalhousie and why this arc contributes to our knowledge of the history of higher education more broadly. Then, the authors might continually remind us of the arc they present at the outset of their paper – especially when they are highlighting a piece of evidence that illustrates their central argument. To me, the quotes from students and faculty responding to grading changes are among the most interesting parts of the paper and placing these in additional context should make them shine even more brightly!

Our Response: Thank you so much for your thoughtful review. We have added a larger new introduction section of the paper (paragraphs 1-5 in the latest draft are new) that outlines the general importance of the topic, the Canadian context, details on Dalhousie University, and our overall thesis statement (i.e., most decisions were to improve the external communication value of grades). Moreover, we have added three new student quotes form the Dalhousie Gazette to build a stronger picture for student reactions, and to build a better case for our overall thesis statement (i.e., that changes in grading were often to increase the external communication value of grades). Moreover, throughout we have added some details on the overall funding trajectory for institutions in Canada that created some pressure to standardize grading. We think that these changes have improved the manuscript.

1. I’d like to read a little more about Dalhousie itself – why it is either a remarkable or unremarkable place to study changes in grading policies. Is it representative of most Canadian universities and thus, a good example of how grading changes work in this national context? Is it unlike any other institution of higher education and thus, tells us something important about grades that we could not learn from other case studies? I don’t think this kind of description needs to be particularly long, but it should be a little more involved than the brief sentences the authors currently include (p.3, paragraph 1) and should explain the choice of this case.

Our Response: This comment revealed that two additional pieces of context were needed for the introduction: (a) some national context for higher education policy in Canada and (b) some extended description of Dalhousie University when compared to other universities in Canada. To this end, two new paragraphs have been added to the paper (paragraphs 2 & 3 in the current draft).

Notably, Jones (2014) notes that “Canada may have the most decentralized approach to higher education than any other developed country on the planet” (pg 20). With this in mind, any historical review of education policy is by necessity specific to province and institution – that is, the information can be placed in its context, but resists wide generalization to the country as a whole. In the newest draft, we tried to describe the national, provincial, and institutional context in some more detail in paragraphs 2 & 3.

1. I’d also like to know more about the archival materials the authors used. The authors mention that they drew from “Senate minutes, university calendars, and student newspapers” (p. 3), but what kinds of conversations about grades did these materials include? At various points, the authors engage in “speculation” (e.g. p.4) about why a particular change occurred. This is just fine and, in fact, it’s good of the authors to remind us that they are not really sure why some of these shifts happened. But, they might go one step further and tell us why they have to speculate. Were explicit discussions of grading changes – including in inter- and intradepartmental letters and memo, reports, and other documents – not available in these archives? Why are these important discussions absent from the historical record?

Our Response: We have added a new paragraph (paragraph 4) to the paper discussing the sources in some more detail. It is true that the verbatim discussions are frequently absent from the record, especially earlier in history – or if they exist, we have not found them! Instead, we frequently are reviewing meeting minutes or committee reports, which are summaries of discussions. As we now note in the paper, “Thus, the sources used showed what policy changes were implemented, when they were implemented, and a general sense of whether there was opposition to changes; however, there were notable gaps in faculty and student reactions to grade policy changes, as these reactions were frequently not written down and archived.”

This gap was most apparent in the Senate minutes around the 1940s, where I (the first author) could not find any direct discussions of why changes were implemented. Under the 1937-1947 heading, we more clearly indicate that the rationale for the changes was absent from the Senate minutes during this period. I add some further speculation on why these records might be absent, based on summaries from Waite (1998b); specifically, the university president of the time often made unilateral decisions, circumventing Senate, which might account for why the changes are absent from the records.

This will hopefully make the limitations of what can be learned from this approach more apparent.

1. At various points, the authors make references to the outside world – for example, WWII (p. 5), the Veteran’s Rehabilitation Act (pp. 6-7), and British versus American grading schemas (p. 6). But, these references are brief and seem almost off-handed. I know space is limited, but putting these grading changes in their broader context might help make the case for why this study is interesting and important. Are the changes in the 1940s, for example, related to the ascendance of one national graduate education model over another (e.g. American versus British)? Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time? If so, I would share any information you might have on these broader trends.

Our Response: To our knowledge, there isn’t any comparable report to what we’ve written here documenting the transition from British “divisions” to American “letter grades” in Canadian Universities, making our report novel in this regard. It might well be that a similar historical arc exists in many of the 223 public and private universities in Canada, but we don’t believe such data exists in any readily accessible way – excepting perhaps undergoing a similar deep dive into historical documents at each respective institution! So, we do not have the answer to your question: “Are there any data on how many Canadian undergraduates enrolled in British versus American graduate programs over time?” However, we did add one reference which provided a snapshot point of comparison in 1960, noting in the paper “Baldwin (1960) notes that the criteria for “High First Class” grades in the humanities was around 75-80% at Universities of Toronto, Alberta, and British Columbia in 1960, suggesting that Dalhousie’s system was similar to other research-intensive universities around this time.” That said, there are a few major national events related to the funding of universities in Canada that we have elaborated on in the text to address the spirit of your recommendation for describing the national context:

a) In the “Late 1940s” section of the paper, we added: “Though Dalhousie had an unusually high proportion of veterans enrolled relative to other maritime universities during this period (Turner, 2011), the Veteran’s Rehabilitation Act was a turning point for large increases in enrollment and government funding Canada-wide, at least until the economic recession of the 1970s (Jones, 2014).”

b) In the 1990s, there were major government cuts to funding, creating challenging financial times for the university. We discuss the funding pressures that likely contributed to standardization of grading during this time by saying the following in the 1980s-2000s section: “Starting in in the 1980s-1990s there were major government cuts to university funding nation-wide, with the cuts becoming more severe in the 1990s (Jones, 2014; Higher Education Strategy Associates, 2021). Because of the nature of the funding formulas, cuts in Nova Scotia were especially deep. Beyond tuition increases, university administrators knew that obtaining external research grants, Canada Research Chairs, and scholarship funding was one of the few other ways for a university to balance budgets, so there was extra pressure to be competitive in these pools. […] The increased standardization was likely related to increased financial pressures at this time – standardization is an oft-employed tool to deal with ever-increasing class sizes with no additional resources.”

c) In the 2010s section of the paper, we added context to how universities in country-wide have become increasingly dependent on tuition fees for funding: “Following the 2008 recession, federal funding decreased again (Jones, 2014; Higher Education Strategy Associates, 2021); however, this time universities tended to balance budgets by increasing tuition and international student fees. This trend towards increased reliance on tuition for income is especially pronounced in Nova Scotia, which has the highest tuition rates in the country (Higher Education Strategy Associates, 2021). Thus, the university moved closer to a “consumer” model of education, so it makes sense that a driving force for standardization was student complaints.”

1. This is a very nitpicky concern that doesn’t fit well elsewhere, so please take it with a grain of salt. I was surprised at the length of the reference list – it seemed quite short for a historical piece! I wonder, again, if more description of the archival material - including why you looked at these sources, in particular, and what was missing from the record – would help explain this and further convince the reader that you have all your bases covered.

Our Response: In the introduction section, paragraph 4, we describe our sources in more detail including what is likely missing from the record and why we used them. Regarding the length of the reference list, we did add ~12 new references to the list in the course of making various revisions, which partially addresses your concern. Beyond this though, it’s worth noting that some of the sources more extensive than they seem, even though they don’t take up much space in the reference list (e.g., there is one entry for course calendars, but this covers ~100 documents reviewed!). Moreover, there were many dead-ends in the archives that are not cited (e.g., reviewing 10 years of Senate minutes in the 1940s produced little of relevance), so the reference list is curated to only those sources where relevant materials were found.

Reviewer response to revisions

The new introduction to the piece addresses many of my previous questions about the authors’ general arguments, the Dalhousie context, and the source material. Thank you for addressing these! Reading this version, it is much clearer that the key argument is that standardized, centralized grading practices were “to improve the external communication value of the grades, rather than for pedagogical reasons” (p. 6). I also really enjoyed the added quotes from students in the Dalhousie Gazette.

The authors’ response to Reviewer 2 really gave me a better sense of why they wrote this piece and also helped me to more clearly put my finger on what was troubling me in the first round. It still reads a little like a report for an internal audience – which is just fine and, in fact, can be extremely useful for historians of the future. But, as Reviewer 2 notes, this means it does not really seem like a piece of historical scholarship. I do worry that shaping it into this form would take an extensive revision and might not be in the spirit of what the authors intended to do.

A different version of this article might start with this idea that grades were standardized for external audiences and in response to financial pressures. It would then develop a richer story behind the sudden importance of these external audiences and the nature (i.e. source, type) of financial pressures Dalhousie was facing. It would highlight the impact such changes had on students and their future careers/graduate experiences. It could then connect these trends to other similar changes for external audiences and the increasing interconnectedness of American, Canadian, and British systems through graduate education. It might even turn to sociological theories of organizational change and adaptation and make an argument for when (historically) similar forms of decoupling were likely to occur in the Canadian higher education system. Finally, it might connect these grading changes to current trends – including accusations of grade inflation and accepted best practices for measuring learning outcomes.

But, it doesn’t seem that the authors necessarily want to do this, which I can understand and respect. I think there is enormous value in a piece of scholarship like this existing – both for internal audiences and for future historians. Indeed, imagine if every university had a detailed history of its grading policies like this available somewhere online! Comparing such practices across institutions would certainly tell us a lot about why grading currently looks the way it does.

Decision changed

Verified manuscript: The content is scientifically sound, only minor amendments (if any) are suggested.

Response to Reviewer 2 (Dr. Morris)

The authors dove headfirst into Dalhousie’s archives, unpacking the subtle shifts in grading policy. Their work seems to be comparable to archaeologists, digging deep beneath mountains of primary sources to find nuggets of clues into Dalhousie’s grading evolution. I particularly liked when the authors were able to link these changes to student voices, as seen in moments when they referenced student publications.

Ultimately, I kept coming back to one main comment that I wrote in the margins: “So what?” I would humbly suggest that the authors reflect on why this history matters to them. Granted, they do this in the conclusion, where they touch on Schneider & Hutt’s argument that grades evolved to increasingly be a form of external communication with audiences beyond school communities. Sure. But I want more. I wanted to see a new insight that this microhistory of Dalhousie significant to the history of Canada or the history of education more generally.

If the authors are so inclined, there might be several approaches to transform this manuscript. I would suggest the following. First, instead of tracing the entire history of grading at the institution, choose one moment of change that you think is the most important. Perhaps in the 1920s and the lack of transparency in grading, or the post-war shift toward American grading. Second, show me – don’t tell me – what Dalhousie was like at this moment. Paint a picture of the institution with details about student demographics, curriculum, educational goals, the broader town, etc. Make the community come alive. Show me what makes Dalhousie unique from other institutions of higher ed. Once you establish that picture, perhaps you could link the change in grading practices to subtle changes at the university community, thereby establishing a before and after snapshot. This will require considerable amounts of work, and the skills of a historian. You will have to find primary and secondary sources that go far beyond what you’ve relied on thus far.

In the end, I found myself wanting the authors to humanize this manuscript, meaning I wanted them to show me that changes in grading practices have tangible effects on real-life human beings. A humanization of their research would mean going narrower and deeper; or, in other words, eliminating much of what they have documented.

However, if that is too tall of an order, I would ask that the authors clarify for themselves who this manuscript is for. Is this a chronicling of facts for an internal audience at Dalhousie’s faculty, alumni, and students? Fine. But my guess is that even members of the Dalhousie community want to read something relatable.

I am suggesting revisions, although not because of objective errors. History is more of an art, in my opinion. With that in mind, I would suggest that the authors paint a more vivid picture (metaphorically) of Dalhousie, showing me how changes one moment of change in grading practices impacted the lives of human beings.

Our Response: Thank you very much for taking the time to read our paper and provide your thoughts and recommendations. It may be helpful to begin by describing why I (the first author) decided to write this paper. Ultimately, I wrote this paper to satisfy my own personal curiosity and to connect with other people at my own place of employment by exploring our shared history. At present day, Dalhousie has a letter grading scheme with a standardized percentage conversion scheme that all instructors used. I wanted to know why this particular scheme was used, but I quickly realized that nobody at Dalhousie really knew how we ended up grading this way! There was an institutional memory gap, and a puzzle that was irresistible to me. So, I wrote this paper for the most basic of all academic reasons: Pure curiosity. I do very much recognize that the subject matter is very niche, perhaps too niche for a traditional journal outlet. Thus, my publishing plan is to self-publish a manuscript to the Education Resources Information Center (ERIC) database and a preprint server as a way of sharing my work with others who might be interested in what I found. Nonetheless, I believe in the importance and value of peer review, especially since I am writing in a field different than most of my scholarly work. That is why I chose PeerRef as a place to submit, so that I could undergo rigorous peer review to improve the work while still maintaining the niche subject matter and focus that drives my passion and curiosity for the project. Of course, if you feel the whole endeavor is so flawed that it precludes publication anywhere, then we can consider this a “rejection” and I will not make any further edits through PeerRef.<br /> The core of your critique suggested that I should write a fundamentally different paper on different subject matter. While I don’t necessarily disagree that the kind of paper you describe might have broader appeal, it would no longer answer the core research question I wanted an answer to: How has Dalhousie’s grading changed over time? So, I must decline to rewrite the paper to focus on a single timeframe as recommended. All this said, I did try my best to address the spirit of your various concerns to improve the quality of the manuscript. Below, I will outline the various major changes to the manuscript that we made to improve the manuscript along the lines you described, while maintaining our original vision for the structure and focus of the paper. The specific changes are outline below:

a) Two new paragraphs (now paragraphs 1-2 of the revised manuscript) were added to explain the “so what” part of the question. Specifically, we describe why we think the subject matter might be of interest to others and summarize the general dearth of historical information on grading practices in Canada as a whole.

b) Consistent with recommendations from the other reviewer, we now state a core argument (i.e., that most major grading changes were implemented to improve the external communication value of the grades) earlier in the introduction in paragraph 5 and describe how various pieces of evidence throughout the manuscript tie back to that core theme.

c) In an attempt to “humanize” the manuscript more, we added more student quotes from the Dalhousie Gazette throughout the paper so that readers can get a better sense of how students thought about grading practices at various times throughout history. Specifically, three new quotes were added in the following sections: 1901-1936, late 1940s, 1950s-1970s. We also added this short note about the physical location where grades used to be posted: “Naturally, this physical location was dreaded by students, and was colloquially referred to as “The Morgue” (Anonymous Dalhousie Gazette Author, 1937).”

d) Early in the paper, we describe why we chose Dalhousie and the potential audience of interest: “As employees of Dalhousie, we naturally chose this institution as a case study due to accessibility of records and because it has local, community-level interest. The audience was intended to be members of the Dalhousie community; however, it may also be a useful point of comparison for other institutions, should similar histories be written.”

e) We have described some of the limitations of our sources in paragraph 4, which may explain why the manuscript takes the form it does – it has conformed to the information that is available!

f) We have linked events at Dalhousie to the national context in some more detail, by detailing some national events related to the funding of universities in Canada. See our response to Reviewer 1, #4 above for more details on the specific changes.

g) Consistent with your stylistic recommendations, we have changed various spots throughout the paper from the present tense (e.g., “is”) to the past tense (e.g., “was”), and were careful in our new additions to maintain the past tense, when appropriate. If there are any spots that we missed, let us know the page number / section, and we will make further changes, as necessary.

h) We retained the first person in our writing – this may be discipline-specific, but in Psychology (the first author’s home discipline), first person is acceptable in academic writing. If you feel strongly about this, we can go through the manuscript and remove all instances of the first person, but we would prefer to keep it, if at all possible.

Hopefully this helps address the spirit of your concerns, and I look forward to hearing your thoughts in the second round of reviews.

Decision changed

Verified with reservations: The content is scientifically sound, but has shortcomings that could be improved by further studies and/or minor revisions.

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

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Referee #3

Evidence, reproducibility and clarity

Summary

In this manuscript, Vas and Boulet et al. presents the potential regulatory role of de novo mutations (DNMs) in intellectual disability (ID). They performed whole-genome sequencing in an ID cohort including 21 ID probands and their healthy parents. To study the regulatory DNMs in ID, they combined 17 ID probands without pathogenic coding DNMs with a previous cohort including 30 exome-negative ID cases. Leveraging their DNM dataset with a variety of epigenomic datasets, they observed ID DNMs were enriched more within fetal brain enhancers than adult brain enhancers. They also detected that the enhancers harboring ID DNMs showed promoter-enhancer interactions for the ID-relevant genes. Moreover, they identified recurrent mutations within enhancer clusters associated with CSMD1, OLFM1, and POU3F3 genes, when combining with larger pre-existing databases of genetic variants. Finally, they found that many ID DNMs were predicted to disrupt binding motifs of TFs, and experimentally validated the regulatory function of some of these loci. They showed the allele-specific activity for an enhancer region including an ID DNM for the SOX8 gene via luciferase assay as an episomal assay. They further showed that the same enhancer region regulates SOX8 expression by performing CRISPRi, and proved the allele-specific impact of the same DNM via also genome editing with CRISPR/Cas9.

Major

• The sample size of the Whole Genome Sequencing conducted in this study is extremely limited, and therefore the conclusions that can be drawn from the study are also extremely limited. The authors combined their data with existing cohorts for a subset of analyses, however the novelty and utility of the findings from this cohort alone is limited.
• Multiple testing burden must be considered when conducting enrichment studies in genomic regions using WGS data. Unfortunately, it is not considered here and without this the observed enrichment is not convincing. See for example https://www.nature.com/articles/s41588-018-0107-y.
• The total number of promoter enhancer interactions as shown in Figure 2 is unbelievably high. The number of gene loops previously detected using Hi-C is much lower. This analysis seems to assign every enhancer in the region to the promoter within a TAD, which is much too liberal an analysis and not consistent with number of gene loops detected via Hi-C or eQTL work.
• Because the total number of DNMs are few, I would recommend moving genomic annotations to hg38 rather than losing 123 DNMs via liftover to hg19.
• The source of the neural progenitors used in the experiments are not described.
• The non-targeting or control gRNA is not described.
• It's difficult to transfect both neural progenitors and neurons, it would be useful to see images of GFP expression if this is on the plasmid to know the degree of transfection efficiency and give greater confidence in the results presented in Figure 4.
• The specific instances where a one-tailed statistical test were used need to be highlighted.
• At page 11, the authors stated "As enhancer regions of none of the human brain cell types showed significant enrichment for ID DNMs, we concentrated on DNMs overlapping enhancers from the bulk fetal brain for downstream analysis." However, cell-type-specific enhancer enrichment analysis vs fetal brain enhancer enrichment are two different analyses. The authors did not test if the ID DNMs were enriched more in fetal brain enhancers than control DNMs were. They only compared enrichment of ID DNMs and control DNMs fetal vs adult brain enhancers. Hence, this statement was not clearly justified. It would be improved by performing a fisher's exact test to assess if ID DNMs showed more enrichment within fetal bulk brain enhancers than control DNMs did similar to cell-type-specific enrichment analysis.
• At page 13, the authors indicated that "The fetal brain enhancer DNMs from ID probands frequently disturbed putative binding sites of TFs that were predominantly expressed in neuronal cells (P = 0.022; Table S12b). Our results suggest that the enhancer DNMs from ID probands were more likely to affect the binding sites of neuronal transcription factors and could influence the regulation of genes involved in nervous system development through this mechanism." How this conclusion is drawn is unclear. Table S12b includes three cell-types with identical p-values and odd ratios based on a statistical test. How could the authors get identical parameters for all cell-types? Which dataset was used to compare the expression of these transcription factors? Were transcription factors also expressed in non-neuronal cell-types? I would request the authors to clarify the analysis performed here in the methods section, and to compare the expression of TFs in other cell-types in order to conclude as "TFs that were predominantly expressed in neuronal cells". Also, this analysis would be improved by assessing the overlap of DNMs disturbed putative binding sites within cell-type-specific ATAC-seq peaks i.e. if they were enriched more within neuronal ATAC-seq peaks than non-neuronal ATAC-seq peaks.
• The authors randomly selected DNMs from 11 ID patients that were predicted to alter TFBS affinity for experimental validation in the luciferase assay. Were the allele-specific impacts of DNMs shown in Figure 3 consistent with the predicted impact via motifbreakR? Given that the authors prioritized the regulatory ID DNMs based on motifbreakR results for the experimental validation, I would request the authors to evaluate if the alleles disrupting a TF motif that mainly has activator/repressor function also showed lower/higher luciferase activity. That would help to support the evidence for the regulatory function of other ID DNMs predicted to be TF disruption but which could not be experimentally validated.
• At page 24 in the methods section, the authors defined the control DNMs set as "We downloaded de novo mutations identified in the healthy individuals in genomes of the<br /> Netherland (GoNL) study (21) from the GoNL website". Does DNM set from GoNL also include protein-truncating mutations? If it does, are there any de novo mutations that were previously also found in any other neurodevelopmental condition as being pathogenic or likely pathogenic? If it includes both protein-truncating de novo mutations and noncoding DNMs, the two datasets used for the analysis described in Figure 1 would not be appropriately comparable to conclude that regulatory DNMs in ID were enriched in fetal brain enhancers whereas control DNMs enriched in adult brain enhancers. In which enhancer category (fetal or adult) ID DNMs would be enriched if the same analysis is performed by using both protein-truncating and regulatory DNMs? I would request the authors to evaluate the possibility that regulatory DNMs were enriched more in fetal brain enhancers compared to adult brain regardless of disease status, if the GoNL control group includes both protein-truncating and regulatory DNMs. Also, as described in the previous statement, if control DNMs include only regulatory DNMs or both protein-truncating+regulatory DNMs is not clear. This analysis would also be improved by restricting control DNMs into regulatory DNMs.
• At page 14, the authors indicated that "In the heterozygous mutant clone, the SOX8 gene showed a significant (P = 0.0301) reduction in expression levels, however, no difference was observed in expression levels of the LFM1 gene (P = 0.8641; Fig. 4d), suggesting that the enhancer specifically regulates the SOX8 gene but not the LFM1 gene." based on the knock-in experiment for DNM. However, they did not show how CRISPRi of the enhancer which is the promoter for LFM1 impacted on LFM1 gene expression as they provided for the SOX8 gene in Figure 4b. I would request the authors to rephrase the statement as "the regulatory impact of DNM within the enhancer is specific for SOX8 but not for LFM1", or provide evidence that LFM1 expression levels did not change after the CRISPRi experiment. Also, if the CRISPRi experiment would not show any change in LFM1 expression, I would also request the authors to interpret what could be potential factors for that a regulatory sequence in a gene promoter would not impact its expression.
• The authors utilized neuroblastoma cells for luciferase assay, neuronal progenitor cells for CRISPRi, and HEK293T cells for genome editing CRISPR/Cas9 experiments. Given the cell-type-specificity of active regulatory elements, I would request the authors to provide more justification for the utilization of different cell types for each assay. More specifically, LMF1 gene expression did not alter, albeit DNM's position in the gene promoter in Figure 4d. Could it be due to the low expression level of cell-type-specific transcription factors in HEK cells? Showing that expression levels of TFs whose binding motifs were disrupted via DNM at the region are comparable between HEK cells vs neuronal cells would be helpful here.
• Citation of many datasets are missing throughout the text including the (1) expression data in prefrontal cortex in the sentence at page 9 ".. but also predominantly expressed in the prefrontal cortex", (2) again expression data from neuronal datasets in the sentence at page 13 "The fetal brain enhancer DNMs from ID probands frequently disturbed putative binding sites of TFs that were predominantly expressed in neuronal cells", (3) NPCs in the sentence at page 14 "To investigate whether the putative enhancer of the SOX8/LMF1 gene indeed regulates the expression of the target genes, we performed CRISPR interference (CRISPRi), by guideRNA mediated recruitment of dCas9 fused with the four copies of sin3 interacting domain (SID4x) in the neuronal progenitor cells (NPCs).", and (4) H3K27ac and H3K4me1 datasets used in Figure 4e and described at page 14 in the sentence "Hence, we investigated H3K4me1 and H3K27ac levels at DNM containing SOX8 enhancer.". Adding citations of all external datasets utilized in the paper would be helpful for the reproducibility of the analyses and experiments.
• At page 10, the authors indicated that "We did not find enrichment for ID DNMs in open chromatin regions (ATAC-seq peaks) for any of the developing brain cell types" and on page 11, they stated, "On the contrary, all four developed human brain cell types showed significant enrichment for ID DNMs compared to GoNL DNMs in promoter regions after correcting for multiple tests". Given that ID DNMs were more enriched in fetal brain enhancers than adult brain enhancers in Figure 1, it is important to discuss why ID DNMs were enriched within developed brain cell-type regulatory elements but not in developing brain cell-type specific regulatory elements. I would request the authors to clarify this discrepancy. Could the distance to the gene be a factor in this discrepancy? How do cell-type-specific enrichment results change if ATAC-seq peaks from developing human cortex would be also restricted by chromatin accessibility regions within gene promoters (e.g. within +/- 2kb from TSS)? If ID DNMs within promoter regions were enriched within at least one of the cell-type-specific regulatory elements in both developing and adult brains, re-evaluating the analysis performed in Figure 1 by considering the distance of DNMs to genes would be critical to conclude temporal-specific enrichment of ID DNMs.

Minor

• In general, the study could benefit from more figures rather than providing results with tables to follow and understand them, especially for Table S6 and Table S11.
• At figure 2, the colors of the arcs do not match the colors indicated in the label.
• At tables 11a and 11c, the column names indicated in the E and F columns are the same, it would be good to distinguish them.
• At page 10, the authors indicated that "The IPCs give rise to most neurons (32) hence DMNs in highly connected active promoters and enhancers from IPCs might have a profound impact on neurogenesis." This sentence is not clear.
• Radical glia -> radial glia
• Describe background gene lists used for all hypergeometric/fisher's exact tests.
• In Figure 4a, it would be useful to label the de novo mutation, otherwise it's not clear why a specific region was highlighted. Also to highlight where the gRNA was targeted for the CRISPRi experiment.

Referees cross-commenting

I agree with the other reviewers' comments. I just have one specific comment: Reviewer 1 suggested that RNA-seq would be more accurate than gene expression; however, I feel that this assay is not necessary and may be quite expensive for the targeted gene expression differences measured here.

Significance

Overall this study attempted to identify and validate novel non-coding variants associated with ID. However, given limitations in sample size, statistical testing, and experimental design, as described above, many of these conclusions are limited.

Maths, Logic, Computer Science, Chess, Music, and Dance

A similar observation could be made about mathematics, logic, and computer science. Sadly, public education in the states seems to lose sight that the formalisms in these domains are merely the tools of the trade and not the trade itself (ie, developing an understanding of the fundamental/foundational notions, their relationships, their instantiations, and cultivating how one can develop capacity to "move" in that space).

Similarly, it's as if we encourage children that they need to merely memorize all the movements of chess pieces to appreciate the depth of the game.

Or saying "Here, just memorize these disconnected contortions of the hand upon these strings along this piece of wood. Once you have that down, you've experienced all that guitar, (nay, music itself!) has to offer."

Or "Yes, once, you internalize the words for these moves and recite them verbatim, you will have experienced all the depth and wonder that dance and movement have to offer."

However, none of these examples are given so as to dismiss or ignore the necessity of (at least some level of) formalistic fluency within each of these domains of experience. Rather, their purpose is to highlight the parallels in other domains that may seem (at first) so disconnected from one's own experience, so far from one's fundamental way of feeling the world, that the only plausible reasons one can make to explain why people would waste their time engaging in such acts are 1. folly: they merely do not yet know their activities are absurd, but surely enough time will disabuse them of their foolish ways. 2. madness: they cannot ever know the absurdity of their acts, for "the absurd" and "the astute" are but two names for one and the same thing in their world of chaos. 3. apathy: they in fact do see the absurdity in their continuing of activities which give them no sense of meaning, yet their indifference insurmountably impedes them from changing their course of action. For how could one resist the path of least resistance, a road born of habit, when one must expend energy to do so but that energy can only come from one who cares?

Or at least, these 3 reasons can surely seem like that's all there possibly could be to warrant someone continuing music, chess, dance, maths, logic, computer science, or any apparently alien craft. However, if one takes time to speak to someone who earnestly pursues such "alien crafts", then one may start to perceive intimations of something beyond their current impressions

The contorted clutching of the strings now seems... coordinated. The pensive placement of the pawns now appears... purposeful. The frantic flailing of one's feet now feels... freeing. The movements of one's mind now feels... marvelous.

So the very activity that once seemed so clearly absurd, becomes cognition and shapes perspectives beyond words

Even in this moment of apparent self-reflection, he is only willing to do so after the effects of alcohol have begun to settle in.

Close, but not quite.

For we do not have a high priest who cannot sympathize with our weaknesses, but One who has been tempted in all things just as we are, yet without sin. Therefore let’s approach the throne of grace with confidence, so that we may receive mercy and find grace for help at the time of our need. Hebrews 4:15-16

It is not important for us to see ourselves in Jesus, but to realize that Jesus has seen Himself in our shoes and yet remained without sin, so that we can turn to Him to help us live in accordance with the will of our Heavenly Father.

True BUT majority white ethnicity specific neighbourhods do not experience a sudden decrease in funding/quality of life like in cases of white flight, and Brampton is not adapting to the majority South Asian population through policy, rather holding onto its last few white residents Probably because Brampton has only had white mayors. An example of this is the City of Brampton's history statement

I understand their perspective and I feel like it’s fair to think that filling your personal time with things that can help to bring your mood up and make you feel better. I personally feel like having a nice balance can be helpful as well. I also personally love horror movies and could easily watch them as frequently as comedy movies or positive movies so I feel like it’s just the enjoyment of watching movies that make me cheerful or improve my mood