Reviewer #3 (Public review):

Summary:

In this set of experiments, the authors describe a novel research tool for studying complex cognitive tasks in mice, the HABITS automated training apparatus, and a novel "machine teaching" approach they use to accelerate training by algorithmically providing trials to animals that provide the most information about the current rule state for a given task.

Strengths:

There is much to be celebrated in an inexpensively constructed, replicable training environment that can be used with mice, which have rapidly become the model species of choice for understanding the roles of distinct circuits and genetic factors in cognition. Lingering challenges in developing and testing cognitive tasks in mice remain, however, and these are often chalked up to cognitive limitations in the species. The authors' findings, however, suggest that instead we may need to work creatively to meet mice where they live. In some cases it may be that mice may require durations of training far longer than laboratories are able to invest with manual training (up to over 100k trials, over months of daily testing) but that the tasks are achievable. The "machine teaching" approach further suggests that this duration could be substantially reduced by algorithmically optimizing each trial presented during training to maximize learning.

Weaknesses:

Cognitive training and testing in rodent models fill a number of roles. Sometimes, investigators are interested in within-subjects questions - querying a specific circuit, genetically defined neuron population, or molecule/drug candidate, by interrogating or manipulating its function in a highly trained animal. In this scenario, a cohort of highly trained animals which have been trained via a method that aims to make their behavior as similar as possible is a strength.

However, often investigators are interested in between-subjects questions - querying a source of individual differences that can have long term and/or developmental impacts, such as sex differences or gene variants. This is likely to often be the case in mouse models especially, because of their genetic tractability. In scenarios where investigators have examined cognitive processes between subjects in mice who vary across these sources of individual difference, the process of learning a task has been repeatedly shown to be different. The authors recognize that their approach is currently optimized for testing within-subjects questions, but begin to show how between-subjects questions might be addressed with this system.

The authors have perhaps shown that their main focus is highly-controlled within-subjects questions, as their dataset is almost exclusively made up of several hundred young adult male mice, with the exception of 6 females in a supplemental figure. It is notable that these female mice do appear to learn the two-alternative forced choice task somewhat more rapidly than the males in their cohort, and the authors suggest that future work with this system could be used to uncover strategies that differ across individuals.

Considering the implications for mice modeling relevant genetic variants, it is unclear to what extent the training protocols and especially the algorithmic machine teaching approach would be able to inform investigators about the differences between their groups during training. For investigators examining genetic models, it is unclear whether this extensive training experience would mitigate the ability to observe cognitive differences, or select for the animals best able to overcome them - eliminating the animals of interest. Likewise, the algorithmic approach aims to mitigate features of training such as side biases, but it is worth noting that the strategic uses of side biases in mice, as in primates, can benefit learning, rather than side biases solely being a problem. However, the investigators may be able to highlight variables selected by the algorithm that are associated with individual strategies in performing their tasks, and this would be a significant contribution.

A final, intriguing finding in this manuscript is that animal self-paced training led to much slower learning than "manual" training, by having the experimenter introduce the animal to the apparatus for a few hours each day. Manual training resulted in significantly faster learning, in almost half the number of trials on average, and with significantly fewer omitted trials. This finding does not necessarily argue that manual training is universally a better choice, because it led to more limited water consumption. However, it suggests that there is a distinct contribution of experimenter interactions and/or switching contexts in cognitive training, for example, by activating an "occasion setting" process to accelerate learning for a distinct period of time. Limiting experimenter interactions with mice may be a labor saving intervention, but may not necessarily improve performance. This could be an interesting topic of future investigation, of relevance to understanding how animals of all species learn.

Reviewer #1 (Public review):

Summary:

This study highlights the strengths of using predictive computational models to inform C. elegans screening studies of compounds' effects on aging and lifespan. The authors primarily focus on all-trans retinoic acid (atRA), one of the 5 compounds (out of 16 tested) that extended C. elegans lifespan in their experiments. They show that atRA has positive effects on C. elegans lifespan and age-related health, while it has more modest and inconsistent effects (i.e., some detrimental impacts) for C. briggsae and C. tropicalis. In genetic experiments designed to evaluate contributing mediators of lifespan extension with atRA exposure, it was found that 150 µM of atRA did not significantly extend lifespan in akt-1 or akt-2 loss-of-function mutants, nor in animals with loss of function of aak-2, or skn-1 (in which atRA had toxic effects); these genes appear to be required for atRA-mediated lifespan extension. hsf-1 and daf-16 loss-of-function mutants both had a modest but statistically significant lifespan extension with 150 µM of atRA, suggesting that these transcription factors may contribute towards mediating atRA lifespan extension, but that they are not individually required for some lifespan extension. RNAseq assessment of transcriptional changes in day 4 atRA-treated adult wild type worms revealed some interesting observations. Consistent with the study's genetic mutant lifespan observations, many of the atRA-regulated genes with the greatest fold-change differences are known regulated targets of daf-2 and/or skn-1 signaling pathways in C. elegans. hsf-1 loss-of-function mutants show a shifted atRA transcriptional response, revealing a dependence on hsf-1 for ~60% of the atRA-downregulated genes. On the other hand, RNAseq analysis in aak-2 loss-of-function mutants revealed that aak-2 is only required for less than a quarter of the atRA transcriptional response. All together, this study is a proof of the concept that computational models can help optimize C. elegans screening approaches that test compounds' effects on lifespan, and provides comprehensive transcriptomic and genetic insights into the lifespan-extending effects of all-trans retinoic acid (atRA).

Strengths:

A clearly described and well-justified account describes the approach used to prioritize and select compounds for screening, based on using the top candidates from a published list of computationally ranked compounds (Fuentealba et al., 2019) that were cross-referenced with other bioinformatics publications to predict anti-aging compounds, after de-selecting compounds previously evaluated in C. elegans as per the DrugAge database. 16 compounds were tested at 4-5 different concentrations to evaluate effects on C. elegans lifespan.

Robust experimental design was undertaken evaluating the lifespan effects of atRA, as it was tested on three strains each of C. elegans, C. briggsae, and C. tropicalis, with trial replication performed at three distinct laboratories. These observations extended beyond lifespan to include evaluations of health metrics related to swimming performance.

In-depth analyses of the RNAseq data of whole-worm transcriptional responses to atRA revealed interesting insights into regulator pathways and novel groups of genes that may be involved in mediating lifespan-extension effects (e.g., atRA-induced upregulation of sphingolipid metabolism genes, atRA-upregulation of genes in a poorly-characterized family of C. elegans paralogs predicted to have kinase-like activity, and disproportionate downregulation of collagen genes with atRA).

Weaknesses:

The authors' computational-based compound screening approach led to a ~30% prediction success rate for compounds that could extend the median lifespan of C. elegans. However, follow-up experiments on the top compounds highlighted the fact that some of these observed "successes" could be driven by indirect, confounding effects of these compounds on the bacterial food source, rather than direct beneficial effects on C. elegans physiology and lifespan. For instance, this appeared to be the case for the "top" hit of propranolol. Other compounds were not tested with metabolically inert or killed bacteria to preclude the possibility of bacteria-produced metabolites exerting observed effects; this might be a useful future direction to consider.

Transcriptomic analyses of atRA effects were extensive in this study, but discussions of potential non-transcriptional effects of key proposed regulators (such as AMPK) were limited. For instance, other outputs of aak-2/AMPK (non-transcriptional changes to metabolic balance, autophagy, etc.) might account for its requirement for mediating lifespan extension effects, since aak-2 was not required for a major proportion of atRA transcriptional responses.

Comments on revisions:

In their revisions, the authors resolved all of my initial recommendations, and I have no additional suggestions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Banse et al. experimentally validate the power of computational approaches that predict anti-aging molecules using the multi-species approach of the Caenorhabditis Intervention Testing Program (CITP). Filtering candidate molecules based on transcriptional profiles, ML models, literature searches, and the DrugAge database, they selected 16 compounds for testing. Of those, eight did not affect C. elegans' lifespan, three shortened it, and five extended C. elegans' lifespan, resulting in a hit rate of over 30%. Of those five, they then focused on all-trans-retinoic acid (atRA), a compound that has previously resulted in contradictory effects. The lifespan-extending effect of atRA was consistent in all C. elegans strains tested, was absent in C. briggsae, and a small effect was observed in some C. tropicalis strains. Similar results were obtained for measures of healthspan. The authors then investigated the mechanism of action of atRA and showed that it was only partially dependent on daf-16 but required akt-1, akt-2, skn-1, hsf-1, and, to some degree, pmk-1. The authors further investigate the downstream effects of atRA exposure by conducting RNAseq experiments in both wild-type and mutant animals to show that some, but surprisingly few, of the gene expression changes that are observed in wild-type animals are lost in the hsf-1 and aak-2 mutants

Strengths:

Overall, this study is well-conceived and executed as it investigates the effect of atRA across different concentrations, strains, and species, including life and health span. Revealing the variability between sites, assays, and the method used is a powerful aspect of this study. It will do a lot to dispel the nonsensical illusion that we can determine a per cent increase in lifespan to the precision of two floating point numbers.

An interesting and potentially important implication arises from this study. The computational selection of compounds was agnostic regarding strain or species differences and was predominantly based on observations made in mammalian systems. The hit rate calculated is based on the results of C. elegans and not on the molecules' effectiveness in Briggsae or Tropicalis. If it were, the hit rate would be much lower. How is that? It would suggest that ML models and transcriptional data obtained from mammals have a higher predictive value for C. elegans than for the other two species. This selectivity for C.elegans over C.tropicalis and C.Briggsae seems both puzzling and unexpected. The predictions for longevity were based on the transcriptional data in cell lines. Would it be feasible to compare the mammalian data to the transcriptional data in Fig. 5 and see how well they match? While this is clear beyond the focus of this study, an implied prediction is that running RNAseqs for all these strains exposed to atRA would reveal that the transcriptional changes observed in the strains where it extends lifespan the most should match the mammalian data best. Otherwise, how could the mammalian datasets be used to predict the effects for C.elegans over C.Briggsae or C.Tropicalis have more predictive for one species than the other? There are a lot of IFs in this prediction, but such an experiment would reconsider and validate the basis on which the original predictions were made.

Weaknesses:

Many of the most upregulated genes, such as cyps and pgps are xenobiotic response genes upregulated in many transcriptional datasets from C.elegans drug studies. Their expression might be necessary to deal with atRA breakdown metabolites to prevent toxicity rather than confer longevity. Because atRA is very light sensitive and has toxicity of breakdown, metabolites may explain some of the differences observed with the lifespan of machine effects compared to standard assay practices. However, the authors provide a potential explanation for that observation.

Comments on revisions:

The authors have adequately addressed my concerns and the paper is suitable for publication.

Reviewer #3 (Public review):

Summary:

In this study, Banse et al., demonstrate that combining computer prediction with genetic analysis in distinct Caenorhabditis species can streamline the discovery of aging interventions by taking advantage of the diverse pool of compounds that are currently available. They demonstrate that through careful prioritization of candidate compounds, they are able to accomplish a 30% positive hit rate for interventions that produce significant lifespan extensions. Within the positive hits, they focus on all-trans retinoic acid (atRA) and discover that it modulates lifespan through conserved longevity pathways such as AKT-1 and AKT-2 (and other conserved Akt-targets such as Nrf2/SKN-1 and HSF1/HSF-1) as well as through AAK-2, a conserved catalytic subunit of AMPK. To better understand the genetic mechanisms behind lifespan extension upon atRA treatment, the authors perform RNAseq experiments using a variety of genetic backgrounds for cross comparison and validation. Using this current state-of-the-art approach for studying gene expression, the authors determine that atRA treatment produces gene expression changes across a broad set of stress-response and longevity-related pathways. Overall, this study is important since it highlights the potential of combining traditional genetic analysis in the genetically tractable organism C. elegans with computational methods that will become even more powerful with the swift advancements being made in artificial intelligence. The study possesses both theoretical and practical implications not only in the field of aging, but also in related fields such as health and disease. Most of the claims in this study are supported by solid evidence, but the conclusions can be refined with a small set of additional experiments or re-analysis of data.

Strengths:

(1) The criteria for prioritizing compounds for screening are well-defined and is easy to replicate (Figure 1), even for scientists with limited experience in computational biology. The approach is also adaptable to other systems or model organisms.

(2) I commend the researchers for doing follow-up experiments with the compound propranolol to verify its effect of lifespan (Figure 2- figure supplement 2), given the observation that it affected the growth of OP50. To prevent false hits in the future, the reviewer recommends the use of inactivated OP50 for future experiments to remove this confounding variable.

(3) The sources of variation (Figure 3-figure supplement 2) are taken into account and demonstrates the need for advancing our understanding of the lifespan phenotype due to inter-individual variation.

(4) The addition of the C. elegans swim test in addition to the lifespan assays provides further evidence of atRA-induced improvement in longevity.

(5) The RNAseq approach was performed in a variety of genetic backgrounds, which allowed the authors to determine the relationship between AAK-2 and HSF-1 regulation of the retinoic acid pathway in C. elegans, specifically, that the former functions downstream of the latter.

Weaknesses:

(1) The authors demonstrate that atRA extends lifespan in a species-specific manner (Figure 3). Specifically, this extension only occurs in the species C. elegans yet, the title implies that atRA-induced lifespan extension occurs in different Caenorhabditis species when it is clearly not the case. While the authors state that failure to observe phenotypes in C. briggsae and C. tropicalis is a common feature of CITP tests, they do not speculate as to why this phenomenon occurs.

(2) There are discrepancies between the lifespan curves by hand (Figure 3-Figure supplement 1) and using the automated lifespan machine (Figure 3-supplement 3). Specifically, in the automated lifespan assays, there are drastic changes in the slope of the survival curve which do not occur in the manual assays and may be suggestive that confounding factors may still operate or produce additional variation in ALM experiments despite relatively well-controlled environmental conditions.

Reviewer #1 (Public review):

Summary:

The authors in this study extensively investigate how telomere length (TL) regulates hTERT expression via non-telomeric binding of the telomere-associated protein TRF2. They conclusively show that TRF2 binding to long telomeres results in a reduction in its binding to the hTERT promoter. In contrast, short telomeres restore TRF2 binding in the hTERT promoter, recruiting repressor complexes like PRC2, and suppressing hTERT expression. The study presents several significant findings revealing a previously unknown mechanism of hTERT regulation by TRF2 in a TL-dependent manner

Strengths:

(1) A previously unknown mechanism linking telomere length and hTERT regulation through the non-telomeric TRF2 protein has been established, strengthening our understanding of telomere biology.

(2) The authors used both cancer cell lines and iPSCs to showcase their hypothesis and multiple parameters to validate the role of TRF2 in hTERT regulation.

(3) Comprehensive integration of the recent literature findings and implementation in the current study.

(4) In vivo validation of the findings.

(5) Rigorous controls and well-designed assays have been used.

Comments on current version:

The current version of the manuscript has addressed all the reviewers' concerns to the best of its ability. However, understanding the limitations of the authors, exploring ALT cell lines for the current mechanism would be desirable in the future.

Reviewer #1 (Public review):

Summary:

In this study, the authors showed that enalapril was able to reduce cellular senescence and improve health status in aged mice. The authors further showed that phosphorylated Smad1/5/9 was significantly elevated and blocking this pathway attenuated the protection of cells from senescence. When middle-aged mice were treated with enalapril, the physiological performance in several tissues, including memory capacity, renal function and muscle strength, exhibited significant improvement.

Strengths:

The strength of the study lies in the identification of pSMAD1/5/9 pathway as the underlying mechanism mediating the anti-senescence effects of enalapril with comprehensive evaluation both in vitro and in vivo.

Weaknesses:

The major weakness of the study is the in vivo data. Despite the evidence shown in the in vitro study, there is no data to show that blocking the pSmad1/5/9 pathway is able to attenuate the anti-aging effects of enalapril in the mice. In addition, the aging phenotypes mitigation by enalapril is not evidenced by the extension of lifespan. If it is necessary to show that NAC is able to attenuate enalapril effects in the aging mice. In addition, it would be beneficial to test if enalapril is able to achieve similar rescue in a premature aging mouse model.

Comments on revisions:

The revised manuscript provided additional in vivo data that addressed my questions accordingly. I think the authors have done an excellent job in demonstrating that enalapril improved physiological phenotypes in aged mice through pSmad1/5/9 pathway.

Their response to my question regarding the test in HGPS mice was not satisfactory. Premature aging and physiological aging share substantial similarities in their pathways. Given that this is not the focus of current study and the manuscript does not provide data on HGPS mice, I think this does not affect the conclusion of the current study.

Reviewer #2 (Public review):

This manuscript presents an interesting study of enalapril for its potential impact on senescence through the activation of Smad1/5/9 signaling with a focus on antioxidative gene expression. Repurposing enalapril in this context provides a fresh perspective on its effects beyond blood pressure regulation. The authors make a strong case for the importance of Smad1/5/9 in this process, and the inclusion of both in vitro and in vivo models adds value to the findings. Below, I have a few comments and suggestions which may help improve the manuscript.

A major finding in the study is that phosphorylated Smad1/5/9 mediates the effects of enalapril. However, the manuscript focused on the Smad pathway relatively abruptly, and the rationale behind targeting this specific pathway is not fully explained. What makes Smad1/5/9 particularly relevant to the context of this study?

Furthermore, their finding that activation of Smad1/5/9 leads to a reduction of senescence appears somewhat contradictory to the established literature on Smad1/5/9 in senescence. For instance, studies have shown that BMP4-induced senescence involves activation of Smad1/5/8 (Smad1/5/9), leading to the upregulation of senescence markers like p16 and p21 (JBC, 2009, 284, 12153). Similarly, phosphorylated Smad1/5/8 has been shown to promote and maintain senescence in Ras-activated cells (PLOS Genetics, 2011, 7, e1002359). Could the authors provide more detailed mechanistic insights into why enalapril seems to reverse the typical pro-senescent role of Smad1/5/9 in their study?

While the authors showed that enalapril increases pSmad1/5/9 phosphorylation, what are the expression levels of other key and related factors like Smad4, pSmad2, pSmad3, BMP2, and BMP4 in both senescent and non-senescent cells? These data will help clarify the broader signaling effects.

They used BMP receptor inhibitor LDN193189 to pharmacologically inhibit BMP signaling, but it would be more convincing to also include genetic validation (e.g., knockdown or knockout of BMP2 or BMP4). This will help confirm that the observed effects are truly due to BMP-Smad signaling and not off-target effects of the pharmacological inhibitor LDN.

I don't see the results on the changes in senescence markers p16 and p21 in the mouse models treated with enalapril. Similarly, the effects of enalapril treatment on some key SASP factors, such as TNF-α, MCP-1, IL-1β, and IL-1α, are missing, particularly in serum and tissues. These are important data to evaluate the effect of enalapril on senescence.

Given that enalapril is primarily known as an antihypertensive, it would be helpful to include data on how it affects blood pressure in the aged mouse models, such as systolic and diastolic blood pressure. This will clarify whether the observed effects are independent of or influenced by changes in blood pressure.

Reviewer #1 (Public review):

Summary:

This fundamental study identifies a new mechanism that involves a mycobacterial nucleomodulin manipulation of the host histone methyltransferase COMPASS complex to promote infection. Although other intracellular pathogens are known to manipulate histone methylation, this is the first report demonstrating the specific targeting of the COMPASS complex by a pathogen. The rigorous experimental design using state-of-the art bioinformatic analysis, protein modeling, molecular and cellular interaction, and functional approaches, culminating with in vivo infection modeling, provides convincing, unequivocal evidence that supports the authors' claims. This work will be of particular interest to cellular microbiologists working on microbial virulence mechanisms and effectors, specifically nucleomodulins, and cell/cancer biologists that examine COMPASS dysfunction in cancer biology.

Strengths:

(1) The strengths of this study include the rigorous and comprehensive experimental design that involved numerous state-of-the-art approaches to identify potential nucleomodulins, define molecular nucleomodulin-host interactions, cellular nucleomodulin localization, intracellular survival, and inflammatory gene transcriptional responses, and confirmation of the inflammatory and infection phenotype in a small animal model.

(2) The use of bioinformatic, cellular, and in vivo modeling that are consistent and support the overall conclusions is a strength of the study. In addition, the rigorous experimental design and data analysis, including the supplemental data provided, further strengthen the evidence supporting the conclusions.

Weaknesses:

(1) This work could be stronger if the MgdE-COMPASS subunit interactions that negatively impact COMPASS complex function were better defined. Since the COMPASS complex consists of many enzymes, examining the functional impact on each of the components would be interesting.

(2) Examining the impact of WDR5 inhibitors on histone methylation, gene transcription, and mycobacterial infection could provide additional rigor and provide useful information related to the mechanisms and specific role of WDR5 inhibition on mycobacterial infection.

(3) The interaction between MgdE and COMPASS complex subunit ASH2L is relatively undefined, and studies to understand the relationship between WDR5 and ASH2L in COMPASS complex function during infection could provide interesting molecular details that are undefined in this study.

(4) The AlphaFold prediction results for all the nuclear proteins examined could be useful. Since the interaction predictions with COMPASS subunits range from 0.77 for WDR5 and 0.47 for ASH2L, it is not clear how the focus on COMPASS complex over other nuclear proteins was determined.

Reviewer #2 (Public review):

Summary:

The manuscript by Chen et al addresses an important aspect of pathogenesis for mycobacterial pathogens, seeking to understand how bacterial effector proteins disrupt the host immune response. To address this question, the authors sought to identify bacterial effectors from M. tuberculosis (Mtb) that localize to the host nucleus and disrupt host gene expression as a means of impairing host immune function.

Strengths:

The researchers conducted a rigorous bioinformatic analysis to identify secreted effectors containing mammalian nuclear localization signal (NLS) sequences, which formed the basis of quantitative microscopy analysis to identify bacterial proteins that had nuclear targeting within human cells. The study used two complementary methods to detect protein-protein interaction: yeast two-hybrid assays and reciprocal immunoprecipitation (IP). The combined use of these techniques provides strong evidence of interactions between MgdE and SET1 components and suggests that the interactions are, in fact, direct. The authors also carried out a rigorous analysis of changes in gene expression in macrophages infected with the mgdE mutant BCG. They found strong and consistent effects on key cytokines such as IL6 and CSF1/2, suggesting that nuclear-localized MgdE does, in fact, alter gene expression during infection of macrophages.

Weaknesses:

There are some drawbacks in this study that limit the application of the findings to M. tuberculosis (Mtb) pathogenesis. The first concern is that much of the study relies on ectopic overexpression of proteins either in transfected non-immune cells (HEK293T) or in yeast, using 2-hybrid approaches. Some of their data in 293T cells is hard to interpret, and it is unclear if the protein-protein interactions they identify occur during natural infection with mycobacteria. The second major concern is that pathogenesis is studied using the BCG vaccine strain rather than virulent Mtb. However, overall, the key findings of the paper - that MgdE interacts with SET1 and alters gene expression are well-supported.

Reviewer #3 (Public review):

In this study, Chen L et al. systematically analyzed the mycobacterial nucleomodulins and identified MgdE as a key nucleomodulin in pathogenesis. They found that MgdE enters into host cell nucleus through two nuclear localization signals, KRIR108-111 and RLRRPR300-305, and then interacts with COMPASS complex subunits ASH2L and WDR5 to suppress H3K4 methylation-mediated transcription of pro-inflammatory cytokines, thereby promoting mycobacterial survival. This study is potentially interesting, but there are several critical issues that need to be addressed to support the conclusions of the manuscript.

(1) Figure 2: The study identified MgdE as a nucleomodulin in mycobacteria and demonstrated its nuclear translocation via dual NLS motifs. The authors examined MgdE nuclear translocation through ectopic expression in HEK293T cells, which may not reflect physiological conditions. Nuclear-cytoplasmic fractionation experiments under mycobacterial infection should be performed to determine MgdE localization.

(2) Figure 2F: The authors detected MgdE-EGFP using an anti-GFP antibody, but EGFP as a control was not detected in its lane. The authors should address this technical issue.

(3) Figure 3C-3H: The data showing that the expression of all detected genes in 24 h is comparable to that in 4 h (but not 0 h) during WT BCG infection is beyond comprehension. The issue is also present in Figure 7C, Figure 7D, and Figure S7. Moreover, since Il6, Il1β (pro-inflammatory), and Il10 (anti-inflammatory) were all upregulated upon MgdE deletion, how do the authors explain the phenomenon that MgdE deletion simultaneously enhanced these gene expressions?

(4) Figure 5: The authors confirmed the interactions between MgdE and WDR5/ASH2L. How does the interaction between MgdE and WDR5 inhibit COMPASS-dependent methyltransferase activity? Additionally, the precise MgdE-ASH2L binding interface and its functional impact on COMPASS assembly or activity require clarification.

(5) Figure 6: The authors proposed that the MgdE-regulated COMPASS complex-H3K4me3 axis suppresses pro-inflammatory responses, but the presented data do not sufficiently support this claim. H3K4me3 inhibitor should be employed to verify cytokine production during infection.

(6) There appears to be a discrepancy between the results shown in Figure S7 and its accompanying legend. The data related to inflammatory responses seem to be missing, and the data on bacterial colonization are confusing (bacterial DNA expression or CFU assay?).

(7) Line 112-116: Please provide the original experimental data demonstrating nuclear localization of the 56 proteins harboring putative NLS motifs.

Reviewer #1 (Public review):

The authors use electrophysiological and behavioral measurements to examine how animals could reliably determine odor intensity/concentration across repeated experience. Because stimulus repetition leads to short-term adaptation evidenced by reduced overall firing rates in the antennal lobe and firing rates are otherwise concentration-dependent, there could be an ambiguity in sensory coding between reduced concentration or more recent experience. This would have a negative impact on the animal's ability to generate adaptive behavioral responses that depend odor intensities. The authors conclude that changes in concentration alter the constituent neurons contributing to the neural population response, whereas adaptation maintains the 'activated ensemble' but with scaled firing rates. This provides a neural coding account of the ability to distinguish odor concentrations even after extended experience. Additional analyses attempt to distinguish hypothesized circuit mechanisms for adaptation. A larger point that runs through the manuscript is that overall spiking activity has an inconsistent relationship with behavior and that the structure of population activity may be the more appropriate feature to consider.

To my knowledge, the dissociation of effects of odor concentration and adaptation on olfactory system population codes was not previously demonstrated. This is a significant contribution that improves on any simple model based on overall spiking activity. The primary result is most strikingly supported by visualization of a principal components analysis in Figure 4. Additional experiments and analysis complement and provide context for this finding regarding the relationship between neural population changes and behavior. There are some natural limitations on the interpretation of these data imposed by the methodology.

(1) Because individual recordings do not acquire a sufficient cell population to carry our population analyses, the cells must be combined into pseudopopulations for many analyses. This is common practice but it limits the ability to test the repeatability of findings across animals or populations. One potential additional solution would be to subsample the pseudopopulation, which would reveal the importance of individual sampled cells in the overall result. The utility of this additional testing is suggested by, for example, the benzaldehyde responses in supplementary figure 5, where two cells differentiate high and low concentration responses and would be expected to strongly impact correlation and classifier analyses.

(2) I do not think the analysis in Figure 2e can be strongly interpreted in terms of the vesicle depletion model. The hard diagonal bound on the lower part of each scatter plot indicates that features of the data/analysis necessarily exclude data in the lower left quadrant. I think this could be possibly explained by a floor effect wherein lower-response neurons cannot possibly express a large deltaResponse. To strengthen this case, one would need to devise a control analysis for the case where neural responses are simply all going as far down as they can go.

(3) Very minor, but it is confusing and not well-described how the error is computed in Figure 1f. One can imagine that the mean p(POR) is arrived at by averaging the binary values across locusts. Is this the case? If so, the same estimation of variance could be applied to Figures 1d and e

Reviewer #3 (Public review):

Summary:

How does the brain distinguish stimulus intensity reduction from response reductions due to adaptation? Ling et al study whether and how the locust olfactory system encodes stimulus intensity and repetition differently. They show that these stimulus manipulations have distinguishable effects on population dynamics.

Strengths:

(1) Provides a potential strategy with which the brain can distinguish intensity decrease from adaptation. -- while both conditions reduce overall spike counts, intensity decrease can also changes which neurons are activated and adaptation only changes the response magnitude without changing the active ensemble.

(2) By interleaving a non-repeated odor, they show that these changes are odor-specific and not a non-specific effect.

(3) Describes how proboscis orientation response (POR) changes with stimulus repetition., Unlike the spike counts, POR increases in probability with stimulus. The data portray the variability across subjects in a clear way.

Weaknesses:

While POR and physiology can show a nice correlation when measured in different animals, additional insight would be gained from acquiring behavior and physiology simultaneously.

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Clarify the outcome: did this lead to improved collaboration or understanding among students?

Quantify the results: what insights were gained or how many users benefited from this app?

Specify the impact: how did this API improve data handling or user experience?

Explain the benefits of this deployment choice, such as improved load times or scalability.

Include user statistics or feedback to illustrate the app's effectiveness and user satisfaction.

Avoid redundancy; clarify unique features or user benefits that distinguish this app from others.

Mention any feedback received from users or improvements in gameplay as a result of your development.

Include the average resolution time or percentage of issues resolved to show effectiveness.

Quantify the number of computers set up or the efficiency gained through your communication efforts.

Clarify the outcome of this exploration; did it lead to any implemented solutions or insights?

Add metrics on user engagement or performance improvements from the app to demonstrate impact.

Specify how the API improved data management or user experience to quantify impact.

Mention any cost savings or performance enhancements achieved through this deployment strategy.

Specify the performance improvement, such as reduced query time or increased data retrieval speed.

Quantify user engagement or efficiency improvements, e.g., 'reduced task completion time by X%'.

Remove 'using with' for clarity. Add impact metrics, such as user adoption rates or performance improvements.

Mention any user feedback or performance metrics that demonstrate the game's success or engagement.

Include metrics on the number of issues resolved or average resolution time to show efficiency.

Quantify the number of computers set up or the time saved through your communication efforts.

Clarify the outcome of this exploration. Did it lead to any implemented solutions or improved learning outcomes?

Add metrics on user engagement or efficiency improvements resulting from the application.

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Provide context on what 'efficiency' means here. What specific tasks were made easier or faster?

Specify how this automation improved deployment frequency or reduced errors in production.

Clarify the baseline metrics for latency and cost to provide context for the improvements made.

Quantify the efficiency gained or time saved through this consolidation to better illustrate the impact.

Explain how the $240,000 savings was calculated and what specific processes were improved to achieve this.

Break down the specific operational problems solved and how they directly impacted user experience or system reliability.

Quantify the reduction in phishing incidents or security breaches to highlight the effectiveness of your solutions.

Clarify how this paradigm shift benefited AWS users or reduced costs. Provide measurable outcomes.

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Specify the impact of alerts (e.g., 'reducing response time to market changes by 50%').

Clarify the original hosting cost for context (e.g., 'from $200/month to $20/month').

Quantify the reduction in clutter (e.g., 'reducing review threads by 50%').

Be specific about the improvements (e.g., 'reducing review time by 30%').

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Quantify the efficiency gained (e.g., 'reducing data retrieval time by 40%').

Clarify the benefit to executives (e.g., 'improving decision-making speed').

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Specify the original coverage percentage for context (e.g., 'from 60% to 75%').

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Include the original hosting cost to emphasize the financial impact of your re-architecture.

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Provide specific user feedback or metrics that demonstrate the improvement in user experience.

Clarify the impact on decision-making or efficiency for the executives who used the dashboards.

Quantify the time saved in testing setup to illustrate the impact of your tool.

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Quantify the exact hours saved to provide a clearer impact of your work.

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Clarify how cost savings were redirected or reinvested, or the impact on project scalability.

Quantify the reduction in time spent on reviews or the increase in review efficiency.

Provide specific outcomes from the feedback integration, such as user adoption rates or satisfaction scores.

Add metrics on user engagement or satisfaction improvements post-redesign.

Quantify the impact of this access on decision-making or operational efficiency for the executives.

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Clarify how this automation improved HR efficiency or employee satisfaction beyond time savings.

Reviewer #2 (Public review):

Summary:

In this manuscript, Weiguang Kong et al. investigate the role of immunoglobulin M (IgM) in antiviral defense in the teleost largemouth bass (Micropterus salmoides). The authors employ an in vivo IgM depletion system and viral infection models, complemented by in vitro assays, histology, and gene expression analysis. Assuming the specificity of the MoAb, their findings demonstrate that largemouth bass IgM functions in both systemic and mucosal immunity and exhibits viral neutralization capabilities by acting on viral particles.

Strengths:

The authors utilize multiple complementary methods, including an innovative teleost immunoglobulin depletion approach, to provide strong evidence for the important and conserved role of IgM in anti-viral resistance. The study also highlights the dual role of teleost IgM at both systemic and mucosal levels, challenging the established idea that IgT primarily mediates mucosal protection. Despite variability in IgM depletion levels, the authors demonstrate that fish with depleted IgM+ B cells exhibit significantly higher viral loads, more severe pathological changes, and increased mortality compared to control fish. These results have evolutionary and practical implications, suggesting that IgM's role as an antiviral effector has been conserved across jawed vertebrates for over 500 million years. Insights into IgM's role could inform vaccine strategies targeting mucosal immunity in fish, addressing a key challenge in aquaculture.

Weaknesses:

While the authors validate the specificity of MoAb against IgM and address most of the aspects suggested by the reviewer. Some aspects are missing, mainly concerning the overstatement of the findings' novelty.

Reviewer #1 (Public review):

Wang, Junxiu et al. investigated the underlying molecular mechanisms of the insecticidal activity of betulin against the peach aphid, Myzus persicae. There are two important findings described in this manuscript: (a) betulin inhibits the gene expression of GABA receptor in the aphid, and (b) betulin binds to the GABA receptor protein, acting as an inhibitor. The first finding is supported by RNA-Seq and RNAi, and the second one is convinced with MST and electrophysiological assays. Further investigations on the betulin binding site on the receptor protein provided a fundamental discovery that T228 is the key amino acid residue for its affinity, thereby acting as an inhibitor, backed up by site-directed mutagenesis of the heterologously-expressed receptor in E. coli and by CRISPR-genome editing in Drosophila.

Although the manuscript does have strengths in principle, the weaknesses do exist: the manuscript would benefit from more comprehensive analyses to fully support its key claims in the manuscript. In particular:

(1) The Western blotting results in Figure 5A & B appear to support the claim that betulin inhibits GABR gene expression (L26), as a decrease in target protein levels is often indicative of suppressed gene expression. The result description for Figure 5A & B is found in L312-L316, within Section 3.6 ("Responses of MpGABR to betulin"), where MST and voltage-clamp assays are also presented. It seems the observed decrease in MpGABR protein content is due to gene downregulation, rather than a direct receptor protein-betulin interaction. However, this interpretation lacks discussion or analysis in either the corresponding results section or the Discussion. In contrast, Figures 5C-F are specifically designed to illustrate protein-betulin interactions. Presenting Figure 5A & B alongside these panels might lead to confusion, as they support distinct claims (gene expression vs. protein binding/inhibition). Therefore, I recommend moving Figure 5A & B either to the end of Figure 3 or to a separate figure altogether to improve clarity and logical flow. A minor point in the Western blotting experiment is that although GAPDH was used as a reference protein, there is no explanation in the corresponding M&M section.

(2) The description of the electrophysiological recording experiment is unclear regarding the use of GABA. I didn't realize that GABA, the true ligand of the GABA receptor, was used in this inhibition experiment until I reached the Results section (L321), which states, "In the presence of only GABA, a fast inward current was generated." Crucially, no details are provided on the experiment itself, including how GABA was applied (e.g., concentration, duration, whether GABA was treated, followed by betulin, or vice versa). This information is essential for reproducibility. Please ensure these details are thoroughly described in the corresponding M&M section.

(3) The phylogenetic analysis, particularly concerning Figures 4 and 6B, needs significant attention for clarity and representativeness. First, your claim that MpGABR is only closely related to CAI6365831.1 (L305-L310) is inconsistent with the provided phylogenetic tree, which shows MpGABR as equally close to Metopolophium dirhodum (XP_060864885.1) and Acyrthosiphon pisum (XP_008183008.2). Therefore, singling out only Macrosiphum euphorbiae (CAI6365831.1) is not supported by the data. Second, the representation of various insect orders is insufficient. All 11 sequences in the Hemiptera category (in both Figure 4 and Figure 6B) are exclusively from the Aphididae family. This small subset cannot represent the highly diverse Order Hemiptera. Consequently, statements like "only THR228 was conserved in Hemiptera" (L338), "The results of the sequence alignment revealed that only THR228 was conserved in Hemiptera" (L430), or "THR228... is highly conserved in Hemiptera" (L486) are not adequately supported. Third, similar concerns apply to the Diptera order, which includes 10 Drosophila and 2 mosquito samples (not diverse or representative enough), and likely to other orders as well. Thereby, the Figure 6B alignment should be revised accordingly to reflect a more accurate representation or to clarify the scope of the analysis. Fourth, there's a discrepancy in the phylogenetic method used: the M&M section (L156) states that MEGA7, ClustalW, and the neighbor-joining method were used, while the Figure 4 caption mentions that MEGA X, MUSCLE, and the Maximum likelihood method were employed. This inconsistency needs to be clarified and made consistent throughout the manuscript. Fifth, I have significant concerns about the phylogenetic tree itself (Figure 4). A small glitch was observed at the Danaus plexippus node, which raises suspicion regarding potential manipulation after tree construction. More critically, the tree, especially within Coleoptera, does not appear to be clearly resolved. I am highly concerned about whether all included sequences are true GABR orthologs or if the dataset includes partial or related sequences that could distort the phylogeny. Finally, for Figure 6B, both protein (XP_) and nucleotide (XM_) sequences were mix used. I recommend using the protein sequences instead of nucleotide sequences in this figure panel, as protein sequences are more directly informative.

(4) The Discussion section requires significant revision to provide a more insightful and interpretative analysis of the results. Currently, much of the section primarily restates findings rather than offering deeper discussion. For instance, L409-L419 restate the results, followed by the short sentence "Collectively, these results suggest that betulin may have insecticidal effects on aphids by inhibiting MpGABR expression". It could be further expanded to make it beneficial to elaborate on proposed mechanisms by which gene expression might be suppressed, including any potential transcription factors involved. In contrast, while L422-L442 also initially summarize results, the subsequent paragraph (L445-L472) effectively discusses the potential mechanisms of inhibitory action and how mortality is triggered, which is a good model for other parts of the section. However, all the discussion ends up with a short statement, "implying that betulin acts as a CA of MpGABR" (L472), which appears to be a leap. The inference that betulin acts as a competitive antagonist (CA) is solely based on the location of its extracellular binding site, which does not exactly overlap with the GABA binding site. It needs stronger justification or actually requires further experimental validation. The authors should consider rephrasing this statement to acknowledge the need for additional studies to definitively confirm this mechanism of action.

Reviewer #2 (Public review):

Summary:

This important study shows that betulin from wild peach trees disrupts neural signaling in aphids by targeting a conserved site in the insect GABA receptor. The authors present a nicely integrated set of molecular, physiological, and genetic experiments to establish the compound's species-specific mode of action. While the mechanistic evidence is solid, the manuscript would benefit from a broader discussion of evolutionary conservation and potential off-target ecological effects.

Strengths:

The main strengths of the study lie in its mechanistic clarity and experimental rigor. The identification of a betulin-binding single threonine residue was supported by (1) site-directed mutagenesis and (2) functional assays. These experiments strongly support the specificity of action. Furthermore, the use of comparative analyses between aphids and fruit flies demonstrates an important effort to explore species specificity, and the integration of quantitative data further enhances the robustness of the conclusions.

Weaknesses:

There are several important limitations that need to be addressed. The manuscript does not explore whether the observed sensitivity to betulin reflects a broadly conserved feature of GABA receptors across animal lineages or a more lineage-specific adaptation. This evolutionary context is crucial for understanding the broader significance of the findings.

In addition, while the compound's aphicidal effect is well established, the potential for off-target effects in non-target organisms - especially vertebrates - remains unaddressed, despite prior evidence that betulin interacts with mammalian GABAa receptors. There is little discussion on the ecological or environmental safety of exogenous betulin application, such as persistence, degradation, or exposure risks.

Add metrics on user engagement or feedback to showcase the app's impact on student productivity.

Quantify user adoption or transaction volume to illustrate the tool's success.

Specify the frequency of alerts or actions taken to demonstrate the system's effectiveness.

Clarify the previous hosting costs to emphasize the financial impact of this change.

Include metrics on how many resumes were improved or the feedback received from users.

Highlight any specific contributions or outcomes from these meetings to show leadership or initiative.

Add metrics on user engagement or satisfaction improvements post-redesign.

Quantify the time saved for executives or any decision-making improvements resulting from this change.

Explain how this time savings affected project timelines or team productivity.

Specify the original coverage percentage to better illustrate the improvement's significance.

Clarify how this automation impacted overall HR efficiency or employee satisfaction.

Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal significant structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are well designed and executed.

Weaknesses:

The last section of cryo-ET analysis is not convincing. "ANKRD5 depletion may impair buffering effect between adjacent DMTs in the axoneme".

"In WT sperm, DMTs typically appeared circular, whereas ANKRD5-KO DMTs seemed to be extruded as polygonal. (Fig. S9B,D). ANKRD5-KO DMTs seemed partially open at the junction between the A- and B-tubes (Fig. S9B,D)." In the TEM images of 4E, ANKRD5-KO DMTs look the same as WT. The distortion could result from suboptimal sample preparation, imaging or data processing. Thus, the subsequent analyses and conclusions are not reliable.

This paper still requires significant improvements in writing and language refinement. Here is an example: "While N-DRC is critical for sperm motility, but the existence of additional regulators that coordinate its function remains unclear" - ill-formed sentences.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Weaknesses:

Limited Citations in Introduction: Key references on the role of N-DRC components (e.g.,DRC2, DRC4) in male infertility are missing, which weakens the contextual background.

Consider shortening for clarity; e.g., 'Developing an AI agent to monitor stablecoin flows and trigger alerts for large movements.'

Break into two sentences for clarity; consider rephrasing 'automated change detection' to 'automated detection of changes'.

Specify 'driving fast and iterative improvements' with measurable outcomes, e.g., 'resulting in 30% faster review times'.

Use active voice: 'Collaborated in daily scrum meetings with a team of 5 developers...' for a stronger impact.

Quantify 'critical responsiveness issues' with specifics to enhance impact; e.g., 'fixed 5 critical responsiveness issues'.

Consider rephrasing 'eliminating the need for 100+ complex spreadsheets' to 'replacing 100+ complex spreadsheets' for stronger impact.

Rephrase to 'reducing developer testing setup time by 86% by eliminating test accounts' for conciseness.

Break into two sentences for clarity; consider rephrasing 'increasing overall coverage by 15%' to 'increasing test coverage by 15%'.

Clarify '50+ hr/month' to '50+ hours/month' for grammatical accuracy.

Add a space after the pipe for better readability: 'Jazz Aviation | React, TypeScript...'.

Use 'May 2025 – Present' for consistency with other dates. Avoid periods after 'May' and 'Present'.

Specify user engagement metrics or feedback to illustrate the bot's effectiveness and popularity.

Clarify the potential impact or use cases of this AI agent to highlight its significance.

Include specific performance metrics or improvements achieved post-rearchitecture for clarity.

Add specific metrics on how many resumes were improved or how quickly to demonstrate impact.

Focus on your contributions or outcomes from these meetings to highlight your role more effectively.

Clarify how this change improved decision-making or efficiency for the executives.

Quantify the time saved in hours or days to provide clearer context for the impact.

Specify the business impact of the feature beyond time savings to enhance significance.

Reviewer #1 (Public review):

Summary:

The major result in the manuscript is the observation of the higher order structures in a cryoET reconstruction that could be used for understanding the assembly of toroid structures. The cross-linking ability of ZapD dimers result in bending of FtsZ filaments to a constant curvature. Many such short filaments are stitched together to form a toroid like structure. The geometry of assembly of filaments - whether they form straight bundles or toroid like structures - depends on the relative concentrations of FtsZ and ZapD.

Strengths:

In addition to a clear picture of the FtsZ assembly into ring-like structures, the authors have carried out basic biochemistry and biophysical techniques to assay the GTPase activity, the kinetics of assembly, and the ZapD to FtsZ ratio.

Weaknesses:

Future scope of work includes the molecular basis of curvature generation and how molecular features of FtsZ and ZapD affect the membrane binding of the higher order assembly.

Reviewer #3 (Public review):

Summary:

Previous studies have analyzed the binding of ZapD to FtsZ and provided images of negatively stained toroids and straight bundles, where FtsZ filaments are presumably crosslinked by ZapD dimers. Toroids without ZapD have also been previously formed by treating FtsZ with crowding agents. The present study is the first to apply cryoEM tomography, which can resolve the structure of the toroids in 3D. This shows a complex mixture of filaments and sheets irregularly stacked in the Z direction and spaced radially. The most important interpretation would be to distinguish FtsZ filaments from ZapD crosslinks, This is less convincing. The authors seem aware of the ambiguity: "However, we were unable to obtain detailed structural information about the ZapD connectors due to the heterogeneity and density of the toroidal structures, which showed significant variability in the conformations of the connections between the filaments in all directions." Therefore, the reader may assume that the crosslinks identified and colored red are only suggestions, and look for their own structural interpretations.

Strengths:

This is the first cryoEM tomography to image toroids and straight bundles of FtsZ filaments bound to ZapD. A strength is the resolution, which. at least for the straight bundles. is sufficient to resolve the ~4.5 nm spacing of ZapD dimers attached to and projecting subunits of an FtsZ filament. Another strength is the pelleting assay to determine the stoichiometry of ZapD:FtsZ (although this also leads to weaknesses of interpretation).

Reviewer #1 (Public review):

Summary:

In this study, Lamberti et al. investigate how translation initiation and elongation are coordinated at the single-mRNA level in mammalian cells. The authors aim to uncover whether and how cells dynamically adjust initiation rates in response to elongation dynamics, with the overarching goal of understanding how translational homeostasis is maintained. To this end, the study combines single-molecule live-cell imaging using the SunTag system with a kinetic modeling framework grounded in the Totally Asymmetric Simple Exclusion Process (TASEP). By applying this approach to custom reporter constructs with different coding sequences, and under perturbations of the initiation/elongation factor eIF5A, the authors infer initiation and elongation rates from individual mRNAs and examine how these rates covary.

The central finding is that initiation and elongation rates are strongly correlated across a range of coding sequences, resulting in consistently low ribosome density ({less than or equal to}12% of the coding sequence occupied). This coupling is preserved under partial pharmacological inhibition of eIF5A, which slows elongation but is matched by a proportional decrease in initiation, thereby maintaining ribosome density. However, a complete genetic knockout of eIF5A disrupts this coordination, leading to reduced ribosome density, potentially due to changes in ribosome stalling resolution or degradation.

Strengths:

A key strength of this work is its methodological innovation. The authors develop and validate a TASEP-based Hidden Markov Model (HMM) to infer translation kinetics at single-mRNA resolution. This approach provides a substantial advance over previous population-level or averaged models and enables dynamic reconstruction of ribosome behavior from experimental traces. The model is carefully benchmarked against simulated data and appropriately applied. The experimental design is also strong. The authors construct matched SunTag reporters differing only in codon composition in a defined region of the coding sequence, allowing them to isolate the effects of elongation-related features while controlling for other regulatory elements. The use of both pharmacological and genetic perturbations of eIF5A adds robustness and depth to the biological conclusions. The results are compelling: across all constructs and conditions, ribosome density remains low, and initiation and elongation appear tightly coordinated, suggesting an intrinsic feedback mechanism in translational regulation. These findings challenge the classical view of translation initiation as the sole rate-limiting step and provide new insights into how cells may dynamically maintain translation efficiency and avoid ribosome collisions.

Weaknesses:

A limitation of the study is its reliance on exogenous reporter mRNAs in HeLa cells, which may not fully capture the complexity of endogenous translation regulation. While the authors acknowledge this, it remains unclear how generalizable the observed coupling is to native mRNAs or in different cellular contexts.

Additionally, the model assumes homogeneous elongation rates and does not explicitly account for ribosome pausing or collisions, which could affect inference accuracy, particularly in constructs designed to induce stalling. While the model is validated under low-density assumptions, more work may be needed to understand how deviations from these assumptions affect parameter estimates in real data.

Furthermore, although the study observes translation "bursting" behavior, this is not explicitly modeled. Given the growing recognition of translational bursting as a regulatory feature, incorporating or quantifying this behavior more rigorously could strengthen the work's impact.

Assessment of Goals and Conclusions:

The authors successfully achieve their stated aims: they quantify translation initiation and elongation at the single-mRNA level and show that these processes are dynamically coupled to maintain low ribosome density. The modeling framework is well suited to this task, and the conclusions are supported by multiple lines of evidence, including inferred kinetic parameters, independent ribosome counts, and consistent behavior under perturbation.

Impact and Utility:

This work makes a significant conceptual and technical contribution to the field of translation biology. The modeling framework developed here opens the door to more detailed and quantitative studies of ribosome dynamics on single mRNAs and could be adapted to other imaging systems or perturbations. The discovery of initiation-elongation coupling as a general feature of translation in mammalian cells will likely influence how researchers think about translational regulation under homeostatic and stress conditions.

The data, models, and tools developed in this study will be of broad utility to the community, particularly for researchers studying translation dynamics, ribosome behavior, or the effects of codon usage and mRNA structure on protein synthesis.

Context and Interpretation:

This study contributes to a growing body of evidence that translation is not merely controlled at initiation but involves feedback between elongation and initiation. It supports the emerging view that ribosome collisions, stalling, and quality control pathways play active roles in regulating initiation rates in cis. The findings are consistent with recent studies in yeast and metazoans showing translation initiation repression following stalling events. However, the mechanistic details of this feedback remain incompletely understood and merit further investigation, particularly in physiological or stress contexts.

In summary, this is a thoughtfully executed and timely study that provides valuable insights into the dynamic regulation of translation and introduces a modeling framework with broad applicability. It will be of interest to a wide audience in molecular biology, systems biology, and quantitative imaging.

Reviewer #2 (Public review):

Summary:

This manuscript uses single-molecule run-off experiments and TASEP/HMM models to estimate biophysical parameters, i.e., ribosomal initiation and elongation rates. Combining inferred initiation and elongation rates, the authors quantify ribosomal density. TASEP modeling was used to simulate the mechanistic dynamics of ribosomal translation, and the HMM is used to link ribosomal dynamics to microscope intensity measurements. The authors' main conclusions and findings are:

(1) Ribosomal elongation rates and initiation rates are strongly coordinated.

(2) Elongation rates were estimated between 1-4.5 aa/sec. Initiation rates were estimated between 0.5-2.5 events/min. These values agree with previously reported values.

(3) Ribosomal density was determined below 12% for all constructs and conditions.

(4) eIF5A-perturbations (KO and GC7 inhibition) resulted in non-significant changes in translational bursting and ribosome density.

(5) eIF5A perturbations resulted in increases in elongation and decreases in initiation rates.

Strengths:

This manuscript presents an interesting scientific hypothesis to study ribosome initiation and elongation concurrently. This topic is highly relevant for the field. The manuscript presents a novel quantitative methodology to estimate ribosomal initiation rates from Harringtonine run-off assays. This is relevant because run-off assays have been used to estimate, exclusively, elongation rates.

Weaknesses:

The conclusion of the strong coordination between initiation and elongation rates is interesting, but some results are unexpected, and further experimental validation is needed to ensure this coordination is valid.

(1) eIF5a perturbations resulted in a non-significant effect on the fraction of translating mRNA, translation duration, and bursting periods. Given the central role of eIF5a, I would have expected a different outcome. I would recommend that the authors expand the discussion and review more literature to justify these findings.

(2) The AAG construct leading to slow elongation is very surprising. It is the opposite of the field consensus, where codon-optimized gene sequences are expected to elongate faster. More information about each construct should be provided. I would recommend more bioinformatic analysis on this, for example, calculating CAI for all constructs, or predicting the structures of the proteins.

(3) The authors should consider using their methodology to study the effects of modifying the 5'UTR, resulting in changes in initiation rate and bursting, such as previously shown in reference Livingston et al., 2023. This may be outside of the scope of this project, but the authors could add this as a future direction and discuss if this may corroborate their conclusions.

(4) The mathematical model and parameter inference routines are central to the conclusions of this manuscript. In order to support reproducibility, the computational code should be made available and well-documented, with a requirements file indicating the dependencies and their versions.

Reviewer #3 (Public review):

Disclaimer:

My expertise is in live single-molecule imaging of RNA and transcription, as well as associated data analysis and modeling. While this aligns well with the technical aspects of the manuscript, my background in translation is more limited, and I am not best positioned to assess the novelty of the biological conclusions.

Summary:

This study combines live-cell imaging of nascent proteins on single mRNAs with time-series analysis to investigate the kinetics of mRNA translation.

The authors (i) used a calibration method for estimating absolute ribosome counts, and (ii) developed a new Bayesian approach to infer ribosome counts over time from run-off experiments, enabling estimation of elongation rates and ribosome density across conditions.

They report (i) translational bursting at the single-mRNA level, (ii) low ribosome density (~10% occupancy {plus minus} a few percents), (iii) that ribosome density is minimally affected by perturbations of elongation (using a drug and/or different coding sequences in the reporter), suggesting a homeostatic mechanism potentially involving a feedback of elongation onto initiation, although (iv) this coupling breaks down upon knockout of elongation factor eIF5A.

Strengths:

(1) The manuscript is well written, and the conclusions are, in general, appropriately cautious (besides the few improvements I suggest below).

(2) The time-series inference method is interesting and promising for broader applications.

(3) Simulations provide convincing support for the modeling (though some improvements are possible).

(4) The reported homeostatic effect on ribosome density is surprising and carefully validated with multiple perturbations.

(5) Imaging quality and corrections (e.g., flat-fielding, laser power measurements) are robust.

(6) Mathematical modeling is clearly described and precise; a few clarifications could improve it further.

Weaknesses:

(1) The absolute quantification of ribosome numbers (via the measurement of $i_{MP}$​) should be improved. This only affects the finding that ribosome density is low, not that it appears to be under homeostatic control. However, if $i_{MP}$​ turns out to be substantially overestimated (hence ribosome density underestimated), then "ribosomes queuing up to the initiation site and physically blocking initiation" could become a relevant hypothesis. In my detailed recommendations to the authors, I list points that need clarification in their quantifications and suggest an independent validation experiment (measuring the intensity of an object with a known number of GFP molecules, e.g., MS2-GFP MS2-GFP-labeled RNAs, or individual GEMs).

(2) The proposed initiation-elongation coupling is plausible, but alternative explanations, such as changes in abortive elongation frequency, should be considered more carefully. The authors mention this possibility, but should test or rule it out quantitatively.

(3) The observation of translational bursting is presented as novel, but similar findings were reported by Livingston et al. (2023) using a similar SunTag-MS2 system. This prior work should be acknowledged, and the added value of the current approach clarified.

(4) It is unclear what the single-mRNA nature of the inference method is bringing since it is only used here to report _average_ ribosome elongation rate and density (averaged across mRNAs and across time during the run-off experiments - although the method, in principle, has the power to resolve these two aspects).

(5) I did not find any statement about data availability. The data should be made available. Their absence limits the ability to fully assess and reproduce the findings.

Reviewer #1 (Public review):

Summary:

In this revised report, Yamanaka and colleagues investigate a proposed mechanism by which testosterone modulates seminal plasma metabolites in mice. The authors have made improvements from the previous version by softening the claim that oleic acid derived from seminal vesicle epithelium strongly affects linear progressive motility in isolated cauda epididymal sperm in vitro. They have also addressed the ambiguous references to the strength of the relationship between fatty acids and sperm motility, making the manuscript more balanced and nuanced.

Strengths:

This study addresses an important gap in our understanding of how testosterone influences seminal plasma metabolites and, in turn, sperm motility. The findings provide valuable insights into the sensitivity of seminal vesicle epithelial cells to testosterone, which could improve in vitro conditions for studying sperm motility. The authors have added methodological details and re-performed experiments with more appropriate control groups, enhancing the robustness of the study. These revisions, along with more carefully modified language reflecting measurement nuances, add significant value to the field. The study's detailed exploration of the physiological role of reproductive tract glandular secretions in modulating sperm behaviors is likely to be of broad interest, providing a strong foundation for future research on the relationship between fatty acid beta-oxidation and sperm motility patterns.

Weaknesses:

While the connection between media fatty acids and sperm motility patterns is still not fully conclusive, the authors have taken substantial steps to clarify and tone down their conclusions. The revised manuscript presents a more balanced view, acknowledging the complexity of the relationship and providing a more solid basis for follow-on studies.

Reviewer #2 (Public review):

Using a combination of in vivo studies with testosterone-inhibited and aged mice with lower testosterone levels, as well as isolated mouse and human seminal vesicle epithelial cells, the authors demonstrate that testosterone induces an increase in glucose uptake. The study reveals that testosterone triggers differential gene expression, particularly focusing on metabolic enzymes. They specifically identify increased expression of enzymes regulating cholesterol and fatty acid synthesis, leading to heightened production of 18:1 oleic acid. The revised version of the manuscript significantly strengthens the role of ACLY as a central regulator of seminal vesicle epithelial cell metabolic programming. The authors suggest that fatty acids secreted by seminal vesicle epithelial cells are taken up by sperm, resulting in a positive impact on sperm function. While the lipid mixture mimicking the lipids secreted by seminal vesicle epithelial cells shows marginal positive effect on sperm motility, the authors have made considerable progress in refining their conclusions. The revised manuscript acknowledges the complexity of pinpointing the specific seminal vesicle fluid component that potentially positively affects sperm function, providing a more measured and credible interpretation of their findings.

Quantify the number of users or sessions tracked to demonstrate the app's usage and impact.

Include user adoption rates or feedback to illustrate the bot's effectiveness and popularity.

Clarify the potential financial impact or risk reduction achieved through this AI agent's alerts.

Add metrics on how many resumes were improved or user satisfaction ratings to demonstrate impact.

Highlight a specific contribution or idea that led to a significant improvement in team performance.

Specify the user engagement metrics or feedback received post-redesign to showcase impact.

Quantify the decision-making improvements or time saved for executives due to the dashboards.

Consider rephrasing to emphasize how this tool improved support response times or user experience.

Add a specific example of a critical bug found to highlight the importance of your contributions.

Clarify the impact by stating how this improved efficiency or employee satisfaction beyond just time saved.

Reviewer #1 (Public review):

Summary:

The authors aim to explore the effects of the electrogenic sodium-potassium pump (Na+/K+-ATPase) on the computational properties of highly active spiking neurons, using the weakly-electric fish electrocyte as a model system. Their work highlights how the pump's electrogenicity, while essential for maintaining ionic gradients, introduces challenges in neuronal firing stability and signal processing, especially in cells that fire at high rates. The study identifies compensatory mechanisms that cells might use to counteract these effects, and speculates on the role of voltage dependence in the pump's behavior, suggesting that Na⁺/K⁺-ATPase could be a factor in neuronal dysfunctions and diseases

Strengths:

(1) The study explores a less-examined aspect of neural dynamics-the effects of Na⁺/K⁺-ATPase electrogenicity. It offers a new perspective by highlighting the pump's role not only in ion homeostasis but also in its potential influence on neural computation.

(2) The mathematical modeling used is a significant strength, providing a clear and controlled framework to explore the effects of the Na⁺/K⁺+-ATPase on spiking cells. This approach allows for the systematic testing of different conditions and behaviors that might be difficult to observe directly in biological experiments.

(3) The study proposes several interesting compensatory mechanisms, such as sodium leak channels and extracellular potassium buffering, which provide useful theoretical frameworks for understanding how neurons maintain firing rate control despite the pump's effects.

Weaknesses:

(1) While the modeling approach provides valuable insights, the lack of experimental data to validate the model's predictions weakens the overall conclusions.

(2) The proposed compensatory mechanisms are discussed primarily in theoretical terms without providing quantitative estimates of their impact on the neuron's metabolic cost or other physiological parameters.

Comments on revisions:

The revised manuscript is notably improved.

Reviewer #2 (Public review):

Summary:

The paper by Weerdmeester, Schleimer, and Schreiber uses computational models to present the biological constraints under which electrocytes - specialized, highly active cells that facilitate electro-sensing in weakly electric fish-may operate. The authors suggest potential solutions that these cells could employ to circumvent these constraints.

Electrocytes are highly active or spiking (greater than 300Hz) for sustained periods (for minutes to hours), and such activity is possible due to an influx of sodium and efflux of potassium ions into these cells after each spike. The resulting ion imbalance must be restored, which in electrocytes, as with many other biological cells, is facilitated by the Na-K pumps at the expense of biological energy, i.e., ATP molecules. For each ATP molecule the pump uses, three positively charged sodium ions from the intracellular space are exchanged for two positively charged potassium ions from the extracellular space. This creates a net efflux of positive ions into the extracellular space, resulting in hyperpolarized potentials for the cell over time. For most cells, this does not pose an issue, as their firing rate is much slower, and other compensatory mechanisms and pumps can effectively restore the ion imbalances. However, in the electrocytes of weakly electric fish, which spike at exceptionally high rates, the net efflux of positive ions presents a challenge. Additionally, these cells are involved in critical communication and survival behaviors, underscoring their essential role in reliable functioning.

In a computational model, the authors test four increasingly complex solutions to the problem of counteracting the hyperpolarized states that occur due to continuous NaK pump action to sustain baseline activity. First, they propose a solution for a well-matched Na leak channel that operates in conjunction with the NaK pump, counteracting the hyperpolarizing states naturally. Their model shows that when such an orchestrated Na leak current is not included, quick changes in the firing rates could have unexpected side effects. Secondly, they study the implications of this cell in the context of chirps-a means of communication between individual fish. Here, an upstream pacemaking neuron entrains the electrocyte to spike, which ceases to produce a so-called chirp - a brief pause in the sustained activity of the electrocytes. In their model, the authors demonstrate that including the extracellular potassium buffer is necessary to obtain a reliable chirp signal. Thirdly, they tested another means of communication in which there was a sudden increase in the firing rate of the electrocyte, followed by a decay to the baseline. For this to occur reliably, the authors emphasize that a strong synaptic connection between the pacemaker neuron and the electrocyte is necessary. Finally, since these cells are energy-intensive, they hypothesize that electrocytes may have energy-efficient action potentials, for which their NaK pumps may be sensitive to the membrane voltages and perform course correction rapidly.

Strengths:

The authors extend an existing electrocyte model (Joos et al., 2018) based on the classical Hodgkin and Huxley conductance-based models of sodium and potassium currents to include the dynamics of the sodium-potassium (NaK) pump. The authors estimate the pump's properties based on reasonable assumptions related to the leak potential. Their proposed solutions are valid and may be employed by weakly electric fish. The authors explore theoretical solutions to electrosensing behavior that compound and suggest that all these solutions must be simultaneously active for the survival and behavior of the fish. This work provides a good starting point for conducting in vivo experiments to determine which of these proposed solutions the fish employ and their relative importance. The authors include testable hypotheses for their computational models.

Weaknesses:

The model for action potential generation simplifies ion dynamics by considering only sodium and potassium currents, excluding other ions like calcium. The ion channels considered are assumed to be static, without any dynamic regulation such as post-translational modifications. For instance, a sodium-dependent potassium pump could modulate potassium leak and spike amplitude (Markham et al., 2013).

This work considers only the sodium-potassium (NaK) pumps to restore ion gradients. However, in many cells, several other ion pumps, exchangers, and symporters are simultaneously present and actively participate in restoring ion gradients. When sodium currents dominate action potentials, and thus when NaK pumps play a critical role, such as the case in Eigenmannia virescens, the present study is valid. However, since other biological processes may find different solutions to address the pump's non-electroneutral nature, the generalizability of the results in this work to other fast-spiking cell types is limited. For example, each spike could include a small calcium ion influx that could be buffered or extracted via a sodium-calcium exchanger.

Reviewer #1 (Public review):

The manuscript by Long et al. focused on SUL1, a gene encoding a sulfate transporter with signaling roles in yeast. The authors claim that the deletion of SUL1, rather than SUL2 (encoding a similar transporter), extended yeast replicative lifespan independent of sulfate transport. They also show that SUL1 loss-of-function mutants display decreased PKA activity, indicated by stress-protective carbohydrate accumulation, relevant transcription factor relocalization (measured during aging in single cells), and changes in gene expression. Finally, they show that loss of SUL1 increases autophagy, which is consistent with the longer lifespan of these cells. Overall, this is an interesting paper, but additional work should strengthen several conclusions, especially for the role of sulfate transport. Specific points include the following:

What prompted the authors to measure the RLS of sul1 mutants? Prior systematic surveys of RLS in the same strain background (which included the same sul1 deletion strain they used) did not report lifespan extension in sul1 cells (PMID: 26456335).

Cells carrying a mutant Sul1 (E427Q), which was reported to be disrupted in sulfate transport, did not have a longer lifespan (Figure 1), leading them to conclude that "lifespan extension by SUL1 deletion is not caused by decreased sulfate uptake". They would need to measure sulfate uptake in the mutants they test to draw that conclusion firmly.

Related to my previous point, another simple experiment would be to repeat the assays in Figure 1 with exogenous sulfur added to see if the lifespan extension is suppressed.

There needs to be more information in the text or the methods about how they did the enrichment analysis in Figure 2B. P-values are typically insufficient, and adjusted FDR values are reported from standard gene ontology platforms (e.g., PANTHER).

It is somewhat puzzling that relocalization of Msn2 was not seen in very old cells (past the 17th generation), but it was evident in younger cells. The authors could consider another possibility, that it was early and midlife experiences that made those cells live longer. Past that window, loss of Sul1 may have no impact on longevity. A conditional shutoff system to regulate SUL1 expression would be needed to test the above, albeit this is probably beyond the scope of this report.

The connections between glucose restriction, autophagy, and sul1 (Figure 4) could be further tested by measuring the RLS of sul1 cells in glucose-restricted cells. If RLS is further extended by glucose restriction, then whatever effects they see should be independent of glucose restriction.

They made and tested the double (sul1, msn2) mutants, but they should also test the sul1, msn4 combination since Msn4 functions similarly to Msn2.

Comments on revisions:

Overall, this is a somewhat improved manuscript, but some prior concerns about the validity of the conclusions remain unresolved.

Reviewer #2 (Public review):

Summary:

In this study, the authors find that deletion of a sulfate transporter in yeast, Sul1, leads to extension of replicative lifespan. They investigate mechanisms underlying this extension, and claim that the effects on longevity can be separated from sulfate transport, and are instead linked to a previously proposed transceptor function of the Sul1 transporter. Through RNA sequencing analysis, the authors find that Sul1 loss triggers activation of several stress response pathways, and conclude that deletion of two pathways, autophagy or Msn2/4, partially prevents lifespan extension in cells lacking Sul1. Overall, while it is well-appreciated that activation of Msn2/4 or autophagy is beneficial for lifespan extension in yeast, the results of this study would add an important new mechanism by which this could achieved, through perceived sulfate starvation. However, as described below, several of the experiments utilized to support the authors conclusion are not experimentally sound, and significant additional experimentation is required to support the authors claims throughout the manuscript.

Strengths:

The major strength of the study is the robust RNA-seq data that identified differentially expressed genes in cells lacking Sul1. This facilitated the authors focus on two of these pathways, autophagy and the Msn2/4 stress response pathway.

Weaknesses:

Several critical experimental flaws need to be addressed by the authors to more rigorously test their hypothesis.

(1) The lifespan assays throughout the manuscript contain inconsistencies in the mean lifespan of the wild type strain, BY4741. For example, in Figure 1A, the lifespan of BY4741 is 24.3, and the extended lifespan of the sul1 mutant is 31. However, although all mutants tested in Figure 1B also have lifespans close to 30 cell divisions, the wild type control is also at 30 divisions in those experiments as well. This is problematic, as it makes it impossible to conclude anything about the lifespan extension of various mutants with the inconsistencies in the wild type lifespan. Additionally, the mutants analyzed in 1B are what the authors use to claim that loss of the transporter does not extend lifespan through sulfate limitation, but instead through a signaling function. Thus, it remains unclear whether loss of sul1 extends lifespan at all, and if it does, whether this is separable from cellular sulfate levels.

(2) While the authors use mutants in Figure 1 that should have differential effects on sulfate levels in cells, the authors need to include experiments to measure sulfate levels in their various mutant cells to draw any conclusions about their data.

(3) Similar to point 2, the authors focused their RNA sequencing analysis on deletion of sul1 and did not include important RNA seq analysis of the specific Sul1 mutation or other mutants in Figure 1B that do not exhibit lifespan extension. The prediction is that they should not see activation of stress response pathways in these mutants as they do not see lifespan extension, but this needs to be tested.

(4) While the RNA-seq data is robust in Figure 2 as well as the follow up quantitative PCR and trehalose/glycogen assays in 2A-B, the follow-up imaging assays for Msn2/4 localization in Figure 2 are not robust and are difficult to interpret. The authors need to include more high-resolution imaging or at least a close up of the cells in Figure 3C.

(5) The autophagy assays utilized in Figure 4 appear to all be done with a C-terminal GFP-tagged Atg8 protein. As C-terminal GFP is removed from Atg8 prior to conjugation to phosphatidylethanolamine, microscopy assays of this reporter cannot be utilized to report on autophagy activity or flux. Instead, the authors need to utilize N-terminally tagged Atg8, which they can monitor for vacuole uptake as an appropriate readout of autophagy levels. As it stands, the authors cannot draw any conclusions about autophagy activity in their studies.

Comments on revisions:

Their autophagy conclusions are weak at best. As was highlighted in the previous review, they need to use an N-terminal Atg8 fusion for these experiments.

Reviewer #3 (Public review):

Summary:

In the revised manuscript, Long et al., showed that sul1∆ mutants have extended replicative lifespan in budding yeast. In comparison, other mutants that have sulfate transport deficiency did not show extended lifespan, suggesting SUL1 deletion extends lifespan independently of sulfate intake. The authors then explored the transcriptome of sul1∆ mutants by RNA-seq, which suggests that SUL1 deletion impacts common longevity pathways. Furthermore, the authors characterized how the PKA pathway is affected in sul1∆ mutants: SUL1 deletion promotes the nuclear localization of Msn2, as well as autophagy, indicating down-regulation of the PKA pathway.

Strengths:

This study raised an interesting point that inorganic transporters may impact cellular stress response pathways and affect lifespan. Some of the characterizations on the sul1∆ mutants, including the RNA-seq and MSN2 localization could provide valuable sources for people in related fields. Compared with the previous version, the writing is significantly improved, making the manuscript clearer.

Weaknesses:

Several critical flaws have not been revised. The claims are still not well supported by the data.

(1) The revised manuscript still uses Atg8-EGFP, in which GFP is likely tagging at the C-terminus of Atg8. No strain information was provided for this strain, so it is unclear whether it is N- or C- terminal tagged. As pointed by reviewers of the previous version, C-terminal tagged Atg8 is not functional. As a result, the conclusions on autophagy (Figure 4) is questionable.

(2) The nuclear localization of Msn2 is much more convincing after the authors updated Figure 3C. However, the rest of the microscopy images (e.g. Figure 3E, 4B, 4E) are still of low resolution. Again, I suggest to separate the DIC and GFP channels. It is really hard to tell where is the GFP signal from these figures.

(3) In the Kankipati et al. 2015 paper, which is cited by the authors, SUL1E427Q is incorporated on a pRS316 (URA3) plasmic and expressed in sul1∆sul2∆ mutants. In this manuscript, the authors used SUL1E427Q mutants but did not give detailed information on how this construct is expressed. Is it endogenously mutated, incorporated into somewhere in the genome, or expressed from an extrachromosomal plasmid?<br /> In Figure 1B, they simply used BY4741 as a control for the SUL1E427Q mutant. This makes me thinking they are using a SUL1E427Q endogenous point mutation mutant. If so, the authors may want to include the information about this strain in their Supplementary table. Or if it is expressed from an extra copy on chromosomes or extrachromosomal plasmids, the authors would need to express this construct in sul1∆ mutant. In this case, the authors may want to use sul1∆ and sul1∆+empty vector as controls, instead of BY4741. As the authors mentioned in their rebuttal letter, lifespan experiments vary between each individual trials and are not comparable between different trials. Thus proper controls are essential to make the results convincing.

(4) As suggested by reviewers of the previous version, the authors tested the sulfate uptake in different mutants within 10 minute of Na2SO4 addition (Figure 1B). The authors concluded from the data that wild type takes up sulfate faster than the mutants but they reach similar concentrations at the end point (as fast as 10 minutes). Are all these cells sulfate-starved before the experiment? If not, the experiment might be affected by the basal level of sulfate in each mutants.

Reviewer #1 (Public review):

The authors aimed to explore the prognostic and therapeutic relevance of immunogenic cell death (ICD)-related genes in bladder cancer, focusing on a risk-scoring model involving CALR, IL1R1, IFNB1, and IFNG. The research indicates that higher expression of certain ICD-related genes is associated with enhanced immune infiltration, prolonged survival, and improved responsiveness to PD1-targeted therapy in bladder cancer patients.

Major strengths:

• The establishment of an ICD-related gene risk model based on publicly available datasets (TCGA and GEO) and further validated through tissue arrays and preliminary single-cell RNA sequencing data provides potential but weak clinical guidance.

• The integration of multi-dimensional data (gene expression, mutation burden, immune infiltration, and treatment responses) strengthens the clinical applicability of the model.

Key limitations and concerns:

(1) Gene Selection and Novelty:

The selection of genes predominantly reflects known regulators of immune responses, somewhat limiting the novelty. Exploring less-characterized ICD markers or extending validation beyond bladder cancer could improve the model's innovative aspect and wider clinical relevance.

(2) Reliance on RNA-Seq for Immune Infiltration:

Immune infiltration analyses based primarily on bulk RNA-Seq data have inherent methodological limitations, such as inability to distinguish cell subsets accurately. Incorporation of robust single-cell sequencing would significantly enhance the reliability of these findings. Although the authors recognize this limitation, future studies should directly address it.

(3) Drug Sensitivity and Immunotherapy Response Data:

While the authors clarify that the drug sensitivity analysis was performed using established databases (TCGA via pRRophetic), the unexpected correlations between ICD-related genes and various targeted therapies need further mechanistic validation. The observed relationships may reflect indirect associations rather than direct biological relevance, which warrants cautious interpretation.

(4) Presentation and Clarity Issues:

Initially noted formatting inconsistencies across figures compromised professional presentation; these have been corrected by the authors. Additionally, the authors have now provided essential methodological details, including clear sample sizes and database versions, enhancing reproducibility.

(5) Immunotherapy Response Evidence:

Conclusions regarding differences in immunotherapy response rates between patient subgroups, although intriguing, remain based on retrospective database analyses with relatively limited demographic and clinical detail. Future prospective studies or more detailed patient characterization would be required to robustly confirm these associations.

(6) Interpretation of ICD Gene Signatures:

The ICD-related gene set includes many genes broadly associated with immune activation rather than specifically ICD. Although this was addressed by the authors, clearly distinguishing ICD-specific versus general immune-response genes in future studies would help clarify biological implications.

Summary and Recommendations for Readers:

Overall, this study presents an interesting and clinically relevant risk-scoring approach to stratify bladder cancer patients based on ICD-related gene expression profiles. It provides useful information about prognosis, immune infiltration, and potential immunotherapy responsiveness. However, readers should interpret the results within the context of its limitations, notably the need for broader validation and careful consideration of the biological significance underlying the observed associations. This work lays a valuable foundation for further investigation into the integration of ICD and immune response signatures in personalized cancer therapy.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the role of deubiquitinases (DUBs) in modulating the efficacy of PROTAC-mediated degradation of the cell-cycle kinase AURKA. Using a focused siRNA screen of 97 human DUBs, they identify UCHL5 and OTUD6A as negative regulators of AURKA degradation by PROTACs. They further offer a mechanistic explanation of enhanced AURKA degradation in the nucleus via OTUD6A expression being restricted to the cytosol, thereby protecting the cytoplasmic pool of AURKA. These findings provide important insight into how subcellular localization and DUB activity influence the efficiency of targeted protein degradation strategies, which could have implications for therapy.

Strengths:

(1) The manuscript is well-structured, with clearly defined objectives and well-supported conclusions.

(2) The study employs a broad range of well-validated techniques - including live-cell imaging, proximity ligation assays, HiBiT reporter systems, and ubiquitin pulldowns - to dissect the regulation of PROTAC activity.

(3) The authors use informative experimental controls, including assessment of cell-cycle progression effects, rescue experiments with siRNA-resistant constructs to confirm specificity, and the application of both AURKA-targeting PROTACs with different warheads and orthogonal degrader systems (e.g., dTAG-13 and dTAGv-1) to differentiate between target- and ligase-specific effects.

(4) The identification of OTUD6A as a cytosol-restricted DUB that protects cytoplasmic but not nuclear AURKA is novel and may have therapeutic relevance for selectively targeting oncogenic nuclear AURKA pools.

Weaknesses:

(1) Although UCHL5 and OTUD6A are shown to limit AURKA degradation, direct physical interaction was not assessed.

(2) Although the authors identify a correlation between DUB knockdown-induced cell cycle progression and enhanced PROTAC activity, only one DUB (USP36) is excluded on this basis. In addition, one DUB is shown in the correlation plot (Figure 3B) whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression, but it is not identified/discussed.

(3) While the authors suggest that combining PROTACs with DUB inhibition could enhance degradation, this was not experimentally tested.

(4) The study identifies UCHL5 as a general antagonist of CRBN-recruiting PROTACs, yet the ubiquitin pulldown experiments (Figure 5G, H) show no change in AURKA ubiquitination upon UCHL5 knockdown. This raises questions about the precise step or mechanism by which UCHL5 exerts its protective effect.

Reviewer #2 (Public review):

Summary:

In this study, the authors present a screening approach to identify deubiquitylases that may impact PROTAC efficacy/potency, specifically in this case using a previously reported AURKA PROTAC as an initial model. The authors claim that UCHL5 is able to control the level of degradation of both AURKA and dTAG when using CRBN-mediated PROTACs; however, VHL is not impacted by UCHL5 activity. They additionally claim that OTUD6A is able to control the extent of AURKA degradation in a target protein-specific manner and that this effect is specific to cytoplasm-located AURKA.

Overall, whilst the endeavour is of interest and importance, we found that the claims made were overly generalised, the effects observed when knocking down the respective DUBs were very small, the systems used are highly artificial, and the data is not presented in a way that makes understanding absolute changes transparent.

Strengths:

The topic is of high interest and relevance and explores an underappreciated and understudied area of the PROTAC mechanism of action. If findings could be better supported, they would certainly bring value to the field.

Weaknesses:

The overall effects observed are sometimes limited in real terms. Even if statistically significant, the data presented does not fully support that changes in degradation due to UCHL5 activity represent changes of functional relevance. The data provided often omits the absolute changes in protein abundance observed. Data on endogenous/less engineered systems and/or with higher resolution read-outs would greatly strengthen some conclusions.

Reviewer #3 (Public review):

Summary:

Cardno et al. "test the hypothesis that DUBs could oppose PROTAC-mediated degradation of cellular targets, using AURKA as a model target". A screen with a panel of siRNA that depleted 97 DUBs in the presence and absence of AURKA targeted PROTAC-D identified DUBs that regulated AURKA and those that affected the sensitivity of PROTAC-D. Validation studies with DUBs, UCHL5, and OTU6A yielded mixed results. UCHL5 not only affected PROTAC-mediated AURKA degradation but also affected CRBN-associated substrates, OTUD6A, more specifically, affected PROTAC-mediated AURKA degradation, and the effects of OTUD6A were associated with the localisation of AURKA. The findings are interesting; the impact of the findings would be strengthened if the key results are validated in one or more cancer cell lines that have not been modified.

Reviewer #1 (Public review):

Summary:

BK channels are widely distributed and involved in many physiological functions. They have also proven a highly useful tool for studying general allosteric mechanisms for gating and modulation by auxiliary subunits. Tetrameric BK channels are assembled from four separate alpha subunits, which would be identical for homozygous alleles and potentially of five different combinations for heterozygous alleles (Geng et al., 2023, https://doi.org/10.1085/jgp.202213302). Construction of BK channels with concatenated subunits in order to strictly control heteromeric subunit composition had not yet been used because the N-terminus in BK channels is extracellular, whereas the C-terminus is intracellular. In this new work, Chen, Li, and Yan devise clever methods to construct and assemble BK channels of known subunit composition, as well as to fix the number of γ1 axillary subunits per channel. With their novel molecular approaches, Chen, Li and Yan report that a single γ1 axillary subunit is sufficient to fully modulate a BK channel, that the deep conducting pore mutation L312A exhibited a graded effect on gating with each addition mutated subunit replacing a WT subunit in the channel adding an additional incremental left shift in activation, and that the V288A mutation at the selectivity filter must be present on all four alpha subunits in order to induce channel inactivation. Chen, Li, and Yan have been successful in introducing new molecular tools to generate BK channels of known stoichiometry and subunit composition. They validate their methods and provide three examples of their use with useful observations.

Strengths:

Powerful new molecular tools for the study of channel gating have been developed and validated in the study.

Weaknesses:

One example each of auxiliary, deep pore, and selectivity filter allosteric actions is presented, but this is sufficient for the purposes of the paper to establish their methods and present specific examples of applicability.

Reviewer #2 (Public review):

Summary:

This manuscript describes novel BK channel concatemers as a tool to study the stoichiometry of the gamma subunit and mutations in the modulation of the channel. Taking advantage of the modular design of the BK channel alpha subunit, the authors connected S1-S6/1st RCK as two- and four-subunit concatemers and coexpressed with S0-RCK2 to form normal function channels. These concatemers avoided the difficulty that the extracellular N-terminus of S0 was unable to connect with the cytosolic C-terminus of the gamma subunit, allowing a single gamma subunit to be connected to the concatemers. The concatemers also helped reveal the required stoichiometry of mutant BK subunits in modulating channel function. These include L312A in the deep pore region that altered channel function additively with each additional subunit harboring the mutation, and V288A at the selectivity filter that altered channel function cooperatively only when all four subunits were mutated. These results demonstrate that the concatemers are robust and effective in studying BK channel function and molecular mechanisms related to stoichiometry. The different requirement of the gamma subunit and the mutations stoichiometry for altering channel function is interesting, which may relate to the fundamental mechanism of how different motifs of the channel protein control function.

Strengths:

The manuscript presents well-designed experiments with high-quality data, which convincingly demonstrate the BK channel concatemers and their utility. The results are clearly presented.

Weaknesses:

This reviewer did not identify any major concerns with the manuscript.

Reviewer #1 (Public review):

Summary:

This manuscript presents a high-quality, chromosome-level genome assembly of the European cuttlefish (Sepia officinalis), a representative species of the cephalopod lineage. Using state-of-the-art sequencing and scaffolding technologies -including PacBio HiFi long reads and Hi-C chromatin conformation capture - the authors deliver a genome assembly with exceptional contiguity and completeness, as evidenced by high BUSCO scores. This genome resource fills a significant gap in cephalopod genomics and offers a valuable foundation for studies in neurobiology, behavior, and evolutionary biology. However, there are several major aspects that need to be strengthened.

Major Revisions Recommended:

(1) Single-individual genome limitation

The genome assembly is based on a single individual, which appears to be male. While this approach is common in genome projects, it does not capture the full genetic diversity of the species. As S. officinalis exhibits a wide geographical range and possible population structure, future efforts (or discussion in this manuscript) should consider re-sequencing multiple individuals - of both sexes and from diverse geographic origins - to characterize population-level variation, sex-linked features, and structural polymorphisms.

(2) Limited experimental validation of chromosomal inferences

The study reports chromosome-scale scaffolding using Hi-C data and proposes a revised karyotype for S. officinalis. However, these inferences would be significantly strengthened by orthogonal validation methods. In particular, fluorescence in situ hybridization (FISH) or karyotyping from cytogenetic preparations would provide direct confirmation of chromosome number and structural arrangements. The reliance solely on Hi-C contact maps for inferring chromosomal organization should be acknowledged as a limitation or supplemented with such validations.

(3) Shallow discussion of chromosomal evolution

The manuscript briefly mentions chromosomal number differences among cephalopods but does not explore their evolutionary or functional implications. A more thorough comparative analysis - linking chromosomal rearrangements (e.g., fusions, fissions) with ecological adaptation, life history, or neural complexity - would greatly enhance the impact of the findings. Referencing chromosomal dynamics in related taxa and possible links to behavioral innovations would contextualize these results more effectively.

(4) Underdeveloped gene family and pathway analysis

While the authors identify expansions in gene families such as protocadherins and C2H2 zinc finger transcription factors, the functional significance of these expansions remains speculative. The manuscript would benefit from:

a) Functional enrichment analyses (e.g., GO, KEGG) targeting these gene families.

b) Expression profiling across tissues or developmental stages to infer regulatory roles.

c) Comparison with expression or expansion patterns in other cephalopods with known behavioral complexity (e.g., Octopus bimaculoides, Euprymna scolopes).

d) Potential integration of transcriptomic or epigenomic data to support regulatory hypotheses.

Reviewer #2 (Public review):

Summary:

This paper concerns an interesting organism, Sepia officinalis. However, in the opinion of this reviewer, the paper reads somewhat like a genome report. The authors have used 23x PacBio HiFi in conjunction with relatively low coverage (11x) Hi-C to scaffold the genome into a karyotype of 47 chromosomes. They have used a combination of short and long read RNA seq to annotate the genome in what looks like a very good annotation. The paper offers basic analyses of the Busco evaluation, some descriptive analyses of gene family and repeat content, and a bit more focused analysis on synteny among sequenced squids. Generally, the data will be useful.

Strengths:

This is a high-quality annotation, and the data ultimately will be useful to other researchers. I appreciate trying to understand what's happening between assemblies of S. officinalis.

Weaknesses:

I don't believe the data at hand makes a strong case for the argument of 47 chromosomes. This is my biggest sticking point with the paper, and it is for a few reasons:

(1) The authors point to assembly differences between the DToL assembly and the one presented in the manuscript and seem to claim that DToL is incorrect. However, the DToL assembly (xcSepOffi3.1) is based on much deeper HiFi and HiC coverage than the one at hand (51x and 80+x respectively). There are many things to try here, including:

a) Downloading the DToL data and reassembling using a common pipeline.

b) Downsampling the DToL data to similar coverage as what the authors have achieved.

c) Combining your data and that of DToL for even deeper coverage (heterozygosity is low enough that I don't imagine this impeding things too badly).

(2) Looking at Figure 1, there appears to be a misjoin at chromosome 42. Looking carefully at Figure S1, that misjoin does not appear on any of the panels - this is confusing. Given the size of that chromosome and the authors' chromosome numbering, I'm guessing this is a manual merge (as it's larger than most of the chromosomes numerically close (40, 41, 43, etc). Further, staring closely at Figure 1, there appear to be cross-scaffold contacts between 42 and 43 and 42 and 44. Secondarily there are contacts between 43 and 44. This bit of the assembly seems potentially problematic.

Reviewer #3 (Public review):

Summary:

In this study, authors Simone Rencken and co-authors present and investigate the genome of the common cuttlefish Sepia officinalis.

Strengths:

The authors explain in a detailed yet concise manner the main steps for a genome assembly, with very robust methods for validation, and according to current best practices. In addition to the chromosomal assembly, the authors confirmed the presence of 47 chromosomes using Hi-C data and multiple species synteny. They also generated a comprehensive gene annotation, with assessments of gene completeness, providing a useful resource for the community of researchers interested in cuttlefish biology and comparative genomics.

Weaknesses:

While the study touches upon the subjects of gene content, TE activity, or species-level comparisons, the study does not provide in-depth investigations of these.

Reviewer #1 (Public review):

Summary:

This research investigates how the cellular protein quality control machinery influences the effectiveness of cystic fibrosis (CF) treatments across different genetic variants. CF is caused by mutations in the CFTR gene, with over 1,700 known disease-causing variants that primarily work through protein misfolding mechanisms. While corrector drugs like those in Trikafta therapy can stabilize some misfolded CFTR proteins, the reasons why certain variants respond to treatment while others don't remain unclear. The authors hypothesized that the cellular proteostasis network-the machinery that manages protein folding and quality control-plays a crucial role in determining drug responsiveness across different CFTR variants. The researchers focused on calnexin (CANX), a key chaperone protein that recognizes misfolded glycosylated proteins. Using CRISPR-Cas9 gene editing combined with deep mutational scanning, they systematically analyzed how CANX affects the expression and corrector drug response of 234 clinically relevant CF variants in HEK293 cells.

In terms of findings, this study revealed that CANX is generally required for robust plasma membrane expression of CFTR proteins, and CANX disproportionately affects variants with mutations in the C-terminal domains of CFTR and modulates later stages of protein assembly. Without CANX, many variants that would normally respond to corrector drugs lose their therapeutic responsiveness. Furthermore, loss of CANX caused broad changes in how CF variants interact with other cellular proteins, though these effects were largely separate from changes in CFTR channel activity.

This study has some limitations: the research was conducted in HEK293 cells rather than lung epithelial cells, which may not fully reflect the physiological context of CF. Additionally, the study only examined known disease-causing variants and used methodological approaches that could potentially introduce bias in the data analysis.

How cellular quality control mechanisms influence the therapeutic landscape of genetic diseases is an emerging field. Overall, this work provides important cellular context for understanding CF mutation severity and suggests that the proteostasis network significantly shapes how different CFTR variants respond to corrector therapies. The findings could pave the way for more personalized CF treatments tailored to patients' specific genetic variants and cellular contexts.

Strengths:

(1) This work makes an important contribution to the field of variant effect prediction by advancing our understanding of how genetic variants impact protein function.

(2) The study provides valuable cellular context for CFTR mutation severity, which may pave the way for improved CFTR therapies that are customized to patient-specific cellular contexts.

(3) The research provides further insight into the biological mechanisms underlying approved CFTR therapies, enhancing our understanding of how these treatments work.

(4) The authors conducted a comprehensive and quantitative analysis, and they made their raw and processed data as well as analysis scripts publicly available, enabling closer examination and validation by the broader scientific community.

Weaknesses:

(1) The study only considers known disease-causing variants, which limits the scope of findings and may miss important insights from variants of uncertain significance.

(2) The cellular context of HEK293 cells is quite removed from lung epithelia, the primary tissue affected in cystic fibrosis, potentially limiting the clinical relevance of the findings.

(3) Methodological choices, such as the expansion of sorted cell populations before genetic analysis, may introduce possible skew or bias in the data that could affect interpretation.

(4) While the impact on surface trafficking is convincingly demonstrated, how cellular proteostasis affects CFTR function requires further study, likely within a lung-specific cellular context to be more clinically relevant.

Reviewer #2 (Public review):

In this work, the authors use deep mutational scanning (DMS) to examine the effect of the endogenous chaperone calnexin (CANX) on the plasma membrane expression (PME) and potential pharmacological stabilization cystic fibrosis disease variants. This is important because there are over 1,700 loss-of-function mutations that can lead to the disease Cystic Fibrosis (CF), and some of these variants can be pharmacologically rescued by small-molecule "correctors," which stabilize the CFTR protein and prevent its degradation. This study expands on previous work to specifically identify which mutations affect sensitivity to CFTR modulators, and further develops the work by examining the effect of a known CFTR interactor-CANX-on PME and corrector response.

Overall, this approach provides a useful atlas of CF variants and their downstream effects, both at a basal level as well as in the context of a perturbed proteostasis. Knockout of CANX leads to an overall reduced plasma membrane expression of CFTR with CF variants located at the C-terminal domains of CFTR, which seem to be more affected than the others. This study then repeats their DMS approach, using PME as a readout, to probe the effect of either VX-445 or VX-455 + VX-661-which are two clinically relevant CFTR pharmacological modulators. I found this section particularly interesting for the community because the exact molecular features that confer drug resistance/sensitivity are not clear. When CANX is knocked out, cells that normally respond to VX-445 are no longer able to be rescued, and the DMS data show that these non-responders are CF variants that lie in the VX-445 binding site. Based on computational data, the authors speculate that NBD2 assembly is compromised, but that remains to be experimentally examined. Cells lacking CANX were also resistant to combinatorial treatment of VX-445 + VX-661, showing that these two correctors were unable to compensate for the lack of this critical chaperone.

One major strength of this manuscript is the mass spectrometry data, in which 4 CF variants were profiled in parental and CANX KO cells. This analysis provides some explanatory power to the observation that the delF508 variant is resistant to correctors in CANX KO cells, which is because correctors were found not to affect protein degradation interactions in this context. Findings such as this provide potential insights into intriguing new hypothesis, such as whether addition of an additional proteostasis regulators, such as a proteosome inhibitor, would facilitate a successful rescue. Taken together, the data provided can be generative to researchers in the field and may be useful in rationalizing some of the observed phenotypes conferred by the various CF variants, as well as the impact of CANX on those effects.

To complete their analysis of CF variants in CANX KO cells, the research also attempted to relate their data, primarily based on PME, to functional relevance. They observed that, although CANX KO results in a large reduction in PME (~30% reduction), changes in the actual activation of CFTR (and resultant quenching of their hYFP sensor) were "quite modest." This is an important experiment and caveat to the PME data presented above since changes in CFTR activity does not strictly require changes in PME. In addition, small molecule correctors also do not drastically alter CFTR function in the context of CANX KO. The authors reason that this difference is due to a sort of compensatory mechanism in which the functionally active CFTR molecules that are successfully assembled in an unbalanced proteostasis system (CANX KO) are more active than those that are assembled with the assistance of CANX. While I generally agree with this statement, it is not directly tested and would be challenging to actually test.

The selected model for all the above experiments was HEK293T cells. The authors then demonstrate some of their major findings in Fischer rat thyroid cell monolayers. Specifically, cells lacking CANX are less sensitive to rescue by CFTR modulators than the WT. This highlights the importance of CANX in supporting the maturation of CFTR and the dependence of chemical correctors on the chaperone. Although this is demonstrated specifically for CANX in this manuscript, I imagine a more general claim can be made that chemical correctors depend on a functional/balanced proteostasis system, which is supported by the manuscript data. I am surprised by the discordance between HEK293T PME levels compared to the CTFR activity. The authors offer a reasonable explanation about the increase in specific activity of the mature CFTR protein following CANX loss.

For the conclusions and claims relevant to CANX and CF variant surveying of PME/function, I find the manuscript to provide solid evidence to achieve this aim. The manuscript generates a rich portrait of the influence of CF mutations both in WT and CANX KO cells. While the focus of this study is a specific chaperone, CANX, this manuscript has the potential to impact many researchers in the broad field of proteostasis.

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data. In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph.

This work provides a useful resource for studying nitrogenase evolution. However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

Comments on revisions:

I appreciate the authors responding to my comments. I think Fig. S10 helps put the structural data into more context. It would be helpful to make clearer in the legend what proteins are being compared, especially in 10C.

Although I can see why the authors focus on the NifK extension and its potential connection to oxygen protection, I would point out that Vnf and Anf do not have this extension in their K subunit, and you find both Vnf and Anf in aerobic and facultative anaerobic diazotrophs. This is a minor point, but I think it is important to mention in the discussion.

Reviewer #2 (Public review):

Summary:

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in environment, including oxygen levels and changes in metal availability.

The study predicts > 3000 structures of nitrogenases, corresponding to extant, ancestral and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive and admirable undertaking. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes

Reviewer #1 (Public review):

Summary:

In this study, the authors utilized in situ cryo-electron tomography (cryo-ET) to uncover the native thylakoid architecture of spinach chloroplasts and mapped the molecular organization of these thylakoids with single-molecule resolution. The obtained images show the detailed ultrastructural features of grana membranes and highlight interactions between thylakoids and plastoglobules. Interestingly, despite the distinct three-dimensional architecture of vascular plant thylakoids, their molecular organization closely resembles that of green algae. The pronounced lateral segregation of PSII and PSI was observed at the interface between appressed and non-appressed thylakoid regions, without evidence of a specialized grana margin zone where these complexes might intermix. Furthermore, unlike isolated thylakoid membranes, photosystem II (PSII) did not form a semi-crystalline array and was distributed uniformly within the membrane plane and across stacked grana membranes in intact chloroplasts. Based on the above observations, the authors propose a simplified two-domain model for the molecular organization of thylakoid membranes, which can be applied to both green algae and vascular plants. This study suggests that the general understanding of the functional separation of thylakoid membranes in vascular plants requires reconsideration.

Strengths:

By employing and refining AI-driven computational tools for the automated segmentation of membranes and identification of membrane proteins, this study successfully quantifies the spatial organization of photosynthetic complexes both within individual thylakoid membranes and across neighboring stacked membranes.

Weaknesses:

This study's weakness is that it requires the use of chloroplasts isolated from leaves and the need to freeze them on a grid for observation. However, the authors have correctly identified the limitations of this approach and have made some innovations, such as rapid sample preparation. The reliability of the interpretation of the results in light of previous results can be evaluated as high.

Comments on revised version:

The author has responded appropriately to the peer review comments and revised the paper.

Reviewer #2 (Public review):

Summary:

For decades, the macromolecular organization of photosynthetic complexes within the thylakoids of higher plant chloroplasts has been a topic of significant debate. Using focused ion beam milling, cryo-electron tomography, and advanced AI-based image analysis, the authors compellingly demonstrate that the macromolecular organization in spinach thylakoids closely mirrors the patterns observed in their earlier research on Chlamydomonas reinhardtii. Their findings provide strong evidence challenging long-standing assumptions about the existence of a 'grana margin'-a region at the interface between grana and stroma lamellae domains that was thought to contain intermixed particles from both areas. Instead, the study establishes that this mixed zone is absent and reveals a distinct, well-defined boundary between the grana and stroma lamellae.

Strengths:

By situating high-resolution structural data within the broader cellular context, this work contributes valuable insights into the molecular mechanisms governing the spatial organization of photosynthetic complexes within thylakoid membranes.

Comments on revised version:

All reviewer comments have been fully addressed, and I have no further comments.

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