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      Reply to the reviewers

      Dear Dr. Sara Monaco,

      Thank you very much for your kind e-mail dated 1-Feb-2024. Please find enclosed our revised version and our point-by-point reply to the comments from the reviewers. We have answered all major and minor points raised by the reviewers. Original Figures and Supplementary Figures were revised and renamed as follows.

      Figure 1 -> Figure 1 and Revised Figure 2

      Figure 2 -> Revised Figure 3 and Supplementary Figure 7

      Figure 3 -> Revised Figure 4

      Figure 4 -> Revised Figure 5 and 6

      Figure 5 -> Revised Figure 7

      Figure 6 -> Revised Figure 8

      Supplementary Figure 1 -> Supplementary Figure 1

      Supplementary Figure 2 -> Revised Supplementary Figure 3

      Supplementary Figure 3 -> Revised Supplementary Figure 4

      Supplementary Figure 4 -> Revised Figure 2

      Supplementary Figure 5 -> Revised Supplementary Figure 6

      Supplementary Figure 6 -> Revised Supplementary Figure 9

      Supplementary Figure 7 -> Revised Supplementary Figure 10

      Supplementary Figure 8 -> Revised Supplementary Figure 11

      Supplementary Figure 9 -> Revised Supplementary Figure 12

      We believe that our revised manuscript has been significantly improved thanks to your help. Thank you very much again for your help.

      Yours sincerely,

      Yosuke Mai and Ken Natsuga

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

      SUMMARY In this study, the researchers investigate the spontaneous patterning of keratinocytes. As model they use HaCaT cells, an immortalized keratinocyte line. The cells exhibit a self-organized pattern of high and low cell density, which is disrupted by medium changes but reappear over time. The researchers find that serum starvation and high calcium concentration are crucial for the formation of these keratinocyte patterns. RNA sequencing analysis of regions of high vs low density indicates enrichment in gene ontology terms related to cell-cell adhesion, mainly adherens junctions (AJs), and keratinocyte differentiation. Experimental manipulations, such as inhibiting E-cadherin- or α-catenin-mediated adhesion, and disrupting myosin-II activity, all interfer with the formation of keratinocyte patterns, emphasizing the importance of AJs. Mathematical modeling suggests that cell-cell adhesion alone is sufficient for the emergence of density patterns. Keratinocyte patterns have spatial regulation of keratinocyte differentiation and proliferation. Differentiated cells are abundant in areas of high cell density, while proliferative cells are in areas of low cell density. The authors verify that YAP activity regulates pattern-dependent differentiation and proliferation. The role of serum starvation and cell-cell adhesion through AJs in the differentiation of keratinocytes are supported by epidermal stratification experiments in 3D culture, and ex vivo experiments on mouse skin suction blister wounds. In conclusion, the study provide insights into the spatial regulation of differentiation and proliferation in epidermal cells. MAJOR COMMENTS Although not novel, given that it has been already demonstrated with several other epithelial cell monolayers and in vivo in Drosophila, the conclusions that serum starvation facilitates epidermal stratification through cell-cell adhesion is convincing. It is unclear whether the cell patterning the authors are describing is a real patterning, defined in biology as any regularly repeated cell or structural arrangement or simply an inhomogeneous distribution of cell densities.*

      We have addressed this issue by analyzing our images with the autocorrelation function (see Fig. 1g, 1h, and Supplementary Fig. 5) and confirmed that the distribution of high/low cell density is patterned with the average nearest neighbor distance between areas of high cell density being approximately 300 µm. We have incorporated these new data into the revised manuscript.

      The conclusion that the cell-cell adhesion signaling pathway identified in the paper "might promote wound healing in clinical settings" (last sentence of the abstract) is not substantiated by the results.

      We agree with the reviewer's point and have deleted the sentence in the abstract, accordingly.

      It would be opportune to better describe the type of "cell patterning" that the authors are seeing in their experiments. In my opinion the effect seen in the described experiment is not a "patterning" but a difference in cell density which can be less or more homogeneous in an HaCat monolayer.

      Please see the answer above on our analysis using the autocorrelation function.

      Importantly, it is unclear whether the "cell patterning" is a subsequent consequence or proceed stratification.

      As the mathematical modeling indicated patterning without the need for stratification steps, we believe that cell patterning is not a direct consequence of stratification. However, it is technically difficult to differentiate whether patterning developed prior to stratification in our experimental settings. We have added this limitation to the Discussion of the revised manuscript.

      It is unclear how starvation relates to the increased adhesions and YAP signaling.

      As the reviewer pointed out, we could not address what molecules in the serum are responsible because the serum is a complex mixture of biomolecules that includes hormones, growth factors, vitamins, and other nutrients. We have added this limitation of our study to the revised manuscript.

      The authors conclude the discussion section proposing "that molecules involved in cell-cell adhesion-induced patterning are suitable target candidates to facilitate wound healing". None the experiments done in the wound healing setting are addressing the role of any molecules described in the paper. I would suggest the authors to remove this last claim from the manuscript. Alternatively, the authors should provide evidence that targeting some of the molecules described in the manuscript are accelerating wound healing in a clinically relevant model of wound healing.

      We agree with the reviewer's point and have deleted the passage in the revised manuscript, accordingly.

      I would request the authors to provide the following essential data to substantiate their experiments: - Provide a full gene list related to Figure 2a.

      We have provided the gene list (Supplementary Table 1), accordingly.

      - In relation to Figure 2c, stain for a-catenin and quantify the intensity ration of a-catenin vs a-18-catenin as proper readout of adhesion strength (see Yonemura et al., Nat Cell Biol 2010).

      As the reviewer pointed out, the intensity ratio of α-catenin vs. α18 is a general readout of cell adhesion strength. However, this ratio should be based on similar intensity of alpha catenin between two groups for comparison. In contrast, the intensity of α-catenin itself was weaker in the area with low cell density compared with in that with high cell density in our experimental setting (Supplementary Fig. 8d, e, g), which could greatly affect the ratio. To overcome this problem, we have reanalyzed line plots of α-catenin immunofluorescence, picked up the α18 intensity at the peaks (corresponding to cell-cell adhesion) of α-catenin, and compared that of high and low cell density area. As expected, α18 was more pronounced in the area with high cell density. We have added the data to Supplementary Fig. 8d-h in the revised manuscript.

      - Properly quantify nuclear vs cytoplasmic localization of YAP in low vs high density areas in Figure 4f.

      According to the reviewer's suggestion, we have quantified nuclear/cytoplasmic YAP and added the data (Revised Fig. 6b (original Fig. 4)) to the revised manuscript.

      • The nuclear localization of YAP is not sufficient to demonstrate activation of the YAP signaling. The authors should provide evidence of YAP activity in low vs high density areas looking for example at known downstream target genes in epithelial cells (see Zhao et al., Genes Dev 2007; Yu et al., Cell 2012; Aragona et al., Cell 2013).

      We have analyzed ANKRD1 (Yu et al., Cell 2012) as a YAP readout molecule and confirmed that, in line with YAP dynamics, ANKRD1 was localized in the nucleus of high cell density area. We have provided the data (Revised Fig. 6c, d (original Fig. 4)) for the revised manuscript.

      • The activity of PY-60 in Figure 4g and XAV939 in Figure 4i as YAP activator and repressor respectively, should be controlled against YAP localization and activity.

      We have quantitatively analyzed YAP and ANKRD1 localization upon chemical treatment and added the data (Supplementary Fig. 1a-d, g-j (original Supplementary Fig. 8)) to the revised manuscript.

      • In Figure 5a a quantification of the numbers of cell layers should be used instead of the thickness and a staining and quantification of K14 and K10 should be added to formally address stratification.

      As expected, the number of K10-positive cell layers was larger in serum-starved conditions than in serum-rich conditions, while the number of K14-positive cell layer was comparable between the two groups. We have provided the quantification data (Supplementary Fig. 12 c-e (original Supplementary Fig. 9)) to the revised manuscript accordingly.

      *Most of the proposed experiments are simply additional quantifications of images or adjustments of data that are already available to the authors. I estimate that the remaining experiments can be done in less than a month and will not require additional expertise.

      The methods, figures presentation and legends, and the statistical analysis are adequate, clear and accurate.

      MINOR COMMENTS There are three fundamental studies that the authors should discuss: - Saw, Doostmohammadi et al., Nature 2017. Topological defects in epithelia govern cell death and extrusion. Here, the role of topological defects (see also Bonn et al., Phys Res E 2022) and a-catenin-dependent cell-cell interactions are connected to cell extrusion and Yap activity in epithelial monolayers including HaCat cells. - Miroshnikova et al., Nat Cell Biol 2018. Adhesion forces and cortical tension couple cell proliferation and differentiation to direct epidermal stratification. Here, the authors demonstrated that the increase of cell-cell adhesion couples with a decrease of cortical tension triggers stratification in the skin epidermis. - Boocock et al., Nature Physics 2021. Theory of mechanochemical patterning and optimal migration in cell monolayers. Here, cell density and ERK activity are formalized to be key players in patterning formation in a cell monolayer. In addition, several components of the Hippo-YAP pathway are known regulators of cell-cell adhesion (e.g. AMOT and NF2) and should be discussed (for reference see reviews on the topic Zheng & Pan, Dev Cell 2019; Karaman & Halder Cold Spring Harb Perspect Biol 2018; Gumbiner & Kim, J Cell Sci 2014) as important molecules implicated in the biological phenomena described in the manuscript.0*

      We appreciate the reviewer's suggestion and have cited and discussed these seminal papers in the revised manuscript.

      Reviewer #1 (Significance (Required)):

      The study aims at understanding spontaneous patterning of keratinocytes. The authors nicely employ various experimental approaches, including cell imaging, RNA sequencing, cell manipulation by genetic engineering and pharmacological treatments, and mathematical modeling, to elucidate the underlying cellular and molecular mechanisms regulating this proces. However, several of the conclusions presented in the manuscript do not present any conceptual advance to the field of self-organization of cell density patterns or epithelial biology.

      The role of starvation in effecting epithelial growth is very well known. The role of AJ in pattern formation has been described previously in epithelial monolayers (Saw, Doostmohammadi et al., Nature 2017) and in vivo in Drosophila (Mao et al., Genes Dev 2011; Mao et al., EMBO J 2013). The effect of cell density on YAP signaling is known (Zhao et al., Genes Dev 2007; Aragona et al., Cell 2013). The importance of AJ for keratinocytes differentiation and stratification has been demonstrated in vitro and in vivo (Miroshnikova et al., Nat Cell Biol 2018). The role of a-catenin upstream of YAP activity in regulating interfollicular epidermis stem cells self-renewal and wound healing has been demonstrated in vitro and in vivo by the group of Fernando Camargo in Cell 2011.

      The manuscript could be of interest for researchers interested in basic cell biology and a specialised audience in cell self-organisation.

      My field of expertise: epithelial biology, stem cell biology, skin homeostasis and wound healing, mechanobiology, YAP signaling. I do not have sufficient expertise to evaluate the mathematical modelling.

      We appreciate the reviewer's constructive comments.

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

      Summary:

      Mai et al. reported an interesting observation that serum starvation induced the keratinocytes, a type of epithelial cells, to form a pattern characterized by regions with high and low densities. They showed that this patterning processing depends on cell-cell adhesion using a series of pharmacological treatment and a CRISPR knockout of alpha-catenin. They used mathematical modeling to demonstrate that cell-density dependent stress can sufficiently generate patterns of high and low cell densities, but the interpretation of the modeling is questionable (see below). They showed correlation of a differentiated keratinocyte marker, keratin 10, with the high-density region, but over claimed this result as patterning modulates differentiation. They also showed correlation of YAP activity (cytoplasmic to nuclear ratio) to the high vs. low-density regions. Interestingly, treatment with a YAP activator PY-60 disrupted pattern formation, while the YAP inhibitor XAV939 barely affected pattern formation. Finally, the authors demonstrated that serum starvation increased the thickness of keratinocytes cultured in a trans-well system (which they called 3D culture), and a mouse back skin explant compared to serum-rich culture conditions. In the former system, they showed dependence on alpha-catenin using the CRISPR knockout.

      Major comments:

      The conclusion that "mathematical modeling indicates that cell-cell adhesion alone is sufficient to form regions with high/low cell density" is misleading. The key assumption of the modeling is that the time derivative of stress (d_sigma/dt) is proportional to the cell density (rho), where the proportion parameter (beta) was interpreted as cell adhesion strength. However, beta could be interpreted as any general attractor proportional to the cell density, such as a chemoattractant.*

      Our purpose here is to demonstrate that the model based on the assumption of cell-cell adhesion as a mere source of attractive forces can reproduce the experimentally observed spatial patterning. As the referee rightly points out, the term beta*rho in the second equation allows different interpretations such as the effect of attractant proportional to cell density. Therefore, our mathematical model cannot be used as a proof of the existence of cell-cell adhesion. We have reduced the tone in the revised manuscript.

      In addition, it is unclear why the time derivative of stress (d_sigma/dt) instead of stress itself (sigma) proportional to the cell density. The authors should further clarify the meanings of modeling parameters and be more careful with their conclusions.

      If the system is in the steady state (d_sigma/dt = 0) with no spatial variations (nabla^2 \sigma = 0), then the second equation reduces to sigma = (beta/alpha) rho, namely that the cell density is proportional to stress, as pointed out by the referee.

      Our model, which describes temporal and spatial variations, generalizes this situation. The spatial dependence represented by nabla^2 sigma was introduced according to the Reference 72 (original Reference 51). Furthermore, we introduced the time derivative d_sigma/dt to account for the fact that the system should relax into the steady state described above. We have included these into the revised manuscript.

      Related to above, the authors should revise the title to reflect that the patterning depends on cell-cell adhesion instead of claiming that cell-cell adhesion drives patterning. This would require experimentally demonstrating sufficiency, for example, showing that increasing adhesion in a cell line with low adhesion that does not show patterning can sufficiently induce patterning.

      We agreed with the reviewer and have revised the title into "Patterning in stratified epithelia depends on cell-cell adhesion" and reduce the tone of the final sentence of the Discussion section accordingly.

      The conclusion that "patterning modulates differentiation" is not supported by evidence. Differentiation as evidenced by the presence of keratin 10 occurred as early as day 2 before any signs of patterning (Fig. 4A). When patterning was completely disrupted by alpha-catenin KO, there are still many keratin 10 positive cells. The apparent higher proportion of keratin 10+ cells in the wild type seems to be merely reflecting the higher cell density - if the quantification were normalized by the cell number, they are probably comparable. Overall, the presented data only supports a correlation of the differentiation marker keratin 10 with high-density regions.

      According to the reviewer's suggestion, we have reduced the tone of the title of Revised Fig. 4 (original Fig. 3) and changed it into "Patterning correlates with differentiation and proliferation markers in keratinocytes".

      The choice of RNA-seq comparison groups (high-density vs. low-density culture) is puzzling, since the effects caused by culture density changes may not be related to the high vs. low-density regions in the patterned cultures. There are so many changes there and the rationale of following up on cell adhesion was unclear. In fact, it seems that the RNA-seq data didn't help the logic flow of the paper at all.

      Although we believe that comparison between high-density and low-density culture partly recapitulates high/low cell density regions in our study, the comparison is not identical to patterned cultures as the reviewer pointed out. We have moved RNA-seq data to the Supplementary Information (Supplementary Fig. 7) and added more analysis to address that cell adhesion and differentiation are major differences between high-density and low-density culture, supporting further analysis on this matter in our study.

      The claim of 3D culture of keratinocytes is confusing. The culture in the trans-well insert is still on the flat 2D surface, why should it be called 3D culture? If the point is to culture at air/liquid interface, that should instead be emphasized instead of calling it 3D.

      We have changed "3D culture" into air-liquid interface culture, accordingly.

      Reviewer #2 (Significance (Required)):

      The observation that serum starvation and replenishment induced reversible patterning of the keratinocytes is quite interesting. However, the biological relevance is unclear - isn't all skin stratified? The evidence supporting the dependence of this patterning on adherens junction by disrupting E-cadherin, myosin, or alpha-catenin is convincing, although not surprising. The involvement of YAP in differentiation vs. proliferation is interesting, but it's in line with the known functions of YAP. The modeling part, with some clarification, can be quite insightful. Overall, this research could be interesting to those working in epithelial morphogenesis, if further developed.

      My expertise is in epithelial tissue morphogenesis, mechanobiology, and extracellular matrix biology.

      We appreciate the reviewer's constructive and thoughtful comments.

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

      In this manuscript the authors aim to understand the signals that coordinate spatial patterns of keratinocyte proliferation and differentiation. To address this question the authors use the HaCaT keratinocyte cell line that upon serum starvation forms spatially separated domains of proliferation and differentiation. The data presented in this manuscript potentially suggest that serum starvation works through adherens junctions to create differentially dense fields within the cultures which determines whether cells proliferate or differentiate. The authors then perform experiments to show that junction formation with starvation drive keratinocyte differentiation potentially through YAP signaling. However, these experiments are rather loosely connected and their results often do not support the conclusions drawn by the authors. However, the not well supported conclusion the form the basis for a fact statement, but their data really did not show that. For example, the authors state: "By contrast, YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation (Fig. 4i, j)," . However, their data do not show that proliferation is pattern-dependent but is nevertheless used to connect to and draw a conclusion about YAP signaling. The data itself appear to be of high quality, figures are well organized and statistics of quantification seem appropriate, but it somewhat problematic that throughout the manuscript it remains unclear if certain statements are hypotheses or conclusions on real data. Pattern formation as a requirement for differentiation is an interesting concept. However, the presented study lacks proper conclusive data how these patterns may contribute to proliferation and differentiation and remains rather short on what exactly is the instructive nature of these patterns, as they only use high density and are not generating own patterns with defined cues that explore what cues contribute. Major points: The statement "According to the RNA-seq data, AJ molecules, such as E-cadherin and actin, were localized at intercellular junctions in areas of high cell density" is not correct. RNA-seq does not allow conclusions about protein localization. Instead, the GO-Term analysis shown in Figure 2b shows downregulation of "cell-adhesion" in dense areas. *

      According to the reviewer's suggestion, we have corrected the sentence. We have reanalyzed our RNA-seq data and confirmed that GO-term "cell-adhesion" was in the top list of both high and low cell density regions. We have provided more data to the revised manuscript (Supplementary Fig. 7 and Supplementary Table 1).

      *Consistently the E-cadherin staining presented in Figure2c suggest lower intercellular E-cadherin levels in the most dense areas. However, any statement about junctional localization of adhesion components requires e.g. intensity quantification at junctions vs. cytoplasm or else to discriminate from intense overall staining due to high cell density and thus high overall junction numbers. *

      Actually, junctional E-cadherin was more pronounced in the high cell density area. We have provided line plot data to confirm this and also added a quantification data (Supplementary Fig. 8).

      Hence, even though potentially true, the statement: "These data suggest that cells in regions of high cell density form AJs in response to intercellular forces" is not fully supported by the data shown so far.

      Please see the answer to the comments of Reviewer 1 on the quantification of α18. We believe that, as α18 intensity is more pronounced in the cell-cell junction of high cell density area compared with low cell density regions, our claim is experimentally supported.

      The authors suggest a pattern of high and low density that is formed over time. However, at the same time high density areas show formation of a second layer. Hence, "denser" areas as observed by phase contrast images or DAPI positive nuclei may either represent dense or stratified cells. What is missing is an analysis of cell density before cells started to stratify making sure only cells in the basal layer are analyzed. Otherwise, density and stratification which are perhaps interdependent in this system cannot be discriminated.

      As the reviewer pointed out, the patterning was analyzed at the level of basal layer. In addition to Figure 1c, we have provided another plane cut immunofluorescence data (Supplementary Fig. 2) to the revised manuscript to address this issue.

      *The mathematical model does not include stratification and it is thus not clear to what extend it may explain the observed patterns. *

      It is true that the model does not account for stratification. It focuses solely on the patterning of cell density in the basal layer. We have incorporated this notion into the revised manuscript as a limitation of this study.

      *Moreover, the model appears to assume variables that have not been determined or cited. This reviewer is not an expert in modeling and thus cannot fully judge the math behind the model. However, the model appears to be biased if it assumes, as mentioned, that cell-adhesion increases with density. *

      The first equation, describing the time evolution of rho (the cell density), incorporates diffusion, collective cell movement due to stress from adjacent cells, and random fluctuations. Each of these terms comes from a general consideration of density dynamics. The second equation, describing stress balance, is a generalization of Reference 72 (originally Reference 51). The crucial assumption here is that the cell-cell adhesion increases with density, which corresponds to the experimental findings (Revised Fig. 3a, b, Supplementary Figure 8a-h).

      What we have demonstrated here is that we only need cell-cell adhesion as a source of attractive interactions for cells to form the density patterning as observed in the experiment. Since it is not self-evident whether the assumption of the density-dependent adhesion entail the emergence of density patterns, we do not believe that our model is begging the question or biased.

      *If low adhesion forces do not produce patterns, what is the counterforce in the model? Are cells allowed to change size to enable low density areas or do cells lose contact with neighbors despite high adhesion strength? *

      Our model does not have a variable corresponding to cell-shape change, which is considered only implicitly: Cells in the low density region (small rho) are regarded as flattened, whereas those in the high density region (large rho) as compressed (though not stratified) (Fig 1c).

      The behavior of the model is controlled by the parameter beta: a smaller beta means that density variations have little effect on stress, whereas a larger beta leads to significant stress changes with density variation. Since stress increases as beta*rho (in the second equation), stress in the low density region remains low even when the parameter beta is large.

      Overall, it appears that the model is set up such, that it tends to reproduce what was observed in experiment. This conclusion, however, may result of an incomplete understanding of the model parameters.

      The model setup, the assumption on the relationship between density and cell-cell adhesion in particular, does not inherently dictate the emergence of high/low density patterns: It might be the case that cell density is uniformly distributed everywhere with uniformly strong adhesion among cells. What our computer simulations have shown, however, is that the model exhibits spatially heterogeneous density patterns for sufficiently high beta values. The emergence of such spatial patterns is not a predefined aspect of the mathematical model itself.

      In the revised manuscript, the non-triviality of the spatial patterning has been made clear in the Results, and more explanations on the mathematical model to address the above points have been added to the Methods section.

      If dense areas do actually represent stratified areas it may not be surprising that the GO analysis indicates an increase in differentiation. A requirement for AJ or intercellular junctions in general is less surprising as stratification requires cell-cell adhesion. The observation that AJ are essential for intercellular junction formation in keratinocytes or in other epithelial cells is not new (e.g. Michels et al. JID 2009).

      We agree with the reviewer in the point that the role of AJ is not new. We have incorporated the notion into the Discussion of the revised manuscript and cited the paper the reviewer indicated.

      The part of the paper addressing the role of YAP suffers from a number of potentially mislead assumptions/conclusions based on a previous experiment which then did not properly supported that conclusion (see also overall comments). For example, the statement "YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation" contains interdependencies that have not been show [sic]. XAV939 may just inhibit proliferation which is not necessarily pattern dependent. Too much speculation confuses data and hypotheses.

      We agree with the reviewer to point out that pattern-dependency was not supported by our results. We have reduced the tones and corrected these terms in the revised manuscript.

      The 3D HaCaT cultures are performed on transwell filters with medium supply above and below cells, with the assumption that organizing patterns are also formed under these conditions. However, this has not been shown by the authors. Their suggestion that serum starvation may increases thickness of cultures through alterations in the organization of [sic]

      We showed the patterning in air-liquid interface culture in the Supplementary Data (Supplementary Fig. 12a, b, original Supplementary Fig. 9a, b), which presents starvation-induced pattering even in such condition.

      Reviewer #3 (Significance (Required)):

      The mechanisms that drive self-organization of epithelial cells to spatially separate domains of proliferation and differentiation is in principle a very interesting topic of high interest to the cell and mechanobiology community, [sic]

      We appreciate the reviewer's constructive and thoughtful comments.

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

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      Referee #3

      Evidence, reproducibility and clarity

      In this manuscript the authors aim to understand the signals that coordinate spatial patterns of keratinocyte proliferation and differentiation. To address this question the authors use the HaCaT keratinocyte cell line that upon serum starvation forms spatially separated domains of proliferation and differentiation. The data presented in this manuscript potentially suggest that serum starvation works through adherens junctions to create differentially dense fields within the cultures which determines whether cells proliferate or differentiate. The authors then perform experiments to show that junction formation with starvation drive keratinocyte differentiation potentially through YAP signaling. However, these experiments are rather loosely connected and their results often do not support the conclusions drawn by the authors. However, the not well supported conclusion the form the basis for a fact statement, but their data really did not show that. For example, the authors state: "By contrast, YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation (Fig. 4i, j)," . However, their data do not show that proliferation is pattern-dependent but is nevertheless used to connect to and draw a conclusion about YAP signaling. The data itself appear to be of high quality, figures are well organized and statistics of quantification seem appropriate, but it somewhat problematic that throughout the manuscript it remains unclear if certain statements are hypotheses or conclusions on real data. Pattern formation as a requirement for differentiation is an interesting concept. However, the presented study lacks proper conclusive data how these patterns may contribute to proliferation and differentiation and remains rather short on what exactly is the instructive nature of these patterns, as they only use high density and are not generating own patterns with defined cues that explore what cues contribute.

      Major points:

      The statement "According to the RNA-seq data, AJ molecules, such as E-cadherin and actin, were localized at intercellular junctions in areas of high cell density" is not correct. RNA-seq does not allow conclusions about protein localization. Instead, the GO-Term analysis shown in Figure 2b shows downregulation of "cell-adhesion" in dense areas. Consistently the E-cadherin staining presented in Figure2c suggest lower intercellular E-cadherin levels in the most dense areas. However, any statement about junctional localization of adhesion components requires e.g. intensity quantification at junctions vs. cytoplasm or else to discriminate from intense overall staining due to high cell density and thus high overall junction numbers. Hence, even though potentially true, the statement: "These data suggest that cells in regions of high cell density form AJs in response to intercellular forces" is not fully supported by the data shown so far. The authors suggest a pattern of high and low density that is formed over time. However, at the same time high density areas show formation of a second layer. Hence, "denser" areas as observed by phase contrast images or DAPI positive nuclei may either represent dense or stratified cells. What is missing is an analysis of cell density before cells started to stratify making sure only cells in the basal layer are analyzed. Otherwise, density and stratification which are perhaps interdependent in this system cannot be discriminated. The mathematical model does not include stratification and it is thus not clear to what extend it may explain the observed patterns. Moreover, the model appears to assume variables that have not been determined or cited. This reviewer is not an expert in modeling and thus cannot fully judge the math behind the model. However, the model appears to be biased if it assumes, as mentioned, that cell-adhesion increases with density. If low adhesion forces do not produce patterns, what is the counterforce in the model? Are cells allowed to change size to enable low density areas or do cells lose contact with neighbors despite high adhesion strength? Overall, it appears that the model is set up such, that it tends to reproduce what was observed in experiment. This conclusion, however, may result of an incomplete understanding of the model parameters. If dense areas do actually represent stratified areas it may not be surprising that the GO analysis indicates an increase in differentiation. A requirement for AJ or intercellular junctions in general is less surprising as stratification requires cell-cell adhesion. The observation that AJ are essential for intercellular junction formation in keratinocytes or in other epithelial cells is not new (e.g. Michels et al. JID 2009).

      The part of the paper addressing the role of YAP suffers from a number of potentially mislead assumptions/conclusions based on a previous experiment which then did not properly supported that conclusion (see also overall comments). For example, the statement "YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation" contains interdependencies that have not been show. XAV939 may just inhibit proliferation which is not necessarily pattern dependent. Too much speculation confuses data and hypotheses.

      The 3D HaCaT cultures are performed on transwell filters with medium supply above and below cells, with the assumption that organizing patterns are also formed under these conditions. However, this has not been shown by the authors. Their suggestion that serum starvation may increases thickness of cultures through alterations in the organization of

      Significance

      The mechanisms that drive self-organization of epithelial cells to spatially separate domains of proliferation and differentiation is in principle a very interesting topic of high interest to the cell and mechanobiology community,

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

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      Referee #2

      Evidence, reproducibility and clarity

      Summary:

      Mai et al. reported an interesting observation that serum starvation induced the keratinocytes, a type of epithelial cells, to form a pattern characterized by regions with high and low densities. They showed that this patterning processing depends on cell-cell adhesion using a series of pharmacological treatment and a CRISPR knockout of alpha-catenin. They used mathematical modeling to demonstrate that cell-density dependent stress can sufficiently generate patterns of high and low cell densities, but the interpretation of the modeling is questionable (see below). They showed correlation of a differentiated keratinocyte marker, keratin 10, with the high-density region, but over claimed this result as patterning modulates differentiation. They also showed correlation of YAP activity (cytoplasmic to nuclear ratio) to the high vs. low-density regions. Interestingly, treatment with a YAP activator PY-60 disrupted pattern formation, while the YAP inhibitor XAV939 barely affected pattern formation. Finally, the authors demonstrated that serum starvation increased the thickness of keratinocytes cultured in a trans-well system (which they called 3D culture), and a mouse back skin explant compared to serum-rich culture conditions. In the former system, they showed dependence on alpha-catenin using the CRISPR knockout.

      Major comments:

      The conclusion that "mathematical modeling indicates that cell-cell adhesion alone is sufficient to form regions with high/low cell density" is misleading. The key assumption of the modeling is that the time derivative of stress (d_sigma/dt) is proportional to the cell density (rho), where the proportion parameter (beta) was interpreted as cell adhesion strength. However, beta could be interpreted as any general attractor proportional to the cell density, such as a chemoattractant. In addition, it is unclear why the time derivative of stress (d_sigma/dt) instead of stress itself (sigma) proportional to the cell density. The authors should further clarify the meanings of modeling parameters and be more careful with their conclusions.

      Related to above, the authors should revise the title to reflect that the patterning depends on cell-cell adhesion instead of claiming that cell-cell adhesion drives patterning. This would require experimentally demonstrating sufficiency, for example, showing that increasing adhesion in a cell line with low adhesion that does not show patterning can sufficiently induce patterning.

      The conclusion that "patterning modulates differentiation" is not supported by evidence. Differentiation as evidenced by the presence of keratin 10 occurred as early as day 2 before any signs of patterning (Fig. 4A). When patterning was completely disrupted by alpha-catenin KO, there are still many keratin 10 positive cells. The apparent higher proportion of keratin 10+ cells in the wild type seems to be merely reflecting the higher cell density - if the quantification were normalized by the cell number, they are probably comparable. Overall, the presented data only supports a correlation of the differentiation marker keratin 10 with high-density regions.

      The choice of RNA-seq comparison groups (high-density vs. low-density culture) is puzzling, since the effects caused by culture density changes may not be related to the high vs. low-density regions in the patterned cultures. There are so many changes there and the rationale of following up on cell adhesion was unclear. In fact, it seems that the RNA-seq data didn't help the logic flow of the paper at all.

      The claim of 3D culture of keratinocytes is confusing. The culture in the trans-well insert is still on the flat 2D surface, why should it be called 3D culture? If the point is to culture at air/liquid interface, that should instead be emphasized instead of calling it 3D.

      Significance

      The observation that serum starvation and replenishment induced reversible patterning of the keratinocytes is quite interesting. However, the biological relevance is unclear - isn't all skin stratified? The evidence supporting the dependence of this patterning on adherens junction by disrupting E-cadherin, myosin, or alpha-catenin is convincing, although not surprising. The involvement of YAP in differentiation vs. proliferation is interesting, but it's in line with the known functions of YAP. The modeling part, with some clarification, can be quite insightful. Overall, this research could be interesting to those working in epithelial morphogenesis, if further developed.

      My expertise is in epithelial tissue morphogenesis, mechanobiology, and extracellular matrix biology.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary

      In this study, the researchers investigate the spontaneous patterning of keratinocytes. As model they use HaCaT cells, an immortalized keratinocyte line. The cells exhibit a self-organized pattern of high and low cell density, which is disrupted by medium changes but reappear over time. The researchers find that serum starvation and high calcium concentration are crucial for the formation of these keratinocyte patterns. RNA sequencing analysis of regions of high vs low density indicates enrichment in gene ontology terms related to cell-cell adhesion, mainly adherens junctions (AJs), and keratinocyte differentiation. Experimental manipulations, such as inhibiting E-cadherin- or α-catenin-mediated adhesion, and disrupting myosin-II activity, all interfer with the formation of keratinocyte patterns, emphasizing the importance of AJs. Mathematical modeling suggests that cell-cell adhesion alone is sufficient for the emergence of density patterns. Keratinocyte patterns have spatial regulation of keratinocyte differentiation and proliferation. Differentiated cells are abundant in areas of high cell density, while proliferative cells are in areas of low cell density. The authors verify that YAP activity regulates pattern-dependent differentiation and proliferation. The role of serum starvation and cell-cell adhesion through AJs in the differentiation of keratinocytes are supported by epidermal stratification experiments in 3D culture, and ex vivo experiments on mouse skin suction blister wounds.

      In conclusion, the study provide insights into the spatial regulation of differentiation and proliferation in epidermal cells.

      Major comments

      Although not novel, given that it has been already demonstrated with several other epithelial cell monolayers and in vivo in Drosophila, the conclusions that serum starvation facilitates epidermal stratification through cell-cell adhesion is convincing. It is unclear whether the cell patterning the authors are describing is a real patterning, defined in biology as any regularly repeated cell or structural arrangement or simply an inhomogeneous distribution of cell densities. The conclusion that the cell-cell adhesion signaling pathway identified in the paper "might promote wound healing in clinical settings" (last sentence of the abstract) is not substantiated by the results.

      It would be opportune to better describe the type of "cell patterning" that the authors are seeing in their experiments. In my opinion the effect seen in the described experiment is not a "patterning" but a difference in cell density which can be less or more homogeneous in an HaCat monolayer.

      Importantly, it is unclear whether the "cell patterning" is a subsequent consequence or proceed stratification. It is unclear how starvation relates to the increased adhesions and YAP signaling. The authors conclude the discussion section proposing "that molecules involved in cell-cell adhesion-induced patterning are suitable target candidates to facilitate wound healing". None the experiments done in the wound healing setting are addressing the role of any molecules described in the paper. I would suggest the authors to remove this last claim from the manuscript. Alternatively, the authors should provide evidence that targeting some of the molecules described in the manuscript are accelerating wound healing in a clinically relevant model of wound healing.

      I would request the authors to provide the following essential data to substantiate their experiments:

      • Provide a full gene list related to Figure 2a.
      • In relation to Figure 2c, stain for a-catenin and quantify the intensity ration of a-catenin vs a-18-catenin as proper readout of adhesion strength (see Yonemura et al., Nat Cell Biol 2010).
      • Properly quantify nuclear vs cytoplasmic localization of YAP in low vs high density areas in Figure 4f.
      • The nuclear localization of YAP is not sufficient to demonstrate activation of the YAP signaling. The authors should provide evidence of YAP activity in low vs high density areas looking for example at known downstream target genes in epithelial cells (see Zhao et al., Genes Dev 2007; Yu et al., Cell 2012; Aragona et al., Cell 2013).
      • The activity of PY-60 in Figure 4g and XAV939 in Figure 4i as YAP activator and repressor respectively, should be controlled against YAP localization and activity.
      • In Figure 5a a quantification of the numbers of cell layers should be used instead of the thickness and a staining and quantification of K14 and K10 should be added to formally address stratification.

      Most of the proposed experiments are simply additional quantifications of images or adjustments of data that are already available to the authors. I estimate that the remaining experiments can be done in less than a month and will not require additional expertise.

      The methods, figures presentation and legends, and the statistical analysis are adequate, clear and accurate.

      Minor comments

      There are three fundamental studies that the authors should discuss:

      • Saw, Doostmohammadi et al., Nature 2017. Topological defects in epithelia govern cell death and extrusion. Here, the role of topological defects (see also Bonn et al., Phys Res E 2022) and a-catenin-dependent cell-cell interactions are connected to cell extrusion and Yap activity in epithelial monolayers including HaCat cells.
      • Miroshnikova et al., Nat Cell Biol 2018. Adhesion forces and cortical tension couple cell proliferation and differentiation to direct epidermal stratification. Here, the authors demonstrated that the increase of cell-cell adhesion couples with a decrease of cortical tension triggers stratification in the skin epidermis.
      • Boocock et al., Nature Physics 2021. Theory of mechanochemical patterning and optimal migration in cell monolayers. Here, cell density and ERK activity are formalized to be key players in patterning formation in a cell monolayer.

      In addition, several components of the Hippo-YAP pathway are known regulators of cell-cell adhesion (e.g. AMOT and NF2) and should be discussed (for reference see reviews on the topic Zheng & Pan, Dev Cell 2019; Karaman & Halder Cold Spring Harb Perspect Biol 2018; Gumbiner & Kim, J Cell Sci 2014) as important molecules implicated in the biological phenomena described in the manuscript.

      Significance

      The study aims at understanding spontaneous patterning of keratinocytes. The authors nicely employ various experimental approaches, including cell imaging, RNA sequencing, cell manipulation by genetic engineering and pharmacological treatments, and mathematical modeling, to elucidate the underlying cellular and molecular mechanisms regulating this proces. However, several of the conclusions presented in the manuscript do not present any conceptual advance to the field of self-organization of cell density patterns or epithelial biology.

      The role of starvation in effecting epithelial growth is very well known. The role of AJ in pattern formation has been described previously in epithelial monolayers (Saw, Doostmohammadi et al., Nature 2017) and in vivo in Drosophila (Mao et al., Genes Dev 2011; Mao et al., EMBO J 2013). The effect of cell density on YAP signaling is known (Zhao et al., Genes Dev 2007; Aragona et al., Cell 2013). The importance of AJ for keratinocytes differentiation and stratification has been demonstrated in vitro and in vivo (Miroshnikova et al., Nat Cell Biol 2018). The role of a-catenin upstream of YAP activity in regulating interfollicular epidermis stem cells self-renewal and wound healing has been demonstrated in vitro and in vivo by the group of Fernando Camargo in Cell 2011.

      The manuscript could be of interest for researchers interested in basic cell biology and a specialised audience in cell self-organisation.

      My field of expertise: epithelial biology, stem cell biology, skin homeostasis and wound healing, mechanobiology, YAP signaling. I do not have sufficient expertise to evaluate the mathematical modelling.

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      Reply to the reviewers

      We thank all reviewers for their thorough assessment and constructive comments. We are glad that the reviewers appreciate that our findings are of interest to the nuclear transport field and that our extension of the use of the RITE methodology can be a valuable tool for the further characterization of NPCs that differ in composition and potentially function. In response to the reviewers’ comments, we have revised the text to incorporate their suggestions and improve overall readability and clarity. Furthermore, we propose to perform a set of additional experiments to address the reviewers’ most important critiques. Below we list our response with the reviewer comments reprinted in dark grey and our response in blue for easier orientation. We have added numbering of the comments for easier orientation.

      Many of the comments made by the reviewers have already been implemented, additional points will be addressed in a revised version of the manuscript as detailed below.

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

      The authors extended the existing recombination-induced tag exchange (RITE) technology to show that they can image a subset of NPCs, improving signal-to-noise ratios for live cell imaging in yeast, and to track the stability or dynamics of specific nuclear pore proteins across multiple cell divisions. Further, the authors use this technology to show that the nuclear basket proteins Mlp1, Mlp2 and Pml39 are stably associated with "old NPCs" through multiple cell cycles. The authors show that the presence of Mlp1 in these "old NPCs" correlates with exclusion of Mlp1-positive NPCs from the nucleolar territory. A surprising result is that basket-less NPCs can be excluded from the non-nucleolar region, an observation that correlates with the presence of Nup2 on the NPC regardless of maturation state of the NPC. In support of the proposal that retention of NPCs via Mlp1 and Nup2 in non-nucleolar regions, simulation data is presented to suggest that basket-less NPCs diffuse faster in the plane of the nuclear envelope.

      However, there are some points that do need addressing:

      Major Points 1. Taking into account that the Nup2 result in Figure 4B forms the basis for one half of the proposed model in Figure 6 regarding the exclusion of NPCs from the nucleolar region of the NE, there is a relatively small amount of data in support of this finding and this proposed model. For example, the only data for Nup2 in the manuscript is a column chart in Figure 4B with no supporting fluorescence microscopy examples for any Nup2 deletion. Further, the Nup60 deletion mutant will have zero basket-containing NPCs, whereas the Nup2 deletion will be a mixture of basket-containing and basket-less NPCs. The only support for the localization of basket-containing NPCs in the Nup2 deletion mutant is through a reference "Since Mlp1-positive NPCs remain excluded from the nucleolar territory in nup2Δ cells (Galy et al., 2004), the homogenous distribution observed in this mutant must be caused predominantly by the redistribution of Mlp-negative NPCs into the nucleolar territory."

      We have already added fluorescent images of the nup2d strain to figure 4A in the preliminary revision.

      In addition, we will repeat the experiment from Galy et al. 2004 to test whether Mlp-positive NPCs are excluded from nucleoli in our hands as well.

      Furthermore, we propose to carry out more experiments to pinpoint which domains of Nup2 contribute to nucleolar exclusion, which will provide more insight into the mechanism behind this effect. We propose to do this by analyzing NPC localization in mutants expressing truncations of Nup2 with deletions for individual domains as their only copy of Nup2. Regardless of whether we find a single domain of Nup2 responsible of a combinatorial action, this experiment will indicate a potential molecular mechanism for nucleolar exclusion.

      1. The authors could consider utilizing this opportunity to discuss their technological innovations in the context of the prior work of Onischenko et al., 2020. This work is referenced for the statement "RITE can be used to distinguish between old and new NPCs" Page 2, Line 43. However, it is not referenced for the statement "We constructed a RITE-cassette that allows the switch from a GFP-labelled protein to a new protein that is not fluorescently labelled (RITE(GFP-to-dark))" despite Onischenko et al., 2020 having already constructed a RITE-cassette for the GFP-to-dark transition. The authors could consider taking this opportunity to instead focus on their innovative approach to apply this technology to decrease the number of fluorescently-tagged NPCs by dilution across multiple cell divisions and to interpret this finding as a measure of the stability of nuclear pore proteins within the broader NPC.

      We apologize for this imprecise citation. We have modified the text to indicate that our RITE cassette was previously used in two publications. It now reads: “We used a RITE-cassette that allows the switch from a GFP-labelled protein to a new protein that is not fluorescently labelled (RITE(GFP-to-dark)) (Onischenko et al., 2020, Kralt et al., 2022). “

      1. The authors could also consider taking this opportunity to discuss their results in the context of the Saccharomyces cerevisiae nuclear pore complex structures published e.g. in Kim et al., 2018, Akey et al., 2022, Akey et al., 2023 in which the arrangement of proteins in the nuclear basket is presented, and also work from the Kohler lab (Mészáros et al., 2015) on how the basket proteins are anchored to the NPC. There is additional literature that also might help provide some perspective to the findings in the current manuscript, such as the observation that a lesser amount of Mlp2 to Mlp1 observed is consistent with prior work (e.g. Kim et al., 2018) and that intranuclear Mlp1 foci are also formed after Mlp1 overexpression (Strambio-de-Castillia et al., 1999).

      Following the reviewer’s suggestion, we extended our discussion of basket Nup stoichiometry and organization in the discussion section including several of the citations mentioned. At this point, we did not see a good way to incorporate discussion about the nuclear Mlp1 foci formed after Mlp1 overexpression. However, this observation is in line with the foci formed in cells lacking Nup60, suggesting that Mlp1 that cannot be incorporated into NPCs forms nuclear foci.

      Minor Points 1. What is the "lag time" of the doRITE switching? Do the authors believe that it is comparable to the approximate 1-hour timeframe following beta-estradiol induction as shown previously in Chen et al. Nucleic Acids Research, Volume 28, Issue 24, 15 December 2000, Page e108, https://doi.org/10.1093/nar/28.24.e108

      Our data (e.g. newRITE, Figure S3B) suggest that the switch occurs on a similar timeframe at

      1. The authors could consider a brief explanation of radial position (um) for the benefit of the reader, in Figures 1E (right panel) and 2B (right panel), perhaps using a diagram to make it easier to understand the X-axis (um).

      To address this, we have now included a diagram and refer to it in the figure legend.

      1. In Figure 1G, would the authors consider changing the vertical axis title and the figure legend wording from "mean number of NPCs per cell" to "mean labeled NPC # per cell" to reflect that what is being characterized are the remaining GFP-bearing NPCs over time?

      Thank you for spotting this inaccuracy. We have changed the label to “mean # of labeled NPCs per cell”.

      1. In Figure 2C, the magenta-labeled protein in the micrographs is not described in the figure or the legend.

      As requested, a description has been added in figure and legend.

      1. In Figure S2A, there is an arrow indicating a Nup159 focus, but this is not described in the figure legend, as is done in Figure 2C.

      A description has been added to the legend.

      1. In Figure S3C, the figure legend does not match the figure. Was this supposed to be designed like Figure 3C and is missing part of the figure? Or is the legend a typographical error?

      We apologize for this error and thank the reviewer for spotting it. The legend has been corrected.

      1. In Figure S4B, the spontaneously recombined RITE (GFP-to-dark) Nup133-V5 appears in the western blot as equally abundant to pre-recombined Nup133-V5-GFP. In the figure legend, this is explained as cells grown in synthetic media without selection to eliminate cells that have lost their resistance marker from the population. In Cheng et al. Nucleic Acids Res. 2000 Dec 15; 28(24): e108, Cre-EBD was not active in the absence of B-estradiol, despite galactose-induced Cre-EBD overexpression. Would the authors be able to comment further on the Cre-Lox RITE system in the manuscript?

      We note that also in the cited publication, cells are grown in the presence of selection to select (as stated in this publication) “against pre-excision events that occur because of low but measurable basal expression of the recombinase”. Although the authors report that spontaneous recombination is reduced with the b-estradiol inducible system (compared to pGAL expression control of the recombinase only), they show negligible spontaneous recombination only within a two-hour time window. Indeed, we also observe low levels of uninduced recombination on a short timeframe, but occasional events can become significant in longer incubation times (e.g. overnight growth) in the absence of selection. It should be noted that in our system, Cre expression is continuously high (TDH3-promoter) and not controlled by an inducible GAL promoter. We have added the information about the promoter controlling Cre-expression in the methods section.

      1. In Figure 6, the authors may want to consider inverting the flow of the cartoon model to start from the wild type condition and apply the deletion mutations at each step to "arrive" at the mutant conditions, rather than starting with mutant conditions and "adding back" proteins.

      Following the suggestions of the reviewer, we have modified our model to more clearly represent the contributions of the different basket components.

      Reviewer #1 (Significance (Required)):

      Recent work has drawn attention to the fact that not all NPCs are structurally or functionally the same, even within a single cell. In this light, the work here from Zsok et al. is an important demonstration of the kind of methodologies that can shed light on the stability and functions of different subpopulations of NPCs. Altogether, these data are used to support an interesting and topical model for Nup2 and nuclear-basket driven retention of NPCs in non-nucleolar regions of the nuclear envelope.

      We thank the reviewer for this positive assessment of our work.

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

      In this study, Zsok et al. develop innovative methods to examine the dynamics of individual nuclear pore complexes (NPCs) at the nuclear envelope of budding yeast. The underlying premise is that with the emergence of biochemically distinct NPCs that co-exist in the same cell, there is a need to develop tools to functionally isolate and study them. For example, there is a pool of NPCs that lack the nuclear basket over the nucleolus. Although the nature of this exclusion has been investigated in the past, the authors take advantage of a modification of recombination induced tag exchange (RITE), the slow turnover of scaffold nups, the closed mitosis of budding yeast, and extensive high quality time lapse microscopy to ultimately monitor the dynamics of individual NPCs over the nucleolus. By leveraging genetic knockout approaches and auxin-induced degradation with sophisticated quantitative and rigorous analyses, the authors conclude that there may be two mechanisms dependent on nuclear basket proteins that impact nucleolar exclusion. They also incorporate some computational simulations to help support their conclusions. Overall, the data are of the highest quality and are rigorously quantified, the manuscript is well written, accessible, and scholarly - the conclusions are thus on solid footing.

      We thank the reviewer for this assessment.

      Reviewer #2 (Significance (Required)):

      I have no concerns about the data or the conclusions in this manuscript. However, the significance is not overly clear as there is no major conceptual advance put forward, nor is there any new function suggested for the NPCs over nucleoli. As NPCs are immobile in metazoans, the significance may also be limited to a specialized audience.

      We respectfully disagree with this assessment. It is becoming increasingly clear that NPC variants are also present in other model systems. We characterize the interaction between conserved nuclear components, the NPC, the nucleolus and chromatin. While the specific architecture of the nucleus varies between species, many of these interactions are conserved. For example, Nup50, the homologue of Nup2, interacts with chromatin also in other systems including mammalian cells and thus may contribute to regulating the interplay between the nuclear basket and adjoining chromatin. Furthermore, our work demonstrates the use of a novel approach in the application of RITE that can be useful for other researchers in the field of NPC biology and beyond. For example, doRITE could be applied to study the properties of aged NPCs in the context of young cells. In the revised manuscript, we attempt to better highlight and discuss the conceptual advances of our manuscript.

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

      The manuscript of Zsok et al. describes the role of nuclear basket proteins in the distribution and mobility of nuclear pore complexes in budding yeast. In particular, the authors showed that the doRITE approach can be used for the analysis of stable and dynamically associated NUPs. Moreover, it can distinguish individual NUPs and follow the inheritance of individual NPCs from mother to daughter cells. The author's findings highlight that Mlp1, Mlp2, and Pml39 are stably associated with the nuclear pore; deletion of Mlp1-Mlp2 and Nup60 leads to the higher NPC density in the nucleolar territory; and NPCs exhibit increased mobility in the absence of the nuclear basket components.

      The manuscript contains most figures supporting the data, and data supports the conclusions. However, authors need to include better explanations for figures in the text and figure legends. Lack of detailed explanation can pose challenges for non-experts. In addition, the authors jump over figures and shuffle them through the manuscript, which disrupts the flow and coherence of the manuscript.

      We thank the reviewer for pointing this out. We have modified the figure legends throughout the manuscript in an attempt to make them more accessible to the reader. In addition, we will revise the figure order and text as suggested to improve the flow of the manuscript.

      Major comments: 1) The nuclear basket contains Nup1, Nup2, Nup60, Mlp1, and Mlp2 in yeast. Nup60 works as a seed for Mlp1/Mlp2 and Nup2 recruitment and plays a key role in the assembly of nuclear pore basket scaffold (PMID: 35148185). Logically, the authors focused primarily on Nup60 in the current manuscript. However, NUP153 has another ortholog of yeast - Nup1, which has not been studied in this work. I recommend adjusting the title of the manuscript to: Nup60 and Mlp1/Mlp2 regulate the distribution and mobility of nuclear pore complexes in budding yeast. I also suggest discussing why work on Nup1 was not included/performed in the manuscript.

      We have changed the title to “Nuclear basket proteins regulate the distribution and mobility of nuclear pore complexes in budding yeast”. We think that this better captures the essence of our manuscript than listing all four proteins (Mlp1/2, Nup60 and Nup2) in the title.

      We initially focused on the network that is involved in Mlp1/2 interaction at the NPC. However, we agree that it would be interesting to test, whether Nup1 plays a role in the analyzed processes as well. Since Nup1 is essential in our yeast background, we will use auxin-inducible degradation of Nup1 to test its involvement in NPC distribution.

      2) Figure 2B: I suggest choosing a more representative image for Pml39. It looks not like a stable component but rather dynamic as NUP60 or Gle1 based on figure showed in Figure 2B.

      Due to its lower copy number, Pml39 is much more difficult to visualize than the other Nups. To guide the reader, we have now added arrow heads to point to remaining Pml39 foci at the 14 hour timepoint. The 11 hour time point most clearly show that Pml39 is less dynamic than other Nups such as Nup116, Nup60 or Gle1. At this time point, clear dots for Pml39 can be detected, while e.g. Nup116 in the same figure exhibits a more distributed signal and the signal for Nup60 and Gle1 is no longer visible. We will describe this more clearly in our revised manuscript as well.

      3) Depletion of AID-tagged proteins needs to be supported by Western blot analysis with protein-specific antibodies, and PCR results should be included in supplementary data to demonstrate the homozygosity of the strains.

      The correct genomic tagging of the depleted proteins by AID was confirmed by PCR. We will include this PCR analysis in the supplemental data. Please note that we are working with haploid yeast cells. Therefore, all strains only carry a single copy of the genes. Unfortunately, we do not have protein-specific antibodies against the depleted proteins. However, the Mlp1-mislocalization phenotype demonstrates that depletion of Nup60 is successful and the depletion strain for PolII depletion was used and characterized previously (PMID: 31753862, PMID: 36220102).

      4) Figure 5B: Snapshots of images from the movie are required. There are no images, only quantifications.

      We have replaced the supplemental movie with a movie showing the detection by Trackmate as well as overlaid tracks. As requested, a snapshot of this movie was inserted in figure 5B. We have also moved the example tracks from the supplement to the main figure. Furthermore, we will deposit the tracking dataset in the ETH Research Collection to make it available to the community.

      5) Description of figure legends is more technical than supporting/explaining the figure. For example, below my suggestions for Figure 1D. Please, consider more detailed explanation for other figures. (D) Left: Schematic of the RITE cassette. NUP of interest is tagged with V5 tag and eGFP fluorescent protein where LoxP sites flank eGFP. Before the beta-estradiol-induced recombination, the old NPCs are marked with eGFP signal, whereas new NPCs lack an eGFP signal after the recombination. ORF: open reading frame; V5: V5-tag; loxP: loxP recombination site; eGFP: enhanced green fluorescent protein. Right: doRITE assay schematic of stable or dynamic Nup behavior over cell divisions in yeast after the recombination.

      We have modified the figure legends throughout the manuscript to make them more explanatory and helpful for the reader.

      In addition, I recommend highlighting the result in the title of the figures. Please, re-consider titles for Figure S3.

      We have revised the title for Figure S3 to state a result. It now reads: “Mlp1 truncations localize preferentially to non-nucleolar NPCs.”

      Minor: i) P.1 Line 31. Extra period symbol before the "(Figure 1A)".

      Fixed

      ii) P.2 Line 10. Inconsistent writing of PML39 and MLP1. Both genes are capitalized. The same for P.4 Line 16. In some cases all letters are capitalized in other only the first one.

      We are following the official yeast gene nomenclature by spelling gene names in italicized capitals and protein names with only the first letter capitalized. We are sorry that this can be confusing for readers more familiar with other model systems but we adhere to the accepted yeast nomenclature standards.

      iii) P.2 Line 18-22. The sentence is too long and hard to read. I recommend splitting it into two sentences.

      We agree and have fixed this.

      iv) P.2-3 Line 46-47. The sentence is unclear. Suggestion: We expected that successive cell divisions would dilute the signal of labelled and stably associated with the NPC nucleoporins. By contrast, ...

      We have modified the sentence to read: “When tagging a Nup that stably associates with the NPC, we expected that successive cell divisions would dilute labelled NPCs by inheritance to both mother and daughter cells leading to a low density of labelled NPCs. By contrast,…”

      v) P.4 Line 17-21. Please, consider adding extra information and clarifying lines 19-21. For example, in Line 19 Figure 2B you can add that the reader needs to compare row 1 and row 4.

      Thank you, we have fixed this as suggested.

      vi) P. 5 Line 15. When a number begins a sentence, that number should always be spelled out. You can pe-phrase the sentence to avoid it. Also, I recommend adding an explanation/hypothesis of why new NPCs are less frequently detected in nucleolar territory.

      We have formatted the text. Interestingly, new NPCs are more frequently detected in the nucleolar territory. We have reformulated this section to make it clearer, also in response to the next comment.

      vii) P.5 Line 17-22. I recommend re-phrasing these two sentences. Logically, it is clear that Mlp1/Mlp2 loss mimics "old NPCs" to look more like "new NPCs", and for that reason, they are more frequently included in the nucleolar territory, but it is not clear when you read these two sentences from the first time.

      We have reformulated this section to make it clearer.

      viii) P6. Line 16. No figure supporting data on graph (Figure 3B).

      We have added fluorescent images of the nup2d strain to figure 4A.

      ix) P.7 Line 10-13. The sentence is unclear.

      We have shortened the sentence and moved part of the content to the discussion in the next paragraph.

      x) P.13,14 etc. If 0h timepoint has been used for normalization, why is it present on the graph?

      The 0h timepoint is shown for comparison and to illustrate the standard deviation in the data.

      xi) P.15. Line 32-33. There is no image here. Potentially wrong description of the figure.

      Thank you for spotting this. This was fixed.

      xii) Figures: - Inconsistent labeling of figures. For example, Fig.1, Fig.1S, Figure 2 etc.

      Thank you, this has been corrected.

      • Inconsistent labeling of figures. For example, Fig.1 G "mean number of NPCs per cell" - no capitalization of the first letter. Fig.1S "Fraction in population" is capitalize d. In general, titles of axis should be capitalized.

      Thank you for spotting this. This was fixed.

      Suggestions for Figure 1D and Figure 6 are attached as a separate file.

      We thank the reviewer for their suggestions to improve these figures. We have taken their recommendation and revised the figures accordingly (see also response to reviewer 1, minor point 8).

      Reviewer #3 (Significance (Required)):

      Zsok et al. used the recombination-induced tag exchange (RITE) approach, which is an interesting and powerful method to follow individual NUPs over time with respect to their localization and abundance. This approach has been used before in PMID: 36515990 to distinguish pre-existing and newly synthesized Nup2 populations and has been extended to other basket NUPs in this work. Using this method, the authors support the earlier data on basket nucleoporins and highlight new insights on how basket nucleoporins regulate NPCs distribution and mobility. Overall, the manuscript provides new details on the stability of nucleoporins in yeast and how these data align with the mass spectrometry and FRAP data performed earlier in other studies. The limitation of this study is the absence of data on Nup1. It was unclear why these data were not present. Additional data can be included on the dynamics of Pml39, for example, using the FRAP method. The dynamic of Pml39 at the pore was shown only using the doRITE method.

      As suggested, we propose to test whether Nup1 influences NPC organization (see also above). Unfortunately, we are not able to provide orthologous data for the dynamics of Pml39. As we have discussed in the manuscript, FRAP is not suitable for the analysis of the dynamics of most nucleoporins in yeast due to the high lateral mobility of NPCs in the nuclear envelope and has previously generated misleading results for Mlp1. Furthermore, the low expression levels of Pml39 will make it difficult to obtain reliable FRAP curves for this protein. We therefore do not think that adding FRAP experiments with Pml39 will provide valuable insight.

      However, in addition to the Pml39 doRITE result itself, our observation that the Pml39-dependent pool of Mlp1 exhibits stable association with the NPC supports the interpretation of Pml39 as a stable protein as well.

      In general, this study represents a unique research study of basic research on nuclear pore proteins that will be of general interest to the nuclear transport field.

      Field of expertise: nuclear-cytoplasmic transport, nuclear pore, inducible protein degradation. I do not have sufficient expertise in ExTrack.

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      Referee #3

      Evidence, reproducibility and clarity

      The manuscript of Zsok et al. describes the role of nuclear basket proteins in the distribution and mobility of nuclear pore complexes in budding yeast. In particular, the authors showed that the doRITE approach can be used for the analysis of stable and dynamically associated NUPs. Moreover, it can distinguish individual NUPs and follow the inheritance of individual NPCs from mother to daughter cells. The author's findings highlight that Mlp1, Mlp2, and Pml39 are stably associated with the nuclear pore; deletion of Mlp1-Mlp2 and Nup60 leads to the higher NPC density in the nucleolar territory; and NPCs exhibit increased mobility in the absence of the nuclear basket components.

      The manuscript contains most figures supporting the data, and data supports the conclusions. However, authors need to include better explanations for figures in the text and figure legends. Lack of detailed explanation can pose challenges for non-experts. In addition, the authors jump over figures and shuffle them through the manuscript, which disrupts the flow and coherence of the manuscript.

      Major comments:

      • The nuclear basket contains Nup1, Nup2, Nup60, Mlp1, and Mlp2 in yeast. Nup60 works as a seed for Mlp1/Mlp2 and Nup2 recruitment and plays a key role in the assembly of nuclear pore basket scaffold (PMID: 35148185). Logically, the authors focused primarily on Nup60 in the current manuscript. However, NUP153 has another ortholog of yeast - Nup1, which has not been studied in this work. I recommend adjusting the title of the manuscript to: Nup60 and Mlp1/Mlp2 regulate the distribution and mobility of nuclear pore complexes in budding yeast. I also suggest discussing why work on Nup1 was not included/performed in the manuscript.
      • Figure 2B: I suggest choosing a more representative image for Pml39. It looks not like a stable component but rather dynamic as NUP60 or Gle1 based on figure showed in Figure 2B.
      • Depletion of AID-tagged proteins needs to be supported by Western blot analysis with protein-specific antibodies, and PCR results should be included in supplementary data to demonstrate the homozygosity of the strains.
      • Figure 5B: Snapshots of images from the movie are required. There are no images, only quantifications.
      • Description of figure legends is more technical than supporting/explaining the figure. For example, below my suggestions for Figure 1D. Please, consider more detailed explanation for other figures. (D) Left: Schematic of the RITE cassette. NUP of interest is tagged with V5 tag and eGFP fluorescent protein where LoxP sites flank eGFP. Before the beta-estradiol-induced recombination, the old NPCs are marked with eGFP signal, whereas new NPCs lack an eGFP signal after the recombination. ORF: open reading frame; V5: V5-tag; loxP: loxP recombination site; eGFP: enhanced green fluorescent protein. Right: doRITE assay schematic of stable or dynamic Nup behavior over cell divisions in yeast after the recombination.

      In addition, I recommend highlighting the result in the title of the figures. Please, re-consider titles for Figure S3.

      Minor:

      P.1 Line 31. Extra period symbol before the "(Figure 1A)".

      P.2 Line 10. Inconsistent writing of PML39 and MLP1. Both genes are capitalized. The same for P.4 Line 16. In some cases all letters are capitalized in other only the first one.

      P.2 Line 18-22. The sentence is too long and hard to read. I recommend splitting it into two sentences.

      P.2-3 Line 46-47. The sentence is unclear. Suggestion: We expected that successive cell divisions would dilute the signal of labelled and stably associated with the NPC nucleoporins. By contrast, ...

      P.4 Line 17-21. Please, consider adding extra information and clarifying lines 19-21. For example, in Line 19 Figure 2B you can add that the reader needs to compare row 1 and row 4.

      P. 5 Line 15. When a number begins a sentence, that number should always be spelled out. You can pe-phrase the sentence to avoid it. Also, I recommend adding an explanation/hypothesis of why new NPCs are less frequently detected in nucleolar territory.

      P.5 Line 17-22. I recommend re-phrasing these two sentences. Logically, it is clear that Mlp1/Mlp2 loss mimics "old NPCs" to look more like "new NPCs", and for that reason, they are more frequently included in the nucleolar territory, but it is not clear when you read these two sentences from the first time.

      P6. Line 16. No figure supporting data on graph (Figure 3B).

      P.7 Line 10-13. The sentence is unclear.

      P.13,14 etc. If 0h timepoint has been used for normalization, why is it present on the graph?

      P.15. Line 32-33. There is no image here. Potentially wrong description of the figure.

      Figures:

      • Inconsistent labeling of figures. For example, Fig.1, Fig.1S, Figure 2 etc.
      • Inconsistent labeling of figures. For example, Fig.1 G "mean number of NPCs per cell" - no capitalization of the first letter. Fig.1S "Fraction in population" is capitalized. In general, titles of axis should be capitalized.

      Suggestions for Figure 1D and Figure 6 are attached as a separate file.

      Significance

      Zsok et al. used the recombination-induced tag exchange (RITE) approach, which is an interesting and powerful method to follow individual NUPs over time with respect to their localization and abundance. This approach has been used before in PMID: 36515990 to distinguish pre-existing and newly synthesized Nup2 populations and has been extended to other basket NUPs in this work. Using this method, the authors support the earlier data on basket nucleoporins and highlight new insights on how basket nucleoporins regulate NPCs distribution and mobility. Overall, the manuscript provides new details on the stability of nucleoporins in yeast and how these data align with the mass spectrometry and FRAP data performed earlier in other studies. The limitation of this study is the absence of data on Nup1. It was unclear why these data were not present. Additional data can be included on the dynamics of Pml39, for example, using the FRAP method. The dynamic of Pml39 at the pore was shown only using the doRITE method.

      In general, this study represents a unique research study of basic research on nuclear pore proteins that will be of general interest to the nuclear transport field.

      Field of expertise: nuclear-cytoplasmic transport, nuclear pore, inducible protein degradation. I do not have sufficient expertise in ExTrack.

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      Referee #2

      Evidence, reproducibility and clarity

      In this study, Zsok et al. develop innovative methods to examine the dynamics of individual nuclear pore complexes (NPCs) at the nuclear envelope of budding yeast. The underlying premise is that with the emergence of biochemically distinct NPCs that co-exist in the same cell, there is a need to develop tools to functionally isolate and study them. For example, there is a pool of NPCs that lack the nuclear basket over the nucleolus. Although the nature of this exclusion has been investigated in the past, the authors take advantage of a modification of recombination induced tag exchange (RITE), the slow turnover of scaffold nups, the closed mitosis of budding yeast, and extensive high quality time lapse microscopy to ultimately monitor the dynamics of individual NPCs over the nucleolus. By leveraging genetic knockout approaches and auxin-induced degradation with sophisticated quantitative and rigorous analyses, the authors conclude that there may be two mechanisms dependent on nuclear basket proteins that impact nucleolar exclusion. They also incorporate some computational simulations to help support their conclusions. Overall, the data are of the highest quality and are rigorously quantified, the manuscript is well written, accessible, and scholarly - the conclusions are thus on solid footing.

      Significance

      I have no concerns about the data or the conclusions in this manuscript. However, the significance is not overly clear as there is no major conceptual advance put forward, nor is there any new function suggested for the NPCs over nucleoli. As NPCs are immobile in metazoans, the significance may also be limited to a specialized audience.

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      Referee #1

      Evidence, reproducibility and clarity

      The authors extended the existing recombination-induced tag exchange (RITE) technology to show that they can image a subset of NPCs, improving signal-to-noise ratios for live cell imaging in yeast, and to track the stability or dynamics of specific nuclear pore proteins across multiple cell divisions. Further, the authors use this technology to show that the nuclear basket proteins Mlp1, Mlp2 and Pml39 are stably associated with "old NPCs" through multiple cell cycles. The authors show that the presence of Mlp1 in these "old NPCs" correlates with exclusion of Mlp1-positive NPCs from the nucleolar territory. A surprising result is that basket-less NPCs can be excluded from the non-nucleolar region, an observation that correlates with the presence of Nup2 on the NPC regardless of maturation state of the NPC. In support of the proposal that retention of NPCs via Mlp1 and Nup2 in non-nucleolar regions, simulation data is presented to suggest that basket-less NPCs diffuse faster in the plane of the nuclear envelope.

      However, there are some points that do need addressing:

      Major Points

      1. Taking into account that the Nup2 result in Figure 4B forms the basis for one half of the proposed model in Figure 6 regarding the exclusion of NPCs from the nucleolar region of the NE, there is a relatively small amount of data in support of this finding and this proposed model. For example, the only data for Nup2 in the manuscript is a column chart in Figure 4B with no supporting fluorescence microscopy examples for any Nup2 deletion. Further, the Nup60 deletion mutant will have zero basket-containing NPCs, whereas the Nup2 deletion will be a mixture of basket-containing and basket-less NPCs. The only support for the localization of basket-containing NPCs in the Nup2 deletion mutant is through a reference "Since Mlp1-positive NPCs remain excluded from the nucleolar territory in nup2Δ cells (Galy et al., 2004), the homogenous distribution observed in this mutant must be caused predominantly by the redistribution of Mlp-negative NPCs into the nucleolar territory."
      2. The authors could consider utilizing this opportunity to discuss their technological innovations in the context of the prior work of Onischenko et al., 2020. This work is referenced for the statement "RITE can be used to distinguish between old and new NPCs" Page 2, Line 43. However, it is not referenced for the statement "We constructed a RITE-cassette that allows the switch from a GFP-labelled protein to a new protein that is not fluorescently labelled (RITE(GFP-to-dark))" despite Onischenko et al., 2020 having already constructed a RITE-cassette for the GFP-to-dark transition. The authors could consider taking this opportunity to instead focus on their innovative approach to apply this technology to decrease the number of fluorescently-tagged NPCs by dilution across multiple cell divisions and to interpret this finding as a measure of the stability of nuclear pore proteins within the broader NPC.
      3. The authors could also consider taking this opportunity to discuss their results in the context of the Saccharomyces cerevisiae nuclear pore complex structures published e.g. in Kim et al., 2018, Akey et al., 2022, Akey et al., 2023 in which the arrangement of proteins in the nuclear basket is presented, and also work from the Kohler lab (Mészáros et al., 2015) on how the basket proteins are anchored to the NPC. There is additional literature that also might help provide some perspective to the findings in the current manuscript, such as the observation that a lesser amount of Mlp2 to Mlp1 observed is consistent with prior work (e.g. Kim et al., 2018) and that intranuclear Mlp1 foci are also formed after Mlp1 overexpression (Strambio-de-Castillia et al., 1999).

      Minor Points

      1. What is the "lag time" of the doRITE switching? Do the authors believe that it is comparable to the approximate 1-hour timeframe following beta-estradiol induction as shown previously in Chen et al. Nucleic Acids Research, Volume 28, Issue 24, 15 December 2000, Page e108, https://doi.org/10.1093/nar/28.24.e108
      2. The authors could consider a brief explanation of radial position (um) for the benefit of the reader, in Figures 1E (right panel) and 2B (right panel), perhaps using a diagram to make it easier to understand the X-axis (um).
      3. In Figure 1G, would the authors consider changing the vertical axis title and the figure legend wording from "mean number of NPCs per cell" to "mean labeled NPC # per cell" to reflect that what is being characterized are the remaining GFP-bearing NPCs over time?
      4. In Figure 2C, the magenta-labeled protein in the micrographs is not described in the figure or the legend.
      5. In Figure S2A, there is an arrow indicating a Nup159 focus, but this is not described in the figure legend, as is done in Figure 2C.
      6. In Figure S3C, the figure legend does not match the figure. Was this supposed to be designed like Figure 3C and is missing part of the figure? Or is the legend a typographical error?
      7. In Figure S4B, the spontaneously recombined RITE (GFP-to-dark) Nup133-V5 appears in the western blot as equally abundant to pre-recombined Nup133-V5-GFP. In the figure legend, this is explained as cells grown in synthetic media without selection to eliminate cells that have lost their resistance marker from the population. In Cheng et al. Nucleic Acids Res. 2000 Dec 15; 28(24): e108, Cre-EBD was not active in the absence of B-estradiol, despite galactose-induced Cre-EBD overexpression. Would the authors be able to comment further on the Cre-Lox RITE system in the manuscript?
      8. In Figure 6, the authors may want to consider inverting the flow of the cartoon model to start from the wild type condition and apply the deletion mutations at each step to "arrive" at the mutant conditions, rather than starting with mutant conditions and "adding back" proteins.

      Significance

      Recent work has drawn attention to the fact that not all NPCs are structurally or functionally the same, even within a single cell. In this light, the work here from Zsok et al. is an important demonstration of the kind of methodologies that can shed light on the stability and functions of different subpopulations of NPCs. Altogether, these data are used to support an interesting and topical model for Nup2 and nuclear-basket driven retention of NPCs in non-nucleolar regions of the nuclear envelope.

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      Reply to the reviewers

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

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      Referee #3

      Evidence, reproducibility and clarity

      Summary:

      This manuscript examines the effects of fibroblast-derived extracellular vesicles (EV) on axon outgrowth in primary neurons, and investigates potential underlying mechanisms. The authors show that fibroblast EV increase axon outgrowth, which is dependent on components of the Wnt-PCP pathway in neurons. They further show that axonal outgrowth is not affected by neuron- or astrocyte-derived EV and that EV from activated astrocytes inhibit axon elongation. Although several experiments are performed thoroughly, major revision is required to substantiate the main claims of the manuscript.

      Major comments:

      1. Even though the authors made a good effort to characterize fibroblast-derived EV, the data so far does not indicate that strictly 'exosomes' are implicated in axon outgrowth, as it is currently virtually impossible to isolate a pure population of exosomes.
      2. In the methods the authors state that the conditioned media was stored for up to 8 weeks at 4C. As long-term storage of EV was shown to decrease their activity, the authors should specify whether they took steps to test the effect of storage time on EV concentration and activity.
      3. In Fig 3F, the authors claim that Vangl2 re-localizes from proximal to distal end of the axon. However, in the representative image the PBS-treated axon is much shorter, and Vangl2 can also be also detected in the growth cone. Therefore, it is not clear how neurons were classified in the analysis. As this is one of the main claims of the paper, the analysis should be performed in a more quantitative manner, such as quantification of intensity and volume of Vangl2 in the soma, proximal / distal axon and growth cone, while accounting for changes in axon length.
      4. The claim that exosomes mobilize neuronal Wnt to promote axon growth is unsubstantiated. Co-localization between Wnt7b and GFP-CD81 is not convincing given the low magnification and broad distribution of Wnt7b. It is also unclear how EV internalized in the soma would have this effect. Additional experiments to prove the direct influence of fibroblast EV on Wnt-PCP signaling (and optionally, how this is unique for fibroblast EV) would increase the validity of these claims.
      5. Neuronal EV were isolated at a different developmental time point (8DIV), which most likely has an effect on EV composition. Therefore, the claim that neuronal EV do not promote neurite outgrowth is not convincing. In addition, AraC or LPS used to treat neurons and astrocytes respectively, could be co-purified with EV and therefore have adverse effects on recipient neurons.
      6. The authors claim that this effect is unique to fibroblast EV. This claim is not valid without a full characterization of EV derived from multiple cell types and is therefore misleading.

      Minor comments:

      1. As in point 1 above, it is recommended to replace the term 'Exosome' in the figures and refer instead to the method of purification (eg. 100k). The term 'Exosome markers' in the text should also be replaced accordingly, as it is not currently clear whether CD81, TSG101 or Flotilin 1 are strictly on exosomes.
      2. Experiments using EV purified using gradient centrifugation (Fig S3) should be repeated at least once, such that statistical significance is calculated and results are shown as in other functional experiments. Alternatively, experiments could be performed using purified EV treated with a proteinase in the presence or absence of detergent, to verify whether it is EV cargo or extracellular proteins co-isolated with EV that mediate the observed phenotype.
      3. The authors should show that porcupine knockdown results in decreased Wnt secretion in fibroblast EV using western blotting.

      Significance

      Overall, the main take home message of this study is that fibroblast EV could have the common feature of upregulating axon outgrowth in neurons during development. While this is not relevant for CNS development per se, it could ultimately be of interest to a specialized audience investigating the translational relevance of fibroblast-derived EV.

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      Referee #2

      Evidence, reproducibility and clarity

      The definition of axonal fate has been under a lot of scrutiny. However nowadays it is not clear how the axonal fate could be distinguished from axon elongation. In other words, how one the neurite is selected to eventually grow as an axon versus the mechanism sustaining axonal growth once the fate is established. In addition, it is not clear, once the axon is growing, what is the mechanism that precludes the other neurites (future dendrites) from growing.

      The authors of this manuscript claim that fibroblast-derived exosomes promote axon growth; meanwhile, exosomes from activated astrocytes inhibit axon elongation. Moreover, the authors claim that exosomes mediate axon elongation throughout the PCP and Wnts pathways.

      I have several concerns regarding the data and concept presented in this manuscript that I consider precluding its publication in the current form.

      First, the claim that axon growth is promoted by exosomes is not well supported by the quantifications. It will be more convincing to show the frequency distribution for each experiment rather than the average growth per experiment (e.g., Figure 1F). In the way the data is presented now, we do not learn the real effect of the treatment on axonal growth. For instance, how is the variability in axon length in each experiment? the way the data is presented now might mask variability that could reduce the effect of their treatment

      Second, I find it difficult to explain this concept in neurons differentiating in the developing cortex. Which cell type is the source of exosomes mediating axon elongation? Is this cellular mechanism an artifact of the culture condition? In other words, are exosomes relevant for axonal growth in situ?

      In the model presented in Figure 7, authors show that fibroblast might mediate axon extension meanwhile activated astrocytes preclude axon extension. Authors do not consider that in the developing cortex, neurons are formed and elongating an axon (while they migrate to the cortical plate) before astrocytes are produced (Noctor et al Nature Neurosci. 2004). How do the authors reconcile this conceptual discrepancy with her in vitro studies? How do the authors reconcile this discrepancy with their studies in situ?

      Significance

      The authors of this manuscript claim that fibroblast-derived exosomes promote axon growth; meanwhile, exosomes from activated astrocytes inhibit axon elongation. Moreover, the authors claim that exosomes mediate axon elongation throughout the PCP and Wnts pathways.

      I have several concerns regarding the data and concept presented in this manuscript that I consider precluding its publication in the current form.

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      Referee #1

      Evidence, reproducibility and clarity

      Ahmad et al. report that exosomes isolated from fibroblast cell lines stimulate neurite extension in cultured neurons while those from neurons or astrocytes do not. They investigate the role of different components from the planar cell polarity (PCP) pathway and conclude that exosomes stimulate autocrine signaling by Wnts in a PCP pathway-dependent manner. However, the inactivation of most PCP components severely reduces neurite length already in untreated controls suggesting that they are required for neurite growth in general rather than specifically for the response to exosomes. The manuscript reports an extensive set of interesting results but does not provide sufficient evidence for the proposed mechanism.

      Major points:

      1. Knockout, knockdown, pharmacological inhibition or blocking antibodies were used to inactivate multiple components of the PCP pathway in cultured neurons. In most cases, the inactivation resulted in a reduction of neurite length both in control cultures and in cultures with exosomes. Because neurons lack the capacity to extend longer neurites after the inactivation of PCP components, it is not possible to determine if they are required for the response to exosomes. Only Fzd7 appears to be specifically required for the effect of exosomes since its knockdown does not reduce neurite length in controls.
      2. The physiological relevance of the stimulation of neurite growth by exosomes is unclear. It is not explained how cortical neurons come into contact with fibroblasts or the exosomes produced by them as the authors acknowledge at the end of the discussion.
      3. The mechanism how exosomes stimulate neurite growth remains unclear. The authors suggest that exosomes modulate autocrine Wnt signaling but do not provide sufficient evidence for this. The knockdown of Wntless or Wnts and blocking Wnt secretion by inhibiting Porcupine suppress neurite extension. This phenotype could results from a defect in autocrine signaling but also from a reduced secretion of Wnts into the medium.
      4. The authors claim that the cell body takes up exosomes that then acquire endogenous Wnt7b. The co-localization of the signals in Fig. 5H is not informative because Wnt7b shows uniform distribution while the CD81-EYFP signal is present in distinct structures that do not show a stronger EYFP signal.
      5. The specificity of the siRNAs has to be verified by rescue experiments in neurons.

      Minor points:

      It is not explained why the authors tested exosomes from fibroblasts.

      Fig. 7: The graphical summary shows that exosomes somehow enter the soma of neurons. It is not clear if this happens by endocytosis or fusion with the membrane. In either case, the topology of exosomes in the soma is incorrect.

      Significance

      Ahmad et al. report that exosomes isolated from fibroblast cell lines stimulate neurite extension in cultured neurons while those from neurons or astrocytes do not. They investigate the role of different components from the planar cell polarity (PCP) pathway and conclude that exosomes stimulate autocrine signaling by Wnts in a PCP pathway-dependent manner. However, the inactivation of most PCP components severely reduces neurite length already in untreated controls suggesting that they are required for neurite growth in general rather than specifically for the response to exosomes. The manuscript reports an extensive set of interesting results but does not provide sufficient evidence for the proposed mechanism.

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      Reply to the reviewers

      We thank the reviewers for their time and effort in assessing our preprint. We have revised our manuscript and addressed their comments in our point-by-point response as follows:

      Reviewer 1

      The authors should cite the existing mCherry-transgenic quail lines reported by Huss et al. (2015) to compare their performance. The lines developed by Huss et al. carry multiple transgenes, and the transgene-derived fluorescence is detectable under a fluorescent stereomicroscope, which indicates that the expression of substantially high levels of fluorescent proteins in quail cells does not affect quail embryogenesis or growth.

      We have now cited the transgenic mCherry line reported by Huss et al, 2015 as an example of using live imaging of avian embryos to study development but we feel that a direct comparison between the line is invalid as the Tg(PGK1:H2B-chFP) line they report has a nuclear localised fluorescent protein and ours expresses an actin-binding fluorescent protein.

      We note that Huss et al generated three independent transgenic quail lines (Q1-3), but each only contained a single copy of the transgene (as shown in their Fig S2).

      Finally, we would like to highlight that the transgene-derived fluorescence of our Lifeact-EGFP quail line is also easily detectable under a stereomicroscope and we use this method to screen for positive Lifeact-EGFP embryos for experiments. As we show in Figure 1, the Lifeact-EGFP expression does not affect quail embryogenesis or growth.

      Here, the authors developed only a single line of single copy integration of a transgene using a weak promoter. This suggests that the procedure used by the authors to produce transgenic quail may be inefficient and that the transgene expression level is lower. The authors should present an objective measure of the transgene expression levels.

      To generate the transgenic line, we used transfection of primordial germ cells as described previously (Barzilai-Tutsch, Hila et al., eLife, 2022; Serralbo, O et al., eLife, 2020). We deliberately chose to use a UbC promoter to drive moderate expression of Lifeact to avoid potential artifacts relating to Lifeact overexpression (Courtemanche, N. et al., Nature Cell Biology, 2016; Flores, L. R. et al., Sci Rep, 2019; Spracklen, A. J. et al., Developmental Biology, 2014; Xu, R. and Du, S., Front Cell Dev Biol, 2021).

      This methodology not only generates a line with no defects in growth but it also allows us to perform high-resolution imaging and to computationally segment and quantify actin in live embryos. To objectively evaluate the transgene expression level, we have now measured the signal-to-noise ratio of Lifeact-EGFP expression and found it does not differ from that of the standard actin stain Phalloidin. We have included these measurements in Figure S1.

      Although the authors attempted to record philopodial dynamics, the images of philopodia are fuzzy. Sharper philopodial images have been published using the Huss et al. transgenic quail embryos (Sato et al., 2017), where mCherry fluorescence is widespread in the cytoplasm, which indicates no advantage of actin-associated fluorescence. (Sato Y, Nagatoshi K, Hamano A, Imamura Y, Huss D, Uchida S, Lansford R. Basal filopodia and vascular mechanical stress organize fibronectin into pillars bridging the mesoderm-endoderm gap. Development 2017; 144(2):281-291. doi.org/10.1242/dev.141259)

      The mCherry transgenic line reported by Huss et al, 2015 and used by Sato et al, 2017 ubiquitously expresses nuclear-localized mCherry fluorescent protein (Tg(PGK1:H2B-chFP)). It does not label the cytoplasm or the membrane and was used to follow cell nuclei in one set of experiments (Sato et al, 2017, Figure 6).

      The mCherry labelling of filopodia in Sato et al, 2017 was performed by DNA electroporation into wildtype embryos. Our Lifeact-EGFP transgenic line confers an advantage over this approach by 1) removing the need for electroporation to label filopodia and 2) labelling the endogenous actin that forms the filopodial structure. Although we have not optimised the imaging conditions to visualise the somitic filopodia described by Sato et al, nevertheless, we can see them quite clearly in the cross-section of our live imaging of the Lifeact-EGFP quail as demonstrated in the attached response to reviewers document.

      These filopodia, also referred to as filopodia-like protrusions (Sagar et al., Development, 2015), extend from the dorsal surface of the somites towards the ectoderm and can be seen in fixed embryos stained with Phalloidin in Figure S1 in the paper by Sato et al.

      The feasibility of live imaging is, of course, the advantage of Lifeact-EGFP; however, the actin fiber images using Lifeact-EGFP are unclear, partially because Lifeact binds to G-actin with a greater affinity than to F-actin. The authors should compare phalloidin-staining and Lifeac-EGFP on the same high-power fields of fixed specimens. The current manuscript compares staining with Lifeact-EGFP and Phalloidin-568 only under low-power magnification (Figure 1).

      We thank the reviewer for the suggestion. Although the data presented in Figure 1 are tiled images of Phalloidin-568 and Lifeact-EGFP taken on the same fixed specimens on a confocal microscope, we now also include a higher magnification image. This data clearly demonstrates the extensive overlap between Phalloidin, Lifeact-EGFP and SPY650 FastAct dye labelling (Figure 1E).

      Furthermore, we found no significant difference in signal-to-noise ratio of Lifeact-EGFP fluorescence compared to Phalloidin-568 staining (Figure S1).

      Data concerning the apical constriction indicated the versatility and limitations of the Lifeact-EGFP transgenic quail line. The transgenic mouse line carrying ZO1-EGFP transgene, better suited for analyzing the apical constriction issue and employed by Francou et al. (2023), provided cleaner data.

      The dynamics of actin during apical constriction have mostly been studied in invertebrate models where it was revealed that pulsed contractions of a medioapical actomyosin network form a ratchet-like mechanism to drive shrinkage of the apical cell area (Martin, A. C. et al., Nature, 2009; Solon, J. et al., Cell, 2009). More recently, a similar process of pulsatile apical constriction has been demonstrated in Xenopus (Christodoulou, N. and Skourides, P. A., Cell Rep, 2015) and mouse embryos (Francou, A. et al., eLife, 2023). However, the ZO1-EGFP transgenic mouse line labels tight junctions, so the dynamics of actin were inferred from staining of fixed samples by Francou et al. The Lifeact-EGFP transgenic quail line enabled us to both segment the cells and directly measure the intensity and localisation of actin as cells underwent apical constriction in a higher vertebrate embryo, providing direct information about the actin dynamics driving apical area change.

      The significance of the FRAP analysis presented in Figure 4 (F to I) is questionable. (1) The FRAP of Lifeact-EGFP that jumps between G-actin and F-actin was measured. Therefore, the data are a composite of G-actin-bound, F-actin-bound, and free transitory Lifeact-EGFP; the data do not directly reflect actin dynamics. (2) The authors should have measured FRAP at different positions in cells using smaller ROIs at the cell junction, next to the cell junction, and remote from the cell junction. (3) Because the FRAP of their measurements involves different molecular states, the recovery curve should be decomposed into individual components before discussing the difference in the recovery rates. (4) The wide range fluctuation of fluorescence intensity during the recovery process, even using a wide (4 µm × 4 µm) ROI, suggests that the fluorescence level before photobleaching was very low, which indicates a limitation in the use of the transgenic quail line with a single copy of Lifeact-EGFP.

      We apologise if the text was not clear. We did not intend to measure actin dynamics directly, but rather to compare the stability of actin at the vertices of multicellular rosettes of different orders. We used a relatively large ROI (to encompass the vertex) and measured fluorescence recovery at the vertices of lower-order (5-cell) rosettes vs higher-order (8-cell) rosettes to understand if actin stability at the vertex changes as the rosette increases in order. The fluorescence intensity level of the Lifeact-EGFP is high at the vertices of the rosettes (see Fig 4F) and the fluctuation range of fluorescence intensity during recovery was in line with what we have observed previously performing FRAP measurements in living mouse embryos (Samarage*, C.R., White*, M.D., Alvarez*, Y.A et al., Developmental Cell, 2015; Zenker*, J., White*, M. D. et al., Cell, 2018).

      To our knowledge, these are the first FRAP measurements of actin at rosette vertices.

      We have updated the text to clarify as follows:

      "To examine the stability of the actin remaining at the centre of the multicellular rosettes following contraction of the supracellular cables we used Fluorescence Recovery After Photobleaching (FRAP)."

      The authors used three wavelengths to detect fluorescence: DAPI (blue), EGFP (green), and Phaloidin-568 (red). Oddly, the authors presented the EGFP fluorescence in orange and Phaloidin-568 in gray in the pseudocolors.

      We chose to pseudocolour the images to make them accessible to people with colour blindness in accordance with current conventions.

      The data presented indicates that although Lifeact-EGFP-dependent actin labeling is useful for live imaging, its efficacy is restricted by elevated levels of background fluorescence.

      We do not find the live imaging to be restricted by high levels of background noise. Our imaging reveals an average Signal-to-Noise ratio of 1.83 +/- 0.17 (mean +/- sem) in fixed samples in Figure 1. The live imaging revealed a Signal-to-Noise ratio of 1.92 +/- 0.13 for embryos imaged in Figures 2, 3 and 4 which is comparable to the signal in the fixed embryos for both Lifeact-EGFP and Phalloidin-568.

      We can live-image the Lifeact-EGFP embryos at high resolution for extended periods (for example, tiled z-stacks at 40x magnification every 6 - 20 minutes for 4 - 10 hours) with the laser power low enough to avoid phototoxicity. Our imaging data is also of sufficient quality to allow computational segmentation with a high degree of accuracy (as demonstrated in Figures 3 and 4).

      Reviewer 2

      Alvarez and colleagues have generated a transgenic quail line expressing the popular Lifeact-eGFP reporter. This is the first actin reporter line in quail, and enables visualization and characterization of cell shapes and behaviors by following actin-rich structures. The reporter is ubiquitously expressed, and of sufficient brightness to enable high resolution live imaging. To demonstrate its usability, the authors visualized cellular protrusions and actin-rich structures during neural tube closure, migration of cardiac progenitor cells, and examined pulsatile apical constriction in the developing neuroepithelium. These results serve more as a proof-of-principle for the utility of the line rather than an in-depth analysis of any particular cell biology/mechanism, but do contain some insights and avenues for further follow-up. In general this is a nice characterization of a line that I am sure people in the avian embryo field have long been waiting for, and will be in high demand in the future.

      We thank the reviewer for their positive comments and recognition of the usefulness of the Lifeact-EGFP quail as a new model system.

      I have a few minor comments/suggestions:

      1) It would be good if the authors could elaborate on the relative photostability of the line - does it bleach quickly? Show any signs of phototoxicity?

      The photostability is dependent on the imaging conditions. In general, we have not noticed significant bleaching and there are no bleach corrections performed on the movies we show. We do not see signs of phototoxicity with the imaging conditions we are using.

      To address the photostability in more depth we examined our most challenging imaging set-ups. The high spatiotemporal imaging of lamellipodia and actin flow in Figure 1 was performed by imaging a single z-plane at 60x magnification every 5 seconds for 17.25 mins. Despite acquiring over 200 images, there was only a 9.26% loss of Lifeact-EGFP intensity during this intensive imaging.

      For the imaging of apically constricting cells in Figure 3, 4 tiled z-stacks containing 62 z-planes each were taken at 63x magnification every 5.5 mins for 110 mins. We observed an 11.8% loss of Lifeact-EGFP intensity during this time.

      This photostability is comparable to the other transgenic quail lines in our lab (Serralbo, O et al., eLife, 2020) and superior to several zebrafish and genetically modified cell lines we have imaged.

      Additionally, can the animals be maintained as homozygotes?

      The Lifeact-EGFP quails can be maintained as homozygotes and we have now indicated this in the text as follows:

      "The TgT2[UbC:Lifeact-EGFP] quails are viable, phenotypically normal and fertile and can be maintained as heterozygotes or homozygotes."

      2) Did the authors check or are they planning to verify that they did indeed have a single-integration event? Or have bred a sufficient number of generations to eliminate any potential off-target integrations?

      We have bred the Lifeact-EGFP line for enough generations that we are confident we have a single integration event that produces positive transgenics at the expected Mendelian ratio.

      3) In Figure 3: Did Lifeact-eGFP intensity and apical cell area show correlated pulsatile dynamics? They are currently shown separately over the course of constriction but it may be more convincing to show correlation analysis.

      We thank the reviewer for this excellent suggestion. We have revised Figure 3 to overlay the mean Lifeact-EGFP intensity at the apical cortex relative to the cell junctions (medial/junctional Lifeact-EGFP) and apical cell area over time for each embryo. The original separate graphs are still available in the new Figure S3A. We first established that there is a highly significant inverse correlation between medial Lifeact-EGFP intensity and apical cell area in constricting cells in each embryo (Figure S3B). We next examined the correlation between the change in medial Lifeact-EGFP intensity and the change in apical cell area for each constricting cell (Figure S3C). Although there is a high degree of variability between cells, on average we find a moderate, but highly significant correlation of 0.37 +/- 0.05, pWe have now included these results in the new Figure S3 and the text as follows:

      "Measuring the ratio of Lifeact-EGFP signal at the apical cortex relative to the cell junctions revealed an average increase of 71.7%+/- 2.9 % during the first 25% of the reduction in apical cell area (Figs. 3C, S3A-B). The inverse correlation between mean Lifeact-EGFP intensity at the apical cortex and mean apical cell area is highly significant (Fig. S3B). Furthermore, the identified cells did not undergo a constant decrease in apical cell area but instead showed a more pulsatile pattern consistent with a ratchet-like mechanism (Figs. 3C, D). There was a moderate, but highly significant correlation between the rate of change in Lifeact-EGFP intensity at the apical cortex and the change in apical cell area for individual cells (Fig. S3C)."

      4) Did they check for integrins at the filopodia tips?

      We did not check for integrins at the tips of the cardiac progenitor cell filopodia, however, we do see integrins at the tips of filopodia in other cells and these data are part of an ongoing study in our lab.

      5) In Figure 4B it is too hard for the reader to verify that these are indeed actin cables - the overlay interferes with the visualization. Could just be 10 cells coincidentally aligned. Same with Figure 4 J/K

      We have made the overlay partially transparent so that the cables are more visible. The same cable structures are also highlighted without overlays in the blue boxes in Figures 4A and 4J.

      6) Figure 4C and 4L are confusing - what is the repeated number of rosette cells mean? Are these different regions cropped out? What are the rows/columns?

      The images show the computational segmentation of the regions shown in 4A and 4J. Each panel shows the number of rosettes identified of each order (containing 5, 6, 7 or 8 cells) at t = 0h (on the left) and t = 2h (on the right).

      We initially displayed all of the rosettes on a single computational segmentation but felt it was much easier to appreciate the relative number of rosettes of each order when they are presented individually. We have updated the Figure Legend to specify that 4C and 4L show computational segmentations of the images in 4A and 4J.

      7) Time stamps on supplementary movies could be made more visible/better labelled.

      We have enlarged the timestamps on the movies.

      8) Would be helpful to include movies of the processes studied in Figures 3 and 4.

      We have now included movies showing apical constriction (Supplementary Movie 5) and rosette formation (Supplementary Movie 6).

      Reviewer 3

      The manuscript is well-written. The Lifeact-EGFP transgenic quail will be a valuable new amniote model system for in vivo investigations of the actin cytoskeleton to promote cell shape changes and tissue morphogenesis. I recommend that this manuscript be accepted with minor revisions.

      We thank the reviewer for their positive comments and are pleased they view the Lifeact-EGFP quail as a valuable new model system.

      Minor suggestions

      -Please include how many transgenic males and females were obtained from the 50 injections.

      We have now included this in the text as follows:

      "One male and one female founder were identified and mated with wild-type quails to establish lines. After further breeding the lines were indistinguishable and the line from the male founder was selected for long-term maintenance."

      -The authors state, "Cardiac progenitor cell filopodia are on average 9.1μm +/- 0.5μm long and highly dynamic with an average persistence time of 389.1 s +/- 22.9 s (n = 86 filopodia, 4 embryos). Filopodia that contact the surrounding tissues are significantly longer and more persistent than those that do not make contact (11.2μm +/- 0.7μm, n = 42 and 523.6 s +/-34.5 s, n = 37, compared to 7.2μm +/- 0.4μm, n = 44 and 276.0 s +/-20.5 s, n = 44, Fig 2C - E)."

      How does this compare to other similar cells? Does this suggest attraction, repulsion, or nothing? Does the higher filopodia persistence correlate with the cell's persistence, migration velocity or direction?

      The cardiac progenitor cell filopodia are slightly longer and more persistent on average than filopodia detected in other migrating cell types in vivo. For example, neural crest cells form filopodia that are on average 5 - 6um long and persist for 121 s in chick (Genuth, M. A. et al., Developmental Biology, 2018; McLennan, R. et al., Development, 2020) or 10um in length in zebrafish (Boer, E. F. et al., PLoS Genet, 2015). Primordial germ cells in zebrafish extend filopodia which are on average 3.4um long and persist for only 33 +/- 2.5 s (Meyen, D. et al., eLife, 2015). In Xenopus retinal ganglion cells, filopodia were on average 6.7um long and persisted for just 19 s (Blake, T. C. A. et al., Journal of cell science, 2024).

      However, the modes of migration of these cell types are quite distinct with neural crest cells collectively migrating as transiently contacting mesenchymal cells whereas primordial germ cells and retinal ganglion cells migrate individually during the embryonic stages examined. The cardiac progenitor cells form a collectively migrating epithelium which maintains cell-cell contacts and migrates over the endoderm at a speed of 4,99 +/-0.09 um min-1, so it is difficult to draw conclusions about their filopodial dynamics by comparison with other cell types characterised to date.

      The reviewer raises a very interesting question about the relationship between filopodial persistence and the migration behaviour of the individual cell. As the cardiac progenitor cells are migrating as a tightly packed collective, resolving individual cell migration behaviours is very challenging when they are homogenously labelled. To accurately correlate filopodia dynamics with individual cell migration would require highly technically demanding experiments to mosaically label the cardiac progenitor cells and track them and their filopodia dynamics live. While this would undoubtedly be an interesting experiment, we feel it is beyond the scope of the current tools manuscript.

      It is well-known that filopodia are sensors for chemotactic and haptotactic signals, and they set the direction of motility for cells. The authors rightly suggest that actin containing filopodia contact ECM components, but do not support this with any experiments.

      We agree that it would be interesting to investigate the molecular components of the filopodia more thoroughly. However, as a tools paper, our primary motivation was to present the Lifeact-EGFP transgenic quail as a new resource for the scientific community and demonstrate different applications it could be useful for - including as a new model to study filopodia dynamics in vivo.

      Significance

      The manuscript is lacking any novel insights regarding actin dynamics. In general, it would be helpful if the authors discuss the significance of their observations in more detail, especially in their Conclusion, which is brief. By carrying out more creative and insightful experiments, the authors would have offered stronger evidence for the value of the Lifeact-EGFP line to other investigators.

      The primary purpose of this manuscript was to present the Lifeact-EGFP transgenic quail as a new resource for the scientific community and demonstrate different applications it could be useful for. However, we did also make some novel insights:

      • Although neural tube protrusions have been visualised in fixed embryos for many decades, the Lifeact-EGFP transgenic quail enabled us to image them live in high spatiotemporal resolution. This revealed that they are highly dynamic, reach across the open lumen to contact each other and appear to assist in pulling the neural folds together. We also found that neural tube zippering proceeded faster in embryos with more protrusions.
      • We demonstrated that cells in the avian neuroepithelium undergo pulsatile apical constriction associated with the enrichment of medioapical actin.
      • We performed, to our knowledge, the first FRAP of actin at the vertices of multicellular rosettes and found that actin stability increases with higher rosette order.
      • Finally, we confirmed that supracellular actin cable contraction and rosette formation contribute to anisotropic bending of the neural plate during neural tube formation - a prediction made previously based on fixed tissue sections (Nishimura, T. et al., Cell, 2012) but not investigated in living avian embryos. We believe that the range of novel insights we present here demonstrates the significance of the Lifeact-EGFP transgenic quail line as a new tool for investigating vertebrate cytoskeletal dynamics and morphogenesis in vivo.

      References

      An, Y., Xue, G., Shaobo, Y., Mingxi, D., Zhou, X., Yu, W., Ishibashi, T., Zhang, L. and Yan, Y. (2017). Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts. Development 144, 2153-2164.

      Barzilai-Tutsch, H., Morin, V., Toulouse, G., Chernyavskiy, O., Firth, S., Marcelle, C. and Serralbo, O. (2022). Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development. eLife 11, e72098.

      Blake, T. C. A., Fox, H. M., Urbancic, V., Ravishankar, R., Wolowczyk, A., Allgeyer, E. S., Mason, J., Danuser, G. and Gallop, J. L. (2024). Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions. Journal of cell science 137.

      Boer, E. F., Howell, E. D., Schilling, T. F., Jette, C. A. and Stewart, R. A. (2015). Fascin1-dependent Filopodia are required for directional migration of a subset of neural crest cells. PLoS Genet 11, e1004946.

      Christodoulou, N. and Skourides, P. A. (2015). Cell-Autonomous Ca(2+) Flashes Elicit Pulsed Contractions of an Apical Actin Network to Drive Apical Constriction during Neural Tube Closure. Cell Rep 13, 2189-202.

      Courtemanche, N., Pollard, T. D. and Chen, Q. (2016). Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nature Cell Biology 18, 676-83.

      Flores, L. R., Keeling, M. C., Zhang, X., Sliogeryte, K. and Gavara, N. (2019). Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour. Sci Rep 9, 3241.

      Francou, A., Anderson, K. V. and Hadjantonakis, A. K. (2023). A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT. eLife 12.

      Genuth, M. A., Allen, C. D. C., Mikawa, T. and Weiner, O. D. (2018). Chick cranial neural crest cells use progressive polarity refinement, not contact inhibition of locomotion, to guide their migration. Developmental Biology 444 Suppl 1, S252-S261.

      Martin, A. C., Kaschube, M. and Wieschaus, E. F. (2009). Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495-9.

      McLennan, R., McKinney, M. C., Teddy, J. M., Morrison, J. A., Kasemeier-Kulesa, J. C., Ridenour, D. A., Manthe, C. A., Giniunaite, R., Robinson, M., Baker, R. E. et al. (2020). Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia. Development 147.

      Meyen, D., Tarbashevich, K., Banisch, T. U., Wittwer, C., Reichman-Fried, M., Maugis, B., Grimaldi, C., Messerschmidt, E. M. and Raz, E. (2015). Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo. eLife 4.

      Nishimura, T., Honda, H. and Takeichi, M. (2012). Planar cell polarity links axes of spatial dynamics in neural-tube closure. Cell 149, 1084-97.

      Sagar, Prols, F., Wiegreffe, C. and Scaal, M. (2015). Communication between distant epithelial cells by filopodia-like protrusions during embryonic development. Development 142, 665-71.

      Samarage*, C. R., White*, M.D., Alvarez*, Y. D., Fierro-Gonzalez, J. C., Henon, Y., Jesudason, E. C., Bissiere, S., Fouras, A. and Plachta, N. (2015). Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo. Developmental Cell 34, 435-47.

      Serralbo, O., Salgado, D., Véron, N., Cooper, C., Dejardin, M., Doran, T., Gros, J. and Marcelle, C. (2020). Transgenesis and web resources in quail. eLife 9.

      Solon, J., Kaya-Copur, A., Colombelli, J. and Brunner, D. (2009). Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell 137, 1331-42.

      Spracklen, A. J., Fagan, T. N., Lovander, K. E. and Tootle, T. L. (2014). The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis. Developmental Biology 393, 209-226.

      Xu, R. and Du, S. (2021). Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function. Front Cell Dev Biol 9, 746818.

      Zenker*, J., White*, M. D., Gasnier*, M., Alvarez*, Y. D., Lim, H. Y. G., Bissiere, S., Biro, M. and Plachta, N. (2018). Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation. Cell 173, 776-791 e17.

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      Referee #3

      Evidence, reproducibility and clarity

      Alvarez et al. report the generation of a transgenic Lifeact-EGFP quail line to study actin organization and dynamics in living embryos. The authors use the Lifeact-EGFP line to visualize how actin filaments guide coordinate cellular movements across tissues. Their example studies of heart and neural tube morphogenesis reveal the dynamics of cells undergoing apical constriction and the emergence of large-scale actin structures, such as supracellular cables and rosettes within the neuroepithelium.

      The manuscript is well-written. The Lifeact-EGFP transgenic quail will be a valuable new amniote model system for in vivo investigations of the actin cytoskeleton to promote cell shape changes and tissue morphogenesis. I recommend that this manuscript be accepted with minor revisions.

      Minor suggestions

      • Please include how many transgenic males and females were obtained from the 50 injections.
      • The authors state, "Cardiac progenitor cell filopodia are on average 9.1μm +/- 0.5μm long and highly dynamic with an average persistence time of 389.1 s +/- 22.9 s (n = 86 filopodia, 4 embryos). Filopodia that contact the surrounding tissues are significantly longer and more persistent than those that do not make contact (11.2μm +/- 0.7μm, n = 42 and 523.6 s +/-34.5 s, n = 37, compared to 7.2μm +/- 0.4μm, n = 44 and 276.0 s +/-20.5 s, n = 44, Fig 2C - E)."

      How does this compare to other similar cells? Does this suggest attraction, repulsion, or nothing? Does the higher filopodia persistence correlate with the cell's persistence, migration velocity or direction?

      "The tissues surrounding the cardiac progenitor cells are covered in an extracellular matrix rich in fibronectin, which also extends along some of the filopodia (Fig. S2). As integrins are known to be present at filopodial tips (Lagarrigue et al., 2015, Galbraith et al., 2007), the higher persistence of filopodia in contact with surrounding tissues may indicate a force-dependent stabilisation of the filopodia (Alieva et al., 2019). This indicates these filopodia could have signalling roles as proposed previously (Francou et., 2014) and/or mechanical roles during cardiac progenitor cell migration."

      It is well-known that filopodia are sensors for chemotactic and haptotactic signals, and they set the direction of motility for cells. The authors rightly suggest that actin containing filopodia contact ECM components, but do not support this with any experiments.

      Significance

      The manuscript is lacking any novel insights regarding actin dynamics. In general, it would be helpful if the authors discuss the significance of their observations in more detail, especially in their Conclusion, which is brief. By carrying out more creative and insightful experiments, the authors would have offered stronger evidence for the value of the Lifeact-EGFP line to other investigators.

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      Referee #2

      Evidence, reproducibility and clarity

      Alvarez and colleagues have generated a transgenic quail line expressing the popular Lifeact-eGFP reporter. This is the first actin reporter line in quail, and enables visualization and characterization of cell shapes and behaviors by following actin-rich structures. The reporter is ubiquitously expressed, and of sufficient brightness to enable high resolution live imaging. To demonstrate its usability, the authors visualized cellular protrusions and actin-rich structures during neural tube closure, migration of cardiac progenitor cells, and examined pulsatile apical constriction in the developing neuroepithelium. These results serve more as a proof-of-principle for the utility of the line rather than an in-depth analysis of any particular cell biology/mechanism, but do contain some insights and avenues for further follow-up. In general this is a nice characterization of a line that I am sure people in the avian embryo field have long been waiting for, and will be in high demand in the future.

      I have a few minor comments/suggestions:

      1. It would be good if the authors could elaborate on the relative photostability of the line - does it bleach quickly? Show any signs of phototoxicity? Additionally, can the animals be maintained as homozygotes?
      2. Did the authors check or are they planning to verify that they did indeed have a single-integration event? Or have bred a sufficient number of generations to eliminate any potential off-target integrations?
      3. In Figure 3: Did Lifeact-eGFP intensity and apical cell area show correlated pulsatile dynamics? They are currently shown separately over the course of constriction but it may be more convincing to show correlation analysis.
      4. Did they check for integrins at the filopodia tips?
      5. In Figure 4B it is too hard for the reader to verify that these are indeed actin cables - the overlay interferes with the visualization. Could just be 10 cells coincidentally aligned. Same with Figure 4 J/K
      6. Figure 4C and 4L are confusing - what is the repeated number of rosette cells mean? Are these different regions cropped out? What are the rows/columns?
      7. Time stamps on supplementary movies could be made more visible/better labelled.
      8. Would be helpful to include movies of the processes studied in Figures 3 and 4.

      Significance

      Transgenic quail models are still in their relative infancy compared to more traditional/well-established model organisms, yet quail has proven to offer many new insights into developmental processes, and with its flat geometry often offers up a view of tissues and cell behaviors that can be hidden in other species. A live reporter line for actin structures is thus keenly needed by the avian developmental biology field, and this new transgenic model reported here should fill that niche nicely.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary

      In this study, the authors produced a Lifeact-EGFP transgenic quail line to investigate the cellular event dynamics that involve F-actin bundles. No perfect reagents exist to specifically label F-actin in live cells at high sensitivity; currently, Lifeact peptide may be the primary option to label actins using transgenic animals. However, it has the drawback of binding to G-actin in addition to F-actin, which results in a high background of Lifeact-EGFP fluorescence in the cytoplasm.

      A transgenic quail line was produced by lipofection of circulating PGCs with Tol2 transposon-based expression vector and Tol2 transposase expression vector; a single founder male harboring a single copy of the transgene was crossed with wild-type females to generate a transgenic colony.

      To demonstrate the utility of Lifeact-EGFP quail embryos, the authors performed the following descriptive studies: (1) filopodia extrusion; (2) actin bundle dynamics during apical constriction; (3) formation of actin bundles and multicellular rosettes; (4) FRAP analysis of actin mobility at the cellular vertices; and (5) the effect of actin polymerization inhibitors on the multicellular rosettes. The data presented demonstrate the range of utility as well as the limitations of the author's transgenic system.

      Major comments

      The authors should cite the existing mCherry-transgenic quail lines reported by Huss et al. (2015) to compare their performance. The lines developed by Huss et al. carry multiple transgenes, and the transgene-derived fluorescence is detectable under a fluorescent stereomicroscope, which indicates that the expression of substantially high levels of fluorescent proteins in quail cells does not affect quail embryogenesis or growth. Here, the authors developed only a single line of single copy integration of a transgene using a weak promoter. This suggests that the procedure used by the authors to produce transgenic quail may be inefficient and that the transgene expression level is lower. The authors should present an objective measure of the transgene expression levels. (Huss D, Benazeraf B, Wallingford A, Filla M, Yang J, Fraser SE, Lansford R. A transgenic quail model that enables dynamic imaging of amniote embryogenesis. Development 2015; 142:2850-9. doi: 10.1242/dev.121392.)

      Although the authors attempted to record philopodial dynamics, the images of philopodia are fuzzy. Sharper philopodial images have been published using the Huss et al. transgenic quail embryos (Sato et al., 2017), where mCherry fluorescence is widespread in the cytoplasm, which indicates no advantage of actin-associated fluorescence. (Sato Y, Nagatoshi K, Hamano A, Imamura Y, Huss D, Uchida S, Lansford R. Basal filopodia and vascular mechanical stress organize fibronectin into pillars bridging the mesoderm-endoderm gap. Development 2017; 144(2):281-291. doi.org/10.1242/dev.141259)

      The feasibility of live imaging is, of course, the advantage of Lifeact-EGFP; however, the actin fiber images using Lifeact-EGFP are unclear, partially because Lifeact binds to G-actin with a greater affinity than to F-actin. The authors should compare phalloidin-staining and Lifeac-EGFP on the same high-power fields of fixed specimens. The current manuscript compares staining with Lifeact-EGFP and Phalloidin-568 only under low-power magnification (Figure 1).

      Data concerning the apical constriction indicated the versatility and limitations of the Lifeact-EGFP transgenic quail line. The transgenic mouse line carrying ZO1-EGFP transgene, better suited for analyzing the apical constriction issue and employed by Francou et al. (2023), provided cleaner data.

      The significance of the FRAP analysis presented in Figure 4 (F to I) is questionable. (1) The FRAP of Lifeact-EGFP that jumps between G-actin and F-actin was measured. Therefore, the data are a composite of G-actin-bound, F-actin-bound, and free transitory Lifeact-EGFP; the data do not directly reflect actin dynamics. (2) The authors should have measured FRAP at different positions in cells using smaller ROIs at the cell junction, next to the cell junction, and remote from the cell junction. (3) Because the FRAP of their measurements involves different molecular states, the recovery curve should be decomposed into individual components before discussing the difference in the recovery rates. (4) The wide range fluctuation of fluorescence intensity during the recovery process, even using a wide (4 µm × 4 µm) ROI, suggests that the fluorescence level before photobleaching was very low, which indicates a limitation in the use of the transgenic quail line with a single copy of Lifeact-EGFP.

      Minor comment

      The authors used three wavelengths to detect fluorescence: DAPI (blue), EGFP (green), and Phaloidin-568 (red). Oddly, the authors presented the EGFP fluorescence in orange and Phaloidin-568 in gray in the pseudocolors.

      Significance

      The single Lifeact-EGFP transgenic quail line developed in this study may be useful in certain contexts; however, better lines may be obtained by checking additional lines for higher levels of transgene expression.

      The data presented indicates that although Lifeact-EGFP-dependent actin labeling is useful for live imaging, its efficacy is restricted by elevated levels of background fluorescence.

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      Reply to the reviewers

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

      Summary:

      In this study the authors apply a rigorous and thorough combination of approaches including sequence analysis, deep-learning structure predictions, molecular dynamics, cell imaging and mutagenic analyses to identify a short MIM2-mimicking motif in the C-terminal region of the pUL71 protein of HCMV (and homologues in other beta-herpesviruses) that is necessary and sufficient for interaction with the ESCRT terminal ATPase VPS4A. pUL71 uses this motif to recruit, or sequester, VPS4A to the HCMV cytoplasmic viral assembly complex, though this process is dispensable for HCMV morphogenesis or replication. The identified pUL71 sequence functions as a mimic of the MIM2 motif of cellular CHMP subunits since, like MIM2, it directly binds the groove in the MIT domain found at the N-terminus of VPS4.

      Major comments:

      1). There appears to be some confusion in the coip experiment in Figure 5D. From the upper blot in 5D, the "+" above each lane suggests there should be VPS4A-FLAG protein in every sample other than the two lanes at the very left of the gel, however the anti-FLAG ip does not pull down VPS4A-FLAG from every "+" lane, but from alternating ones (and from the next to the leftmost lane, which should lack VPS4A-FLAG). Similarly, the lower "Input" blot shows VPS4A-FLAG present in alternating lanes across the blot, which does not match the "+" and "-" labeling at the top of the figure. Conversely, there is anti-HA signal in most input lanes (lower blot) though the HA-tagged pUL71 homologues should be absent from alternate lanes (top of upper blot).

      We apologise and thank the reviewer for spotting this annotation error. Figure 5D has been updated to correctly show the samples used for each lane of the IP.

      2). The Discussion is an excellent, comprehensive and scholarly assessment of the implications of this work. One appealing hypothesis is that pUL71 may be sequestering VPS4A rather than using it for envelope scission. In this regard, the authors point out that VPS4A sequestration is supported by the finding that the VPS4A MIT domain binds the isolated pUL71 vMIM2 more tightly (~ 5 fold lower Kd) than the MIM2 of CHMP6, and that pUL71 and homologues are highly abundant at later stages of viral infection, allowing them to compete effectively with endogenous CHMP6 for VPS4A. I like the sequestration model very much, but could the authors comment on the fact that this apparent sequestration is seen even in the transfection experiments in Fig. 2A and 3G, where essentially 100% of transfected WT VPS4A-FLAG is recruited to the pUL71 compartment. Even given the increased binding affinity to pUL71, this suggests that in these transfection studies pUL71 must be in excess over the sum of both endogenous and transfected VPS4. Do the authors know if this is the case, and do cells transfected with pUL71 in these experiments exhibit any cytotoxicity, or cell cycle arrest, indicative of a block in normal ESCRT function/cytokinesis?

      *With regards to the transfection experiment, the levels of pUL71 and VPS4A-FLAG expression varied across the fields of view. It is therefore hard to make a definitive statement regards the level of pUL71 expression that gives complete sequestration of VPS4A-FLAG. In transient expression experiment, cells were transfected with equal amounts of DNA for VPS4A-FLAG and pUL71 expression vectors and analysed 20 to 24 hours after transfection. Sequestration of VPS4A-FLAG by pUL71, or lack of sequestration due to mutations, was consistently observed and was thus the predominant phenotype. However, degrees in the appearance of this phenotype were noted, which were likely caused by differences in expression levels. The images in Figs. 1, 2, 3 and 5 are confocal images of selected cells that represented the predominant phenotype. In our opinion, no clear statements can be made about expression levels and their relationship with respect to sequestration of VPS4A, as transient expression gives considerable cell-to-cell variability in expression levels. *

      In MRC-5 cells transiently expressing VPS4A-FLAG under doxycycline control and infected with different strains of HCMV we see strong sequestration of VPS4A-FLAG (Fig. 6B). While VPS4A-FLAG sequestration is not always complete in the context of infection (compare WT infection in Fig. 6C and Fig. 6D), presumably because of differing VPS4A-FLAG levels, it is reasonable to assume that ectopic VPS4A-FLAG expression increases the total pool of VPS4A available. Thus, in the context of infected cells we would expect the vast majority of cellular VPS4A to be sequestered by pUL71, also considering the strong expression of pUL71 during infection. However, we note that evenly distributed signals in the cytoplasm are more difficult to visualize than concentrated signals (such as localization at the Golgi), especially in confocal images, which could contribute to the impression that almost 100% of VPS4A-FLAG is sequestered by pUL71. We have therefore added the following three sentences to the third paragraph of the discussion:

      “In the context of infection, pUL71 yields strong sequestration of ectopically expressed VPS4A-FLAG (Fig. 7A–C). As ectopic expression would be expected to increase the total pool of VPS4A present in cells, we anticipate extensive pUL71-mediated sequestration of endogenous VPS4A in HCMV-infected cells. However, we note that diffuse cytoplasmic signals are more difficult to visualise than organelle-associated signals in confocal microscopy, and it is therefore possible that some VPS4A remains free in the cytoplasm even in the presence of abundant pUL71.”

      Unfortunately, we are unaware of a high quality VPS4A antibody suitable for immunofluorescence microscopy that would allow us to probe the localisation of endogenous VPS4A directly.

      *The reviewer raises an interesting point with regard to the potential blocking of cellular ESCRT functions in the presence of transfected pUL71. We did not find indications of a block in ‘normal’ ESCRT functions like cytokinesis in cells expressing pUL71 (or mutant versions thereof). We therefore investigated the function of ESCRT in pUL71-expressing cells by assessing whether expression of pUL71 can inhibit the function of VPS4 in the release of HIV Gag virus like particles (VLPs). The results of these studies have been added to the manuscript as supplemental figure 7 – they show no evidence for a functional inhibition of VPS4 by co-expression of pUL71. We have added a section to the results describing this experiment, plus the following section in the discussion: *

      “We did not observe any defect in ESCRT-mediated Gag VLP production in the presence of pUL71 (Fig. S7), suggesting that transient expression of pUL71 is not sufficient to inhibit cellular ESCRT activity. However, we note that studies analysing the role of VPS4 in ESCRT-mediated virus budding generally exploit dominant-negative forms of VPS4A or VPS4B (Corless et al., 2010; Horii et al., 2006; Pawliczek and Crump, 2009; Taylor et al., 2007). As human VPS4A and VPS4B interact with each other (Scheuring et al., 2001), overexpressing dominant-negative mutants of either protein would be expected to poison the activity of both via formation of heteromeric APTase hexamers. Studies using CRISPR/Cas9 gene editing show only modest defects in VLP budding when VPS4A or VPS4B are deleted individually, with the VPS4B deletion causing a greater VLP budding defect than VPS4A deletion (Harel et al., 2022), and the MIM2 of CHMP6 has higher affinity for the MIT domain of VPS4A than of VPS4B (Wenzel et al., 2022). While we have not investigated the interaction between pUL71 and the VPS4B MIT domain in this study, it is possible that pUL71 has higher affinity for VPS4A than VPS4B and pUL71 expression may thus lead to selective sequestration of only one VPS4 isoform.”

      *In light of the above new results and discussion, we can neither confirm nor rule out that pUL71 modulates ESCRT functions by sequestration of VPS4. We agree with the reviewer that it is an extremely interesting hypothesis but addressing it properly would require thorough experimental investigation, which we feel is a substantial study in its own right and is beyond the scope of this manuscript. Lastly, we apologise that we had inadvertently included the affinity of GST-tagged pUL71(300–325) for VPS4A in the discussion text, not the data for the pUL71(300–325) peptide. We have updated the text accordingly and confirm that all the data in Table 2 are correct. *

      Minor comments:

      In general, the text and figures are very clear and accurate, the Results section is careful to walk the reader though these studies in a clear and well written fashion and prior studies are referenced appropriately. There are some minor issues that are listed below.

      i). For clarity, please direct the reader to panel 1B when referring to the pp28 data (line 11 of Results section).

      Done

      ii). At the bottom of the page 4, the Results section states "immunoprecipitation experiments show VPS4A-FLAG to be robustly co-precipitated by wild-type pUL71 but not by the PPAA and V317D mutants". However, from Fig. 1E it appears to be the reverse. The wild type pUL71 (but not mutants) is being co-precipitated by VPS4A-FLAG, using an anti-FLAG antibody.

      Corrected – we apologise for this error and thank the reviewer for spotting it.

      iii). In Fig.1D the localization of WT pUL71 and the PPAA and V317D mutants to a juxtanuclear compartment provides a nice internal control demonstrating that the mutant proteins are at least partially functional (able to localize correctly), and the fluorescence intensities of the WT and mutant pUL71 proteins appear comparable. However, do the authors have any additional quantitative or semi-quantitative data (such as from a Western) to confirm similar expression levels for the pUL71 WT and PPAA/V317D mutant proteins?

      The relevant data is shown in Fig. 1E. Specifically, the immunoblot of the input samples shows that pUL71 mutants are expressed at similar levels to the wild-type protein. We have added a note to this effect to the Results.

      “Inspection of the immunoprecipitation input samples confirms that pUL71 mutants are expressed at similar levels to the wild-type protein.”

      *Furthermore, we added to methods following statement: “equal volumes of lysate were used for all samples”, to confirm that the signals in Co-IPs stem from equal amounts of lysates. *

      iv). In Fig. 4, An OPTIONAL experiment, which would add to the paper, would be to test the ability (or rather, lack of the ability) of the pUL71 I307R mutant to coip VPS4A from infected or transfected cells. Such a study would extend the predictive power of the elegant MD simulations and ITC studies to the "gold standard" of testing the phenotype in vivo.

      While we appreciate that this additional experiment would provide further confirmation of our computational analysis in the cellular context, we would argue that ITC is the ‘gold standard’ when it comes to the measurement of protein interaction affinities. We show in Figure 1 that ITC, coIP and immunofluorescence experiments yield the same result (compare WT and PPAA pUL71 in panels D, E and G). We have thus respectfully declined to perform this additional IP experiment as we feel that the ITC data included in the manuscript, combined with the coIP data for the P315A and P318A mutants, are sufficient to prove the predictive power of the model.

      v). The Fig. 6B TB71stop pp28 panel is not referred to in the Fig. 6 legend.

      We apologise for this oversight. We have added a description of this experiment to the Fig. 6 legend:

      “Cells infected with TBstop71 were also stained for tegument protein pp28 to confirm successful cVAC formation (bottom).”

      We have also added the relevance of this image in the Results:

      “Formation of perinuclear cVAC in cells infected with TBstop71 was confirmed via immunostaining for pp28 (Fig. 6C) (Sanchez et al., 2000b; Seo and Britt, 2007).”

      vi). In the second paragraph of the Discussion it is stated that "The pUL71 vMIM2 is necessary and sufficient to recruit VPS4A to specific membranes in co-transfected cells (Fig. 5) and to sites of virus assembly in HCMV infection (Fig. 6)". Strictly speaking, Fig. 5 (panel 5F) shows that the HCMV pUL71 region 283-361 is sufficient to localize VPS4A to a compact juxtanuclear structure in transfected cells, and Fig. 6 (panel 6C) shows that pUL71 residues 315-326 (and the two conserved prolines in this region) are necessary for VPS4A localization to a structure that appears to be the HCMV assembly compartment.

      We thank the reviewer for highlighting that we been imprecise when describing the implications of our results. We have updated the relevant sentence as follows:

      “The pUL71 vMIM2 is necessary and sufficient to recruit VPS4A to juxtanuclear structures in co-transfected cells (Fig. 5) and is necessary for VPS4A recruitment to pUL71-positive structures that have been identified as sites of virus assembly during HCMV infection (Fig. 6) (Dietz et al., 2018).”

      Reviewer #1 (Significance (Required)):

      This is the first report of a virus encoding a MIM-like domain, and of a viral motif that directly binds the VPS4A MIT domain. This will be of broad interest to those studying the cell biology of virus assembly and mechanisms of virus-host cell interaction, as well as to cell biologists and structural biologists studying the ESCRT apparatus. It is striking, and will be illuminating to virologists and ESCRT biologists, that viruses have evolved to mimic MIM2 with a motif that has a lower Kd than a conventional cellular MIM2 motif. The possibility, addressed in the Discussion, that pUL71 may be sequestering VPS4A (rather than using it) is an important issue that virologists should consider.

      This is a rigorous, thorough and well controlled basic science study that elegantly combines a variety of approaches to provide important new insights concerning the biology of pUL71 in HCMV, other human beta-herpesviruses and a large number of mammalian and rodent cytomegaloviruses. The claims and conclusions are thoughtful and measured, and supported by the data. Data and methods are presented in such a way that they can be reproduced, and experiments are adequately replicated with appropriate statistical analyses.

      Reviewers fields of interest: Cell biology, ESCRT function, Virus assembly

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

      Summary

      The manuscript from Butt et al. entitled "Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly" addresses the potential role of ESCRT components in the biogenesis of HCMV virions. The topic is relevant since the ESCRT machinery has been implicated in the propagation of various herpesviruses. However, conflicting results are present in the literature regarding its role and relevance. In the present study, the authors focus on HCMV pUL71, which plays a role during the final envelopment of the viral capsids, and explore the possibility that it acts as an ESCRT-III component by recruiting the VPS4A ATPase (which induces membrane deformation and scission). To this end, they identified a motif in the C-terminal region of HCMV pUL71 that resembles the cellular type 2 MIM (MIM2) consensus sequence that is present in ESCRT-III proteins such as CHMP4B and CHMP6. They show substantial data that delineate an interaction between that pUL71 motif and the cellular ATPase using a panoply of tools (co-IF, co-IP, ITC, bimolecular fluorescence and markerless BAC mutagenesis). They also show that this interaction in present across a wide range of HCMV strains, but is absent in the case of the HSV-1 pUL51 homolog.

      Main Comments

      The manuscript is very well written, albeit line numbers would facilitate reviewing. The plethora of assays used convincingly show an interaction between pUL71 and VPS4A. They also indicate that this interaction relocalizes VPS4A to the TGN and likely the VAC. However, I do have some issues. For instance, using another viral marker, instead of only pUL71, would have been a good idea to distinguish the TGN from the VAC. This is not trivial given the reorganization of the cellular organelles by the virus. For this reason, looking at tegument and envelope viral proteins may not be optimal for this task since potentially on both compartments. However, viral capsid proteins or the viral genome may be useful here. Immuno-EM against VPS4A could also be a useful experiment to show a potential link between the ATPase and re-envelopment.

      It is well established that pUL71 is present at the cVAC of HCMV-infected cells. We apologise that we did not make this clearer in the introduction. We have now updated a sentence in the penultimate paragraph of the introduction to clarify this:

      “pUL71 and pUL103 are present at the cVAC (Ahlqvist and Mocarski, 2011; Dietz et al., 2018; Read et al., 2019; Womack and Shenk, 2010) and deficiencies in these proteins result in an accumulation of nucleocapsids at various advanced stages of envelopment (Ahlqvist and Mocarski, 2011; Schauflinger et al., 2013, 2011; Womack and Shenk, 2010), which is consistent with impaired envelopment and a block in membrane scission at the end of the envelopment process.”

      *Additionally, we have added confocal images of infected cells showing co-staining of pUL71, capsid associated tegument protein pp150, and Golgi maker GM130 in a new figure (Fig. 6A). *

      We are unaware of any commercial antibodies that recognise VPS4A and are suitable for immuno-EM, making such analysis unfeasible. However, we note that our study concurs with the data from Streck et al (2018) that VPS4 activity is not required for virus envelopment (although we do not rule out a contributory role).

      Another issue is the actual pUL71 residues interacting with VPS4A. While substantial efforts were made to map them (truncated constructs, bimolecular assay, viral mutants), the data do not always point toward the exact same residues (for example aa 314-320 by co-IF but aa 300-310 by ITC). This suggests potentially multiple binding sites or conformational issues. Hence, the statement on page 5 "that pUL71 residues 300-310 are necessary for the VPS4A interaction, in addition to the potential MIM2" may be misleading. What happens if one deleted aa 314-320 in the ITC assay? Or aa 300-310 by IF? These findings are further confounded by the lack of impact of the mutations of aa 315 and 318, predicted to be important in silico (p. 6). Moreover, in figure 7, the mutants made were a deletion 315-326 or the double point mutant P315A and P318A (not clear why in light of above results). Would a deletion of aa 300-320 not be a more appropriate and safer one to test for viral propagation?

      We are afraid that the reviewer may have misinterpreted several of our results. In Fig. 2 we demonstrate that residues 1–320 are sufficient for co-localisation of pUL71 with VPS4A-FLAG, but residues 1–314 are not. This implies that residues 314–320 are necessary for the interaction, but it is not evidence that they are sufficient. Similarly, our ITC data shows that a purified peptide spanning residues 300–325 is sufficient for the interaction, but a peptide spanning residues 310–325 is not. From this we can clearly infer that residues 300–310 are necessary for the interaction, as we state on page 5. We have expanded the sentence in question to further clarify our reasoning:

      “Further ITC analysis of pUL71 truncations purified as GST fusions (Fig. S1 and Table 1) demonstrated that pUL71 residues 300–310 are necessary for the VPS4A interaction, in addition to the potential MIM2, as GST-pUL71(300–325) was capable of binding VPS4A while GST-pUL71(310–336) was not.”

      We agree that residues 300–320 might be sufficient for the interaction, as indicated by the immunofluorescence analysis (Fig. 2A). However, out of an abundance of caution we included residues 300–325, spanning the entire MIM2-like sequence, in all of our biophysical analyses as the dynamic range of immunofluorescence experiments is limited and we wanted to avoid removal of residues that are not necessary but nonetheless contribute to the interaction. The similar affinity of GST-pUL71(283–361) and GST-pUL71(300–325) for VPS4A (2.8 and 2.3 µM, respectively) confirms that residues 300–325 contain all residues that contribute to the interaction (Figs 1 and S1, Table 1).

      With regards the mutational data presented in Fig. 4, our molecular dynamics analysis indicates (Fig. 4A–C) that single point mutations P315A and P318A do not disrupt the interaction between pUL71 and VPS4, only the double mutation (P315A+P318A; PPAA) disrupts the interaction. This is consistent with the immunoprecipitation presented in Fig. 4E: The pUL71(P315A) and pUL71(P318A) proteins are efficiently immunoprecipitated by VPS4A-FLAG, while the pUL71(PPAA) mutant is not. We have updated the penultimate sentence of the section “Model of the HCMV pUL71 in complex with VPS4A MIT” to explain this in more detail:

      “Immunoprecipitation of co-transfected pUL71 and VPS4A-FLAG confirmed this surprising result, showing that pUL71(P315A) and pUL71(P318A) are efficiently immunoprecipitated by VPS4A-FLAG whereas pUL71(PPAA) is not (Fig. 4E).”

      Regarding the choice of mutant viruses, we wanted to make the smallest change possible to pUL71 to avoid inadvertent removal of additional (potentially unknown) functional motifs. Both of the viruses we have used show an absence of VPS4A recruitment to the pUL71-positive cVAC in immunofluorescence (Fig. 6D) and, in the case of pUL71(PPAA), we have also shown an absence of VPS4A binding to this mutant in ITC (Fig. 1G) and coIP (Figs 1E and 4E). We feel this is sufficient evidence to confirm that these mutations either severely impair or completely abolish recruitment of VPS4A.

      Given the above, we don’t believe there is any need for additional experimentation or consideration of confounding variables when it comes to the definition of the vMIM2 motif or mutations introduced into HCMV for functional analysis.

      As the identification of the VPS4A binding motif in other herpesviruses appears to only be detected by manual inspection of the protein sequences, I wonder if other HCMV proteins or alpha/gamma viral proteins may interact with VPS4A. A good way to address this would be to do a VPS4A affinity column to see if any other viral proteins can bind. MS analyses may be required to identify the bound viral proteins. This could be a good follow-up paper...

      We thank the reviewer for suggestion and agree that it would form the basis for a good follow-up study.

      I am unfortunately unable to evaluate the outcome of the in silico analyses and cannot therefore judge their relevance or accuracy. Other reviewers can hopefully access this portion of the manuscript.

      Unless mistaken, previous work (Albecka A et al, 2017, JVI) has shown that HSV-1 pUL51 does not require its binding partner pUL7 to reach the TGN. Given that HSV-1 pUL51 does not seem to recruit VPS4A, could the pUL7/pUL51 complex be required for the recruitment of VPS4A to the TGN or VAC? Alternatively, could the lack of pUL51 binding to VPS4A reflect a different re-envelopment mechanism (absence of the CMV onion ring VAC)? These possibilities should be addressed in the manuscript.

      The reviewer is correct that, like pUL71, the HSV-1 protein pUL51 associates with TGN membranes as both proteins are N-terminally palmitoylated. Inspection of HSV-1 pUL7 does identify a potential vMIM2 sequence, spanning residues 221–232 (sequence LANnPpPVlsaL). However, these residues lie in a well-structured region at the interface with pUL51 (helices α8 and α9; see Fig. 2B of Butt et al (2020)) and would thus be unavailable to bind VPS4A. If the pUL7:pUL51 complex were required for VPS4A recruitment to sites of HSV-1 assembly, which has not been shown, then a different mechanism would be required. To test if this was the case, we performed a transfection experiment where pUL51-mCherry, or mTurquoise2-pUL7 +pUL51-mCherry, were co-transfected with GFP-VPS4A into U2-OS cells. As a positive control, we co-transfected pUL71-mCherry and GFP-VPS4A. As shown below, we observe recruitment of GFP-VPS4A to pUL71-mCherry positive membranes but do not see recruitment of GFP-VPS4A to pUL51-mCherry positive TGN membranes in the presence or absence of mTurquoise2-pUL7.

      This experiment has been performed twice with identical results. However, we have declined to include the above figure in the manuscript because our study focusses on the vMIM2 motif and the betaherpesviruses in which it is conserved. We already show that the pUL71 homologue in HSV (pUL51) does not recruit VPS4A to membranes (Fig. 5E). We believe that additional negative data on the lack of VPS4A recruitment by this HSV-1 complex complicates the story and would distract the reader. Identifying and characterising mechanisms via which other herpesvirus subfamilies may (or may not) specifically recruit VPS4A to sites of virus assembly is interesting, but it lies outside the scope of this current manuscript.

      We agree with the reviewer that the HCMV secondary envelopment pathway likely differs from that of HSV. For example, HSV envelopment is severely restricted by dominant negative VPS4 whereas HCMV is not. This indicates that, at a minimum, HCMV must have additional/redundant mechanisms that drive envelopment in the absence of a functioning ESCRT machinery. We have added a comment to this effect to the second paragraph of the discussion:

      “This lack of requirement for ESCRT activity during HCMV secondary envelopment contrasts with the situation for HSV-1, where expression of dominant-negative VPS4 (Calistri et al., 2007; Crump et al., 2007) or CHMP proteins (Calistri et al., 2007; Pawliczek and Crump, 2009) severely restricts virion production. We therefore conclude that either HCMV and HSV-1 utilise different molecular mechanisms for secondary envelopment, or HCMV can exploit additional (redundant) pathways in addition to ESCRT-mediated membrane remodelling to ensure assembly of mature virus particles.”

      Minor

      Fig 3S: I would suggest highlighting the central P residue in the aligned sequence and consensus sequence.

      We thank the reviewer for this helpful suggestion. We have highlighted both the central ‘P’ plus the other conserved hydrophobic residues of the vMIM2 in the aligned and consensus sequence in Figures 5, S3 and S4.

      Reviewer #2 (Significance (Required)):

      Not surprisingly, the biggest issue in the manuscript is that perturbing pUL71 / VPS4A binding has no detectable positive or negative impact on VAC assembly, secondary viral envelopment or viral spread (titre, plaque size). This raises the question as to the relevance of VPS4A for the virus. As mentioned above, it could be relevant to test a viral mutant lacking pUL71 aa 300-320, which may lead to different results.

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

      Summary

      In this study the authors investigate mechanisms by which human cytomegalovirus (HCMV) modulates ESCRT III to facilitate virus maturation. The viral protein pUL71 has been shown previously to be an important viral mediator of the so called "secondary envelopment", that is the process in which the viral capsid is budding into Golgi-derived membranes to acquire its envelope.

      pUL71 was previously shown to recruit VPS4 to the trans-Golgi upon co-transfection. In this study, the authors investigate the structural requirements for this interaction. Sequence comparison prompted the investigation of a short motif in the C-terminus of pUL71 with homology to the Type 2 MIT-Interacting Motif (MIM2) of CHMP6 that is known to bind the MIT of VPS4. Using co-localization, co-immunoprecipitation and isothermal titration calorimetry they show clearly that a peptide spanning amino acids 300-325 of pUL71 is required and sufficient for binding of VPS4A. State of the art modeling of the protein complex identifies the crucial amino acids that define the interaction on both sides. The authors validate these predictions experimentally in transfection and with purified peptides as well as in the context of HCMV infection using bimolecular fluorescence complementation. Furthermore, the authors demonstrate that not only the other human betaherpesviruses but also closely related CMVs of rat and mouse encode viral MIM2-like motifs (vMIM2)that are able to interact with VPS4A. Unexpectedly, albeit in line with previous reports, they find that mutation of this highly conserved vMIM2 domain did not alter viral progeny and focus size largely. The authors further confirm by quantification of high quality electron micrographs, that a large portion of capsids is able to complete the process of budding into and scission from cellular membranes, demonstrating that the ability of pUL71 to bind VPS4A is dispensable for secondary envelopment.

      Taken together, this study demonstrates clearly that a newly defined vMIM2 in the HCVM pUL71 protein binds cellular VPS4A. Yet, it remains unclear in which context the virus requires this novel form of molecular mimicry.

      While we thank the reviewer for summarising our study, highlighting the careful attention they paid to our work, we would like to emphasise that we are unaware of any previous study showing that pUL71 recruits VPS4 to the trans-Golgi upon co-transfection. __It had previously been observed that VPS4 is present at the cytoplasmic virus assembly compartment (cVAC) during infection – as we state in paragraph 4 of the introduction (first sentence). However, none of these studies identified the virus protein responsible for this recruitment nor did they identify the viral motif that mediated this recruitment. __Our identification of pUL71 as the HCMV protein that recruits VPS4A to the cVAC is novel.

      Major comments:

      1. Despite the very thorough analysis and conduction of the study, the presented work does not reveal a phenotype in virus infection. The authors would need to find out what the functional relevance of their discovery is.

      As described by reviewer 1, our data confirms with and extends previous studies to show that ESCRT activity seems not to be essential for HCMV secondary envelopment. This is different from other herpesviruses such as HSV-1, showing that the secondary envelopment process is not universally conserved across the herpesviruses (or that additional redundant processes are encoded by HCMV). We also identify a novel virus-encoded VPS4 recruitment motif, the vMIM2. The fact that this sequence is conserved across beta-herpesvirus pUL71 homologues strongly suggests that it has a conserved and important function in the virus lifecycle, even if we haven’t identified that function in this study. In the discussion we posit multiple hypotheses for how this motif may function during infection, including sequestration of VPS4. As reviewer 1 states, VPS4 sequestration “is an important issue that virologists should consider”. We feel that additional experiments to tease out the precise function conferred by this motif represent important future work but are beyond the scope of this current study.

      Please include statistical analysis of virus release, spread and envelopment (Figure 7 and Table 2). It would be helpful to see if the small differences observed are likely to be random or not.

      We thank the reviewer for this suggestion. We have performed relevant statistical tests for the virus growth data and virus spread (plaque size) assays.

      *A repeated measures two-way ANOVA test of the high MOI (single step) virus growth data shows that there is no significant difference between the viruses tested (P = 0.5824). A repeated measures two-way ANOVA test of the low MOI (multi-step) virus growth shows that there is a difference between viruses. A Dunnett’s multiple comparison test shows that there is no significant difference between the TB71revPPAA and wild-type virus at any time point. There are significant differences between the TB71mutPPAA virus and wild-type at 15 dpi (P “While two-way ANOVA analysis showed significant differences between wild-type and mutant virus yields at late time points in the multi-step growth curve, the TB71mutPPAA mutant had higher titres at 15 dpi whereas TB71del315-326 had lower titres at 15 and 18 dpi. Given the divergence in observed effect between the two mutants, and the fact that these differences were observed only at very late times post-infection, we do not believe they represent biologically meaningful differences in virus release.”

      A Mann Whitney test of the focus expansion assay data, performed instead of a t test because a D'Agostino & Pearson test showed the WT plaque data to not be normally distributed, showed no significant difference between the WT and TB71 mutPPAA virus (P = 0.489), which would agree with our notion that there is no in change in virus growth when interaction with VPS4A is disrupted.

      Concerning quantitative evaluation of secondary envelopment, we have respectfully declined to include statistical analysis in the manuscript. This is because quantitative analysis of virus envelopment via electron microscopy has multiple caveats that complicate robust statistical analysis. The numbers of virus particles in the area of the cVAC in the individual cells is subject to stark variation, only a small part of the cell or the cVAC is analysed, and naked capsids are only rarely observed. Defects in HCMV secondary envelopment, as has been published for pUL71 knock-out viruses, manifest as strong shifts in the proportions of the envelopment stages; see for example Schauflinger et al. (2013). We did perform a two-way ANOVA with Dunnett’s multiple comparison test, which shows that there are significantly fewer enveloped particles (P In summary, none of our data consistently and robustly show involvement of VPS4A for HCMV assembly that could explain the conservation of this interaction among betaherpesviruses, which is consistent with previous publications indicating that HCMV secondary envelopment does not require the cellular ESCRT machinery.*

      One caveat is that the presented study investigates the impact of VPS4A on HCMV only in fibroblasts. However, other studies used epithelial cells to investigate the impact of VPS4 knockout on HCMV and also did not see a reduction in virus titers. Yet, the authors could significantly improve the manuscript by testing for a cell type specific requirement of the vMIM2. The replication of the PPAA mutant virus could be analyzed in additional cell types such as macrophages or endothelial cells and using different experimental systems.

      *We thank the reviewer for this comment. We have evaluated viral growth of the PPAA mutant virus in monocyte-derived macrophages, similar to our analysis of a pp65 stop mutant (Chevillotte et al., JVirol 2009). We specifically tested macrophages as this cell type appears to restrict HCMV growth when compared to released virus yield from fibroblasts and endothelial cells. However, viral growth of the PPAA mutant is similar to that of parental and revertant virus, verifying our growth analysis in fibroblasts. Furthermore, we investigated virion morphogenesis of the PPAA mutant in macrophages by electron microscopy because pUL71 plays an important role in HCMV secondary envelopment. Consistent with our growth analysis, we could not find evidence for a role of VPS4 recruitment by pUL71 for virion morphogenesis. We have added this additional data as a new supplementary figure (Fig. S6). *

      Consider discussing if other viral and cellular proteins could compensate the loss of interaction between pUL71 and VPS4. Is a similar motif found in any other HCMV protein? Could redundancy explain the lack of a consequences for viral growth?

      The short answer is no, it is unlikely that any other HCMV protein could compensate for the loss of pUL71 binding and efficiently recruit VPS4A to the cVAC because we see a complete loss of VPS4A-FLAG recruitment to the cVAC when pUL71 is either absent (Fig. 6C) or has a defective vMIM2 (Fig. 6D). However, it is possible that additional HCMV proteins could interact with VPS4A, for example to enhance its retention at the cVAC by increasing the avidity of binding.

      We used the ScanProsite web server to identify additional proteins encoded by HCMV with the vMIM2 sequence [YLM]-{P}-{P}-x-P-x-[AVP]-[VP]-x-x-x-[LVP]. This sequence corresponds to the residues observed at each position in the vMIM2s of betaherpesvirus pUL71 homologues presented in Figures 5, S3 and S4, where proline is disallowed at the second and third position because of our identification that the first residues of the vMIM2 form an α-helix (proline residues being incompatible with α-helix formation).

      *We identified eight additional HCMV proteins with potential vMIM2 sequences: pUL31, pUL57, pUL72, pp28 (a.k.a. pUL99), pUL141, pUS22, pUS29 and pUS30. Of these, pUL141 could be immediately discounted because the vMIM2 sequence is located in an extracellular portion of the protein and thus would be incapable of binding the cytosolic VPS4A MIT domain. pUL31, pUL57 and pUS22 could similarly be discounted because inspection of AlphaFold2 models of these proteins (https://www.bosse-lab.org/herpesfolds/) reveal the potential vMIM2 sequences to lie within regions of the protein that are predicted to be well-ordered and are buried and/or form secondary structures incompatible with binding the VPS4A MIT domain. The vMIM2 motifs of the remaining four proteins were in regions of the proteins that lacked tertiary structure and were predicted with low confidence, indicating that these regions are likely to have little intrinsic structure in the absence of a binding partner. Additionally, we observed that the potential vMIM2 sequences of pUL72 and pUS29 were predicted to have a helix-then-extended conformation, like pUL71. *

      To probe whether the pUL72, pp28, pUS29 and pUS30 sequences that matched the vMIM2 consensus were likely to bind VPS4A, we used AlphaFold2 to predict structures of the relevant 26 amino acid regions from these proteins in complex with the VPS4A MIT domain. Analysis of the pLDDT scores show that the interaction between VPS4A and pUL72 is plausible, although this interaction is predicted with less confidence than the VPS4A:pUL71(300–325) interaction. The other models are predicted with very low confidence, suggesting that these regions are unlikely to interact. This agrees with our data for pp28, where we demonstrated using transient expression experiments that pUL71 but not pp28 could sequester VPS4A (Fig. 1B).

      Further inspection of the VPS4A:pUL72(potential vMIM2) prediction showed that several residues in the potential interacting region are predicted to contribute to the pUL72 folded domain, forming the final strand of a β-sheet. AlphaFold-Multimer prediction of a complex between the VPS4A MIT domain and full-length pUL72 failed to yield models where the potential vMIM2 interacted with VPS4A, suggesting that steric clashes between VPS4A and the globular domain of pUL72 would prevent pUL72 from binding VPS4A in cells.

      While it is theoretically possible that the potential vMIM2 motifs identified above could interact with VPS4A, the interaction is clearly not sufficient to effectively recruit VPS4A to the cVAC in the absence of pUL71 or in the presence of a pUL71 mutant with a defective vMIM2 (Fig. 6C,D). There are also several HCMV proteins that have ‘late domains’ and could in theory compensate for the absence of pUL71 via recruitment of ‘upstream’ ESCRT machinery components (Streck 2020), but these are similarly incapable of efficiently recruiting VPS4A in the absence of pUL71. It is possible that a small amount of residual VPS4 recruitment via late domain containing proteins could functionally compensate for the absence of the vMIM2 but, given the published evidence that VPS4 activity is dispensable for virus envelopment, it is more likely in our opinion that an alternative non-ESCRT mechanism drives HCMV envelopment.

      We have added a paragraph at the end of the results section “VPS4A binding is conserved amongst cytomegaloviruses and human β-herpesviruses” and a new supplemental figure (Fig. S5) describing the other potential vMIM2 sequences in HCMV. We have also added a section to the discussion where we describe our interpretation of these results, outlining our reasons for concluding that other HCMV sequences that match the vMIM2 consensus are very unlikely to play a role in envelopment (although we admit that we cannot entirely discount this hypothesis):

      “While other HCMV proteins have sequences that match the vMIM2 consensus, none are able to recruit VPS4A to the cVAC when pUL71 is absent (Fig. 6C) or has a defective vMIM2 (Fig. 6D). It is therefore unlikely that these sequences are functionally redundant to the pUL71 vMIM2, although we cannot formally discount this hypothesis.”

      We thank the reviewer for asking this interesting question.

      Would the small differences (if significant) in virus titer be sufficient to provide enough of an evolutionary advantage to explain the sequence conservation? It would be interesting to try an in vitro selection assay and test if wildtype would outcompete the PPAA mutant after some passages.

      This is an interesting suggestion, but it is not really supported by our data, as all our data indicate that sequestration of VPS4 by pUL71 has no growth advantage (see also answer to point 3). This is further supported by our results from electron microscopy. Even the minimal differences in growth are not significant at most time points. In addition, to our knowledge, there is no established assay for HCMV for the proposed analysis and therefore no reliable data regarding the significance.

      Albeit far beyond the original scope of the study:

      In the very thoughtful discussion, the option is discussed that other MIT domain containing proteins could be the actual targets of the pUL71 MIM2-domain. It would be interesting to use the generated expression constructs to identify other cellular targets by co-IP and mass spectrometry.

      We agree this would be an interesting avenue of future work and it is one we intend to pursue in the future. However, identification of novel binding partners for the vMIM2 and biochemical plus functional characterisation of these interactions is a large study and is thus outside the scope of this current manuscript.

      Minor comments: None, the presented experiments are well conducted and presented. The work is adequately discussed.

      Reviewer #3 (Significance (Required)):

      Significance section

      General assessment:

      The performed experiments are well described and the high quality is revealed by the abundant primary data shown. Multiple independent methods were used to investigate the central findings. The claims made are therefore well supported. Especially the data supporting the direct interaction between the MIM2-like domain and the MIT of VPS4 are excellent and unequivocally demonstrate a direct interaction. On the other hand, the lack of effect of this interaction in the context of viral infection questions the significance of the finding. Possibly, by testing additional cell lines to assess virus spread, the authors could increase relevance of the findings.

      Advance:

      The impact of VPS4 and the ESCRT machinery on HCMV secondary envelopment has been a matter of debate since a study by Tandon et al. in 2009 seemed to contradict the first publication on the topic by Fraile-Ramos et al in 2007. The current study by Butt et al. now supports a more recent report by Streck et al., which suggested that VPS4 is not required for secondary envelopment. The fact that the two studies use different experimental systems with similar outcome, suggests that virus maturation is indeed independent of VPS4. However, Streck et al. observe an effect of dominant negative ESCRT mutants on virus spread, suggesting that the interaction of the HCMV tegument with ESCRT is required only under special conditions, which still remain to be defined. Albeit the present study cannot fill all the gaps of our understanding of this topic, the high quality of the data is a good basis for further investigations.

      In addition, the description of a viral MIM2-like motifs might spur the investigation of similar motifs in other viruses, potentially bringing more cases of molecular mimicry to light.

      Audience:

      This study is of interest to basic researchers investigating aspects of modulation of cellular membranes by viruses or interested the cellular components and interactors of the ESCRT complexes.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary

      In this study the authors investigate mechanisms by which human cytomegalovirus (HCMV) modulates ESCRT III to facilitate virus maturation. The viral protein pUL71 has been shown previously to be an important viral mediator of the so called "secondary envelopment", that is the process in which the viral capsid is budding into Golgi-derived membranes to acquire its envelope.

      pUL71 was previously shown to recruit VPS4 to the trans-Golgi upon co-transfection. In this study, the authors investigate the structural requirements for this interaction. Sequence comparison prompted the investigation of a short motif in the C-terminus of pUL71 with homology to the Type 2 MIT-Interacting Motif (MIM2) of CHMP6 that is known to bind the MIT of VPS4. Using co-localization, co-immunoprecipitation and isothermal titration calorimetry they show clearly that a peptide spanning amino acids 300-325 of pUL71 is required and sufficient for binding of VPS4A. State of the art modeling of the protein complex identifies the crucial amino acids that define the interaction on both sides. The authors validate these predictions experimentally in transfection and with purified peptides as well as in the context of HCMV infection using bimolecular fluorescence complementation. Furthermore, the authors demonstrate that not only the other human betaherpesviruses but also closely related CMVs of rat and mouse encode viral MIM2-like motifs (vMIM2)that are able to interact with VPS4A. Unexpectedly, albeit in line with previous reports, they find that mutation of this highly conserved vMIM2 domain did not alter viral progeny and focus size largely. The authors further confirm by quantification of high quality electron micrographs, that a large portion of capsids is able to complete the process of budding into and scission from cellular membranes, demonstrating that the ability of pUL71 to bind VPS4A is dispensable for secondary envelopment. <br /> Taken together, this study demonstrates clearly that a newly defined vMIM2 in the HCVM pUL71 protein binds cellular VPS4A. Yet, it remains unclear in which context the virus requires this novel form of molecular mimicry.

      Major comments:

      1. Despite the very thorough analysis and conduction of the study, the presented work does not reveal a phenotype in virus infection. The authors would need to find out what the functional relevance of their discovery is.
      2. Please include statistical analysis of virus release, spread and envelopment (Figure 7 and Table 2). It would be helpful to see if the small differences observed are likely to be random or not.
      3. One caveat is that the presented study investigates the impact of VPS4A on HCMV only in fibroblasts. However, other studies used epithelial cells to investigate the impact of VPS4 knockout on HCMV and also did not see a reduction in virus titers. Yet, the authors could significantly improve the manuscript by testing for a cell type specific requirement of the vMIM2. The replication of the PPAA mutant virus could be analyzed in additional cell types such as macrophages or endothelial cells and using different experimental systems.
      4. Consider discussing if other viral and cellular proteins could compensate the loss of interaction between pUL71 and VPS4. Is a similar motif found in any other HCMV protein? Could redundancy explain the lack of a consequences for viral growth?
      5. Would the small differences (if significant) in virus titer be sufficient to provide enough of an evolutionary advantage to explain the sequence conservation? It would be interesting to try an in vitro selection assay and test if wildtype would outcompete the PPAA mutant after some passages.

      Albeit far beyond the original scope of the study: 6. In the very thoughtful discussion, the option is discussed that other MIT domain containing proteins could be the actual targets of the pUL71 MIM2-domain. It would be interesting to use the generated expression constructs to identify other cellular targets by co-IP and mass spectrometry.

      Minor comments: None, the presented experiments are well conducted and presented. The work is adequately discussed.

      Significance

      General assessment:

      The performed experiments are well described and the high quality is revealed by the abundant primary data shown. Multiple independent methods were used to investigate the central findings. The claims made are therefore well supported. Especially the data supporting the direct interaction between the MIM2-like domain and the MIT of VPS4 are excellent and unequivocally demonstrate a direct interaction. On the other hand, the lack of effect of this interaction in the context of viral infection questions the significance of the finding. Possibly, by testing additional cell lines to assess virus spread, the authors could increase relevance of the findings.

      Advance:

      The impact of VPS4 and the ESCRT machinery on HCMV secondary envelopment has been a matter of debate since a study by Tandon et al. in 2009 seemed to contradict the first publication on the topic by Fraile-Ramos et al in 2007. The current study by Butt et al. now supports a more recent report by Streck et al., which suggested that VPS4 is not required for secondary envelopment. The fact that the two studies use different experimental systems with similar outcome, suggests that virus maturation is indeed independent of VPS4. However, Streck et al. observe an effect of dominant negative ESCRT mutants on virus spread, suggesting that the interaction of the HCMV tegument with ESCRT is required only under special conditions, which still remain to be defined. Albeit the present study cannot fill all the gaps of our understanding of this topic, the high quality of the data is a good basis for further investigations.<br /> In addition, the description of a viral MIM2-like motifs might spur the investigation of similar motifs in other viruses, potentially bringing more cases of molecular mimicry to light.

      Audience:

      This study is of interest to basic researchers investigating aspects of modulation of cellular membranes by viruses or interested the cellular components and interactors of the ESCRT complexes.

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      Referee #2

      Evidence, reproducibility and clarity

      Summary

      The manuscript from Butt et al. entitled "Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly" addresses the potential role of ESCRT components in the biogenesis of HCMV virions. The topic is relevant since the ESCRT machinery has been implicated in the propagation of various herpesviruses. However, conflicting results are present in the literature regarding its role and relevance. In the present study, the authors focus on HCMV pUL71, which plays a role during the final envelopment of the viral capsids, and explore the possibility that it acts as an ESCRT-III component by recruiting the VPS4A ATPase (which induces membrane deformation and scission). To this end, they identified a motif in the C-terminal region of HCMV pUL71 that resembles the cellular type 2 MIM (MIM2) consensus sequence that is present in ESCRT-III proteins such as CHMP4B and CHMP6. They show substantial data that delineate an interaction between that pUL71 motif and the cellular ATPase using a panoply of tools (co-IF, co-IP, ITC, bimolecular fluorescence and markerless BAC mutagenesis). They also show that this interaction in present across a wide range of HCMV strains, but is absent in the case of the HSV-1 pUL51 homolog.

      Main Comments

      The manuscript is very well written, albeit line numbers would facilitate reviewing. The plethora of assays used convincingly show an interaction between pUL71 and VPS4A. They also indicate that this interaction relocalizes VPS4A to the TGN and likely the VAC. However, I do have some issues. For instance, using another viral marker, instead of only pUL71, would have been a good idea to distinguish the TGN from the VAC. This is not trivial given the reorganization of the cellular organelles by the virus. For this reason, looking at tegument and envelope viral proteins may not be optimal for this task since potentially on both compartments. However, viral capsid proteins or the viral genome may be useful here. Immuno-EM against VPS4A could also be a useful experiment to show a potential link between the ATPase and re-envelopment.

      Another issue is the actual pUL71 residues interacting with VPS4A. While substantial efforts were made to map them (truncated constructs, bimolecular assay, viral mutants), the data do not always point toward the exact same residues (for example aa 314-320 by co-IF but aa 300-310 by ITC). This suggests potentially multiple binding sites or conformational issues. Hence, the statement on page 5 "that pUL71 residues 300-310 are necessary for the VPS4A interaction, in addition to the potential MIM2" may be misleading. What happens if one deleted aa 314-320 in the ITC assay? Or aa 300-310 by IF? These findings are further confounded by the lack of impact of the mutations of aa 315 and 318, predicted to be important in silico (p. 6). Moreover, in figure 7, the mutants made were a deletion 315-326 or the double point mutant P315A and P318A (not clear why in light of above results). Would a deletion of aa 300-320 not be a more appropriate and safer one to test for viral propagation?

      As the identification of the VPS4A binding motif in other herpesviruses appears to only be detected by manual inspection of the protein sequences, I wonder if other HCMV proteins or alpha/gamma viral proteins may interact with VPS4A. A good way to address this would be to do a VPS4A affinity column to see if any other viral proteins can bind. MS analyses may be required to identify the bound viral proteins. This could be a good follow-up paper...

      I am unfortunately unable to evaluate the outcome of the in silico analyses and cannot therefore judge their relevance or accuracy. Other reviewers can hopefully access this portion of the manuscript.

      Unless mistaken, previous work (Albecka A et al, 2017, JVI) has shown that HSV-1 pUL51 does not require its binding partner pUL7 to reach the TGN. Given that HSV-1 pUL51 does not seem to recruit VPS4A, could the pUL7/pUL51 complex be required for the recruitment of VPS4A to the TGN or VAC? Alternatively, could the lack of pUL51 binding to VPS4A reflect a different re-envelopment mechanism (absence of the CMV onion ring VAC)? These possibilities should be addressed in the manuscript.

      Minor

      Fig 3S: I would suggest highlighting the central P residue in the aligned sequence and consensus sequence.

      Significance

      Not surprisingly, the biggest issue in the manuscript is that perturbing pUL71 / VPS4A binding has no detectable positive or negative impact on VAC assembly, secondary viral envelopment or viral spread (titre, plaque size). This raises the question as to the relevance of VPS4A for the virus. As mentioned above, it could be relevant to test a viral mutant lacking pUL71 aa 300-320, which may lead to different results.

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      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      In this study the authors apply a rigorous and thorough combination of approaches including sequence analysis, deep-learning structure predictions, molecular dynamics, cell imaging and mutagenic analyses to identify a short MIM2-mimicking motif in the C-terminal region of the pUL71 protein of HCMV (and homologues in other beta-herpesviruses) that is necessary and sufficient for interaction with the ESCRT terminal ATPase VPS4A. pUL71 uses this motif to recruit, or sequester, VPS4A to the HCMV cytoplasmic viral assembly complex, though this process is dispensable for HCMV morphogenesis or replication. The identified pUL71 sequence functions as a mimic of the MIM2 motif of cellular CHMP subunits since, like MIM2, it directly binds the groove in the MIT domain found at the N-terminus of VPS4.

      Major comments:

      1. There appears to be some confusion in the coip experiment in Figure 5D. From the upper blot in 5D, the "+" above each lane suggests there should be VPS4A-FLAG protein in every sample other than the two lanes at the very left of the gel, however the anti-FLAG ip does not pull down VPS4A-FLAG from every "+" lane, but from alternating ones (and from the next to the leftmost lane, which should lack VPS4A-FLAG). Similarly, the lower "Input" blot shows VPS4A-FLAG present in alternating lanes across the blot, which does not match the "+" and "-" labeling at the top of the figure. Conversely, there is anti-HA signal in most input lanes (lower blot) though the HA-tagged pUL71 homologues should be absent from alternate lanes (top of upper blot).
      2. The Discussion is an excellent, comprehensive and scholarly assessment of the implications of this work. One appealing hypothesis is that pUL71 may be sequestering VPS4A rather than using it for envelope scission. In this regard, the authors point out that VPS4A sequestration is supported by the finding that the VPS4A MIT domain binds the isolated pUL71 vMIM2 more tightly (~ 5 fold lower Kd) than the MIM2 of CHMP6, and that pUL71 and homologues) are highly abundant at later stages of viral infection, allowing them to compete effectively with endogenous CHMP6 for VPS4A. I like the sequestration model very much, but could the authors comment on the fact that this apparent sequestration is seen even in the transfection experiments in Fig. 2A and 3G, where essentially 100% of transfected WT VPS4A-FLAG is recruited to the pUL71 compartment. Even given the increased binding affinity to pUL71, this suggests that in these transfection studies pUL71 must be in excess over the sum of both endogenous and transfected VPS4. Do the authors know if this is the case, and do cells transfected with pUL71 in these experiments exhibit any cytotoxicity, or cell cycle arrest, indicative of a block in normal ESCRT function/cytokinesis?

      Minor comments:

      In general, the text and figures are very clear and accurate, the Results section is careful to walk the reader though these studies in a clear and well written fashion and prior studies are referenced appropriately. There are some minor issues that are listed below.

      i). For clarity, please direct the reader to panel 1B when referring to the pp28 data (line 11 of Results section).

      ii). At the bottom of the page 4, the Results section states "immunoprecipitation experiments show VPS4A-FLAG to be robustly co-precipitated by wild-type pUL71 but not by the PPAA and V317D mutants". However, from Fig. 1E it appears to be the reverse. The wild type pUL71 (but not mutants) is being co-precipitated by VPS4A-FLAG, using an anti-FLAG antibody.

      iii). In Fig.1D the localization of WT pUL71 and the PPAA and V317D mutants to a juxtanuclear compartment provides a nice internal control demonstrating that the mutant proteins are at least partially functional (able to localize correctly), and the fluorescence intensities of the WT and mutant pUL71 proteins appear comparable. However, do the authors have any additional quantitative or semi-quantitative data (such as from a Western) to confirm similar expression levels for the pUL71 WT and PPAA/V317D mutant proteins?

      iv). In Fig. 4, An OPTIONAL experiment, which would add to the paper, would be to test the ability (or rather, lack of the ability) of the pUL71 I307R mutant to coip VPS4A from infected or transfected cells. Such a study would extend the predictive power of the elegant MD simulations and ITC studies to the "gold standard" of testing the phenotype in vivo.

      v). The Fig. 6B TB71stop pp28 panel is not referred to in the Fig. 6 legend.

      vi). In the second paragraph of the Discussion it is stated that "The pUL71 vMIM2 is necessary and sufficient to recruit VPS4A to specific membranes in co-transfected cells (Fig. 5) and to sites of virus assembly in HCMV infection (Fig. 6)". Strictly speaking, Fig. 5 (panel 5F) shows that the HCMV pUL71 region 283-361 is sufficient to localize VPS4A to a compact juxtanuclear structure in transfected cells, and Fig. 6 (panel 6C) shows that pUL71 residues 315-326 (and the two conserved prolines in this region) are necessary for VPS4A localization to a structure that appears to be the HCMV assembly compartment.

      Significance

      This is the first report of a virus encoding a MIM-like domain, and of a viral motif that directly binds the VPS4A MIT domain. This will be of broad interest to those studying the cell biology of virus assembly and mechanisms of virus-host cell interaction, as well as to cell biologists and structural biologists studying the ESCRT apparatus. It is striking, and will be illuminating to virologists and ESCRT biologists, that viruses have evolved to mimic MIM2 with a motif that has a lower Kd than a conventional cellular MIM2 motif. The possibility, addressed in the Discussion, that pUL71 may be sequestering VPS4A (rather than using it) is an important issue that virologists should consider.

      This is a rigorous, thorough and well controlled basic science study that elegantly combines a variety of approaches to provide important new insights concerning the biology of pUL71 in HCMV, other human beta-herpesviruses and a large number of mammalian and rodent cytomegaloviruses. The claims and conclusions are thoughtful and measured, and supported by the data. Data and methods are presented in such a way that they can be reproduced, and experiments are adequately replicated with appropriate statistical analyses.

      Reviewers fields of interest: Cell biology, ESCRT function, Virus assembly

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      Reply to the reviewers

      We thank the reviewers for their valuable comments that we have followed to highly improve our manuscript.

      REVIEWER 1

      Major Comments:

      While the evidence presented supports the application of machine learning in predicting RNA editing events, the paper falls short in justifying its significance within the scope of RNA editing in non-coding regions and Alu repeats, which are typically characterized by low conservation. The paper should provide a more compelling rationale for the method's necessity and potential uses. While it is true that the databases used in mouse and human, as well as the procedures used for the obtention of the mackerel RNA-editing data are rich in Alu repeats and non-coding regions, that is not our focus. We gathered all the available A-to-I editing sites and feed them to our algorithms without distinction. In addition, we are not looking for conservation of the sites themselves yet, but if there is a conservation of the mechanism. This is attempted by assessing the ability of the algorithm trained in one species to predict the editing sites in a different species, a.k.a. cross-training. We already state this in the introduction but we have added an extra sentence in the last paragraph of the introduction.

      A significant limitation of this study is the lack of a thorough comparison with existing methodologies and traditional statistical approaches. Incorporating such analyses would substantially strengthen the validity of the findings.

      We would like the reviewer for pointing this limitation. We have updated the manuscript with a new table in results, and a new discussion segment.

      The descriptions of the machine learning algorithms are insufficiently detailed for replication or thorough comparison. A more comprehensive explanation of the algorithms' parameters and configurations is critical.

      While the main manuscript methods section is short to avoid it to over encumber the manuscript, there is a whole extended methods section with step-by-step instructions to replicate the results, as well as full documentation available in the github at https://github.com/cherrera1990/RNA-editing-pred.

      1. The paper lacks detailed analysis of the prediction accuracy, particularly concerning non-human data and the implications of false positives in unbalanced datasets. A more nuanced interpretation is essential for a comprehensive understanding.

      We have added two discussion segments to address this point. We thank the reviewer for notice this and help us to improve our manuscript.

      The discussion on the evolutionary conservation of RNA editing needs to more explicitly highlight potential practical applications and future research directions. The current treatment of this topic does not offer clear actionable insights.

      While true, we believe that what the reviewer suggests is not the main scope of the paper. We have added and extra sentence at the end to suggests possible doors this work can open.

      Minor Comments:

      The manuscript is marred by grammatical errors and awkward phrasing, including unnecessary references to historical figures like Charles Darwin. A thorough editing and proofreading process would greatly enhance readability. We removed the Charles Darwin reference and proofread the manuscript to correct grammatical errors.

      1. The justification for the selection of statistical tests is unclear, and a more detailed explanation of their relevance to the study's findings would improve the paper's analytical rigor. Incorporating descriptions of the statistical descriptors directly into the main text would remedy this issue.

      We don't exactly know to what the reviewer means with this point. The descriptors used for the random forest are thoroughly described in the extended methods. Besides the tests used for assessing prediction accuracies which are listed in the extended methods section as well as in github, we don't use any other statistical analysis. Nonetheless, we have improved the general methods with an extra paragraph for RF and added reminders of the availability of the extended methods.

      REVIEWER 2

      The main problem of this study is its dependence on computationally predicted RNA secondary structures. To date, algorithms for inferring the secondary structures of polynucleotide chains are affected by considerable errors in several cases. Therefore, there is a high probability that at least part of the training data is largely biased. In this sense it would be appropriate to correlate the performance of the model to that of linearfold used to obtain the secondary structure data. While this is completely true for the RF algorithm and probably the cause of the low accuracy achieved, compared with other methods, that is not the case for the biLSTM algorithm. As we can see in Figure 3 A and Figure 3 B (and Supp. Figure 8 A and 8 B), the accuracy obtained using sequence alone is almost identical to the one obtained using both channels, while the accuracy obtained using just secondary structure is noticeably lower. This most probably means that the biLSTM algorithm is just ignoring the secondary structure channel, so no bias is being introduced in the training dataset.

      Furthermore, it is known that bi-LSTMs trained on large datasets tend to be affected by catastrophic forgetting, therefore it should be evaluated to what extent the performances can be improved by expanding the dataset.

      While true, this can be deal with an attention layer such as the one we use. In addition, we can see (Supp. Figure 5) how the mackerel prediction accuracy decrease when we reduce the database size. This can be marginally observed in human as well.

      It is also notable an inconsistency between the performance summary table and the confusion matrices to which it refers.

      We have corrected Figure 6 showing the proper percentages (the confusion matrices were correct) as well as reordered Supp. Figure 3 in order to be more similar to the Table 2.

      In the end the 3' enrichment of guanosines, which is the typical of the consensus recognized by the ADARs, does not appear to emerge from the sequence logo relating to the training data.

      We did notice this, and while we had already a small comment in the discussion, we expanded it further.

      Point-by-point description of the revisions

      __ Figures and Tables__ - Figure 6 has been corrected with the proper accuracies.

      • Supp. Figure 3 has been reordered to mirror the Table 2 design.

      • Table 1 has been renamed to Table 2.

      • A new table has been added as Table 1 with other analysis of RNA-editing predictions by machine learning.


      __ Introduction__ - Charles Darwin reference has been removed (L11).

      • "independently of the conservation of editing sites" added to last paragraph (L117).

      __ Results__ - New section "Benchmarking the algorithms with previous RNA-editing prediction attempts based on machine learning" added including a new table as Table 1 (L170-178).

      __ Discussion__ - "Random forest" section expanded at the end (L254-258).

      • "biLSTM algorithm" section expanded at the end of paragraph 1 and paragraph 2 (L274-280; L289-295).

      • "Differences in accuracy between human and non-human data" section expanded at the end (L313-316).

      • Additional sentence added at the end of "Cross-training and mechanism conservation" section (L353-355).

      __ Methods__ __- __Reminders of availability of extended methods added at the end of "Origin of the RNA-editing and genomic data", "General pipeline for constructing the Random Forest and Neural networks datasets", and "biLSTM" sections (L375; L390; L429-430).

      • Extra paragraph added for "RF" section (L408-413).

      __ Proofreading and correction of typos__

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      Referee #2

      Evidence, reproducibility and clarity

      This study describes Deep Learning applications aimed at identifying edited sites in different organisms. The method is able, starting from the knowledge of the transcriptome of one organism, to predict RNA editing in another, exploiting the functional conservation of ADAR enzymes throughout the animal kingdom. This study concludes that this approach, within certain limits, is a feasible option and worthy of further development.

      The main problem of this study is its dependence on computationally predicted RNA secondary structures. To date, algorithms for inferring the secondary structures of polynucleotide chains are affected by considerable errors in several cases. Therefore there is a high probability that at least part of the training data is largely biased. In this sense it would be appropriate to correlate the performance of the model to that of linearfold used to obtain the secondary structure data. Furthermore, it is known that bi-LSTMs trained on large datasets tend to be affected by catastrophic forgetting, therefore it should be evaluated to what extent the performances can be improved by expanding the dataset. It is also notable an inconsistency between the performance summary table and the confusion matrices to which it refers. In the end the 3' enrichment of guanosines, which is the typical of the consensus recognized by the ADARs, does not appear to emerge from the sequence logo relating to the training data.

      Significance

      Advance: compare the study to existing published knowledge: does it fil a gap? what kind of advance does it make (conceptual, fundamental, methodological, incremental, ...) The study, although the critical remarks addressed above represents a conceptual advancement

      Audience: which communities will be interested in/influenced, what kind of audience (broad, specialised, clinical, basic research, applied sciences, fields and subfields, ...) This contributions targets a specialised audience even if the potential applications are broad

      Describe your expertise

      Comparative Genomics and Bioinformatics

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      Referee #1

      Evidence, reproducibility and clarity

      Summary: This manuscript presents an approach for assessing the conservation of RNA editing, with a particular focus on non-coding regions and Alu repeats, using machine learning techniques. The goal is to forecast RNA editing occurrences and their evolutionary conservation across different species. However, the paper does not convincingly argue for the importance or the necessity of this method, especially considering the anticipated low conservation levels in the targeted regions.

      Major Comments:

      1. While the evidence presented supports the application of machine learning in predicting RNA editing events, the paper falls short in justifying its significance within the scope of RNA editing in non-coding regions and Alu repeats, which are typically characterized by low conservation. The paper should provide a more compelling rationale for the method's necessity and potential uses.
      2. A significant limitation of this study is the lack of a thorough comparison with existing methodologies and traditional statistical approaches. Incorporating such analyses would substantially strengthen the validity of the findings.
      3. The descriptions of the machine learning algorithms are insufficiently detailed for replication or thorough comparison. A more comprehensive explanation of the algorithms' parameters and configurations is critical.
      4. The paper lacks detailed analysis of the prediction accuracy, particularly concerning non-human data and the implications of false positives in unbalanced datasets. A more nuanced interpretation is essential for a comprehensive understanding.
      5. The discussion on the evolutionary conservation of RNA editing needs to more explicitly highlight potential practical applications and future research directions. The current treatment of this topic does not offer clear actionable insights.

      Minor Comments:

      1. The manuscript is marred by grammatical errors and awkward phrasing, including unnecessary references to historical figures like Charles Darwin. A thorough editing and proofreading process would greatly enhance readability.
      2. The justification for the selection of statistical tests is unclear, and a more detailed explanation of their relevance to the study's findings would improve the paper's analytical rigor. Incorporating descriptions of the statistical descriptors directly into the main text would remedy this issue.

      Significance

      Summary: The manuscript introduces a method to explore the functional conservation of RNA editing. However, it does not adequately justify its significance or practical applicability, particularly in the context of non-coding regions characterized by low conservation. The lack of comparative analysis with existing methods and detailed machine learning methodology explanations detracts from its potential impact. Addressing these issues would greatly enhance the paper's contribution to the scientific community.

      General Assessment: The cornerstone of this study is its approach towards the prediction and evolutionary conservation analysis of RNA-editing events using machine learning techniques. Despite these technical achievements, the study falls short in adequately highlighting the biological significance of RNA editing within non-coding regions and Alu repeats. Additionally, the absence of a comprehensive comparative analysis with pre-existing methods and the lack of detailed algorithmic descriptions somewhat diminish the study's potential influence and applicability in the wider scientific domain. Moreover, there are grammatical errors and awkward phrasings that disrupt the flow of the text (e.g. why are we talking about Charles Darwin?) Please just focus on the method and RNA editing improve the overall readability of the paper!

      Advance: The research notably progresses the field of genomics by harnessing machine learning to investigate RNA editing prediction and conservation, a subject not thoroughly examined in existing literature. Its innovative utilization of advanced computational models sets a new precedent, offering fresh perspectives on the mechanisms of RNA editing and their evolutionary contexts. This study enriches our understanding of genomics by illustrating the applicability of machine learning in unraveling the complexities of biological phenomena, such as RNA editing, thereby expanding the frontier of knowledge in both theoretical and practical aspects of genomics research.

      Audience: A niche audience comprising bioinformatics experts focused on RNA editing, computational biology, and evolutionary genetics.

      My proficiency centers on human genomics, RNA editing biology, and computational methodologies.

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      Reply to the reviewers

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

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      Referee #4

      Evidence, reproducibility and clarity

      Summary:

      In this work, Zemlianski and colleagues exploit S. pombe mutations responsible for catastrophic mitoses, in particular those leading to a cut / cut-like phenotypes, whereby cytokinesis takes place without proper DNA segregation, trapping DNA molecules by septum formation in between the two separating cells. The work builds on the team's previous observation that these defects can be alleviated when cells are grown in a nitrogen-rich medium, and motivate their efforts to understand this better. The manuscript is written in a concise, neat and informative manner, and the results are presented clearly, with consistence in the format and the style all along. The analyses appear to have been, in general, conducted under the best standards. The findings are important and the data are of good quality. I have, however, important concerns that will be detailed below, and which, as I hope will be made clear, question the pertinence of including "TOR signaling" in the title, and making a distinction between "good" and "poor" nitrogen sources in the abstract.

      Major comments:

      Results

      The conclusion that the phenotype is suppressed by "good" but not "poor" nitrogen sources is not sufficiently supported. First, this interpretation is based on comparing only two or three sources of each type; Second, the "good" source glutamate needed to be raised for it to have a significant effect; 3) there is a strange datum, as Glu 100 mM in Graph 1D looks exactly the same as Glu 50 mM in Graph 1E, I guess there is a mistake in the plotting; 4) and, more important, the fact that the authors had the nice initiative of reproducing their YES medium experiments for every graph led to the inevitable fact that slightly different values were obtained every time, which is normal. While the values yield very similar data for panels 1B, 1C and even 1D, the frequency of catastrophic mitoses for the cbf11 mutant in YES in panel 1E is much lower than in panel Figure 1B, for example. This has the consequence of making the suppression obtained when adding 'poor' sources, such as proline or uracil, non-significantly different. Thus, the authors conclude that 'poor' nitrogen sources are not good at suppressing the phenotype. I suggest that the authors pool all their YES data (they will have 12 repeats of their experiment) and plot, in a single graph, all the other treatments. By performing the analyses again, using the appropriate statistical test for that, perhaps they will have a surprise. After which, the question is, is it so important to put the emphasis on whether the source is good or poor? The incontestable observation is that, in general, there is clear trend of suppression of the phenotype.

      In Figure 2, images should be shown as an example of what was seen, what was quantified, how the "decrease in nuclear cross-section area" looked like indeed.

      Also, important for Figure 2, the authors used the nuclear cross-section area as a readout for nuclear envelope expansion versus shrinkage. For that, they did not use a fluorescent marker for the nuclear envelope that is continuous, but a nucleoporin (Cut11-GFP). In my experience, nucleoporins being discontinuously distributed throughout the nuclear envelope, the area encompassed by the signal may be underestimated in the event of a strong nuclear envelope deformation, as I have tried to illustrate in the scheme below: I WILL SEND THE SCHEME BY MAIL TO THE EDITOR, AS I CANNOT COPY-PASTE IT IN THE SYSTEM BOX Given that the photos from which the data were retrieved have not been shown, I cannot at present judge whether the use of a nuclear envelope marker providing continuous signals is absolutely necessary or not, and whether this consideration will affect (or not at all) the conclusions.

      The authors do not seem to comment or pay any attention to a very crucial result they obtain: the addition of ammonium to the WT strain has the effect of also restricting the nuclear cross-section area. They indeed say in their text "we did not observe any differences between cultures grown with or without ammonium supplementation (Fig.2)". I guess they refer here to the cbf11 mutant, in which case the sentence is true (although unfair to the WT). But by neglecting that the supplementation with ammonium had the power of reducing the cross-section area of WT nuclei, they are misled (or misleading) in their interpretation. The same, although milder, is true for Figure 5C, where the addition of ammonium to the WT culture does not alter the median value of prophase + metaphase duration, however has the virtue of very much rendering sharp (less scattered) the population of values, suggesting that the accuracy / control of the process is enhanced. What does this mean? I think it should be carefully thought about and considered as a whole.

      In the same line as above, the authors omit the RNA-seq analysis concerning the treatment of the WT with ammonium (Figure 3). This is very important to understand the standpoint of what this treatment elicits. It would also help unravel the observations I mentioned above that the authors did not assess in their descriptions. Also regarding Figure 3, it is completely obscure why the authors decided to show the genes on the right axis, and not others. Knowing how vast the lipid pathways are, there are likely many other hits that could be relevant. A particular thought goes for the proteins in charge of filling lipid droplets, such as sterol- and fatty acid-esterifying enzymes. Unless a very justified reason is provided, the choice at present seems arbitrary and it would be better to show a more unbiased data representation.

      In the same vein, related to the effect of ammonium onto the WT, in Figure S1 (I want to congratulate the authors for showing their 3 experimental replicates), the results very neatly show that ammonium supplementation to the WT leads to a neat and reproducible increase in TAG, a fact on which the authors do not comment. In the mutant, irrespective of ammonium presence or absence, a huge increase in squalene and steryl esters (SE) are seen. I think the work would benefit from actually quantifying the intensity of these bands and thus materializing this in the form of values. TAG, squalene and SE are all neutral lipids, and are all stored within LD to prevent lipotoxicity if accumulated in the endoplasmic reticulum. While ammonium elicits strong TAG accumulation in the WT, this is not the case in the mutant, likely because the massive occupation of LD storage capacity is overwhelmed with squalene and SE. Could this have something to do with the suppression they are studying?

      In the section of results where the authors comment the TLC analysis, they write "suggesting failed coordination between sterol and TAG lipid metabolism pathway". As it stands, the sentence is rather devoid of real meaning and may be even misleading, when considering what I wrote before.

      My biggest concern has to do with the very last part, when they explore the implications of TOR:

      • First, all the data presented in the two concerned panels of Figure 7 (B and C) and of Figure S3 lack the values obtained for the single mutants with which cbf11 was combined. This is not acceptable from a genetic point of view, and may prevent us from having important information. For example: if the authors were right that Tor2/TORC1 is ensuring successful progression through closed mitosis (last sentence of results), then one would predict that the tor2-S allele leads to an increase, already per se, of the frequency of catastrophic mitoses. However, at present, I cannot check that.
      • the authors turn to use a ∆ssp2 mutant to "increase Tor2 activity". However, this is a pleiotropic strategy, as AMP-kinase is the major sensor and responder to energy depletion, frequently triggered by glucose shortage, thus I am not sure the effects associated to its absence can be unequivocally be ascribed to a Tor2 raise.
      • there is a counterintuitive observation: rapamycin, which mimics nitrogen shortage, has the same effect than ammonium supplementation. This is strangely bypassed in the discussion, where the authors wrote "we showed increased mitotic fidelity in cbf11 cells when the stress-response branch of the TOR network was suppressed, either by ablation of Tor1/TORC2 or by boosting the activity of the pro-growth Tor2/TORC1 branch. These data are in agreement with previous findings that Tor2/TORC1 inhibition mimics nitrogen starvation".
      • last, and irrespective of what was said above, the authors conclude that the phenotype suppression is due to "a role for Tor2/TORC1 in ensuring successful progression through mitosis". If, as stated by the authors, Tor1/TORC2 absence not only abrogates Tor1/TORC2 activity, but it simultaneously raises Tor2/TORC1 activity, and if reciprocally Tor2/TORC1 increased activity concurs with Tor1/TORC2 attenuation, it cannot therefore be discerned if the suppression is due to Tor2/TORC1 raise or to Tor1/TORC2 dampening.

      Discussion

      The authors invoke that TOR controls lipin, despite what they go on to dismiss the link between TOR and lipids by saying "we did not observe any major changes in phospholipid composition when cells were grown in ammonium-supplemented YES medium compared to plain YES (Figure S2)", with this reinforcing their conclusion that ammonium does not suppress lipid-related cut mutants through directly correcting lipid metabolism defects. While I agree with that reasoning, I invoke again that they nevertheless neglected the clear change observed in their three replicates (Figure S2) that ammonium addition to WT cells strongly increases the amount of TAG (esterified fatty acids). Since lipin activity promotes DAG formation, which then leads to TAG accumulation, this aspect should not be neglected.

      The emphasis on TOR, which expands several paragraphs of the Discussion, should be revisited if the evidence provided for this part of the data is not reinforced.

      To finish, if I may provide some personal thoughts that may be useful for the authors, I would first remind that TAG storage prevents the channeling of phosphatidic acid towards novel phospholipid synthesis thus antagonizes NE expansion, which agrees with their neglected observation for the WT in Figure 2A. The antagonization of NE expansion can be achieved through autophagy (DOI 10.1038/s41467-023-39172-3; DOI 10.1177/25152564231157706), and indeed rapamycin addition (a very potent inducer of autophagy) also suppressed the cut phenotype (Figure 7A). What is more, in S. cerevisiae, autophagy has been shown as important to transition through mitosis conveniently and to prevent mitotic aberrations (DOI 10.1371/journal.pgen.1003245), and to impose a "genome instability" intolerance threshold by restricting NE expansion (DOI 10.1177/25152564231157706). In the first mentioned work, the authors proposed that autophagy may help raising aminoacid levels, which could assist cell cycle progression. This would have the virtue of reconciling the otherwise counterintuitive observation of the authors that rapamycin, which mimics nitrogen shortage, has the same effect than ammonium supplementation. It could be that ammonium supplementation mimics the downstream signal of a complex cascade initiated by actual aminoacid shortage, known to elicit autophagy-like processes (thus explaining why TAG raise, why the NE does not expand), and may culminate with launching a program for more accurate mitosis and genome segregation. In further support, TORC1 inhibition (as elicited by +rapamycin) is a central node that integrates multiple cues, not only nitrogen availability, but also carbon shortage (DOI 10.1016/j.molcel.2017.05.027), and even genetic instability cues (DOI 10.1016/j.celrep.2014.08.053), perhaps helping unravel why ammonium (via TOR) suppresses very diverse cut mutants, irrespective of whether they stem from lipid or chromatid cohesion deficiencies. These previous works should be considered by the authors.

      Minor

      There was no speculation about why the suppressions are partial.

      Reference 15, cited in the text, is absent from the references list.

      An explanation of which statistical tests were chosen and why they were chosen would be necessary.

      In particular, for the analyses performed for Figure 5, one-way ANOVA should be applied instead of several t-tests.

      A small section in M&M about how data in general was acquired, quantified, plotted and analyzed would be appropriate.

      In the discussion, the sentence "this could mean that the signaling of availability of a good nitrogen source is by itself more important for mitotic fidelity than the actual physical presence of the nutrients" is a rather void sentence. Because, from the point of view of how a cell "works", the signal is important for the basic reason that it is supposed to represent the actual real cue eliciting it.

      In the second part of Results, when the phenotype of cbf11 mutants concerning LD is mentioned, the authors said "aberrant LD content". It would be good if they can mention already at this stage which type of aberration this was: more LD? less LD? bigger? smaller?

      What is the difference between the two SE bands in Figure S2? What exactly does SE-1 and SE-2 mean?

      In Figure 2, the two graphs, presented side by side, would be more easily comparable if they could be plotted with the same y-axis scale.

      In Figure 1A, it would be useful for non-specialists of this phenotype and non-pombe readers to show a control of how it looks to be "normal".

      Referees cross-commenting

      Overall, there is a striking consensus on the need to either address experimentally or remove the emphasis put on the TOR/mitotic fidelity connection, and of clarifying the counter-intuitive notions associated to the results obtained with rapamycin. Also, the need for revisiting / improving / justifying the means by which nuclear envelope deformation is assessed has been raised at least twice. I therefore guess that the common guidelines for improving this manuscript are clearly established.

      Significance

      In view of all of the above, my feeling is that the authors have put the accent on the TOR message, which is weak, while they have less put the accent on very strong and elegant findings they do: The authors discover that the suppression of cut(-like) mutant phenotype by addition of NH4 is not due to a correction in lipid metabolism defects, suggesting that the effect is indirect. In support, cut-like mutants whose molecular defect stems from lipid-unrelated defects are also suppressed by ammonium addition. What is more, the authors refine the type of cut-like mutants susceptible of being "corrected" by ammonium addition, finding a "novel definition of cuts" that invoke a temporal rule. This important observation has relevant implications:

      • the long-standing interpretation (commented by the authors) that lipid-related cut mutants are defective because of insufficient synthesis of lipids to be able to grow their nuclear envelope membranes seems now inappropriate in light of their data;
      • this has the immediate implication that perhaps the importance of nitrogen supplementation for accurate mitosis is no longer a fact that may apply only to (yeast) organisms performing closed mitosis, which may broaden the implications of their finding substantially;
      • the nature of the temporal ruler they discover that makes defects appearing early susceptible of being suppressed by nitrogen supplementation deserves analysis in further works, thus opening an immediate perspective.
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      Referee #3

      Evidence, reproducibility and clarity

      In this manuscript, Zemlianski et al conducted careful analysis of a group of lipid metabolism mutants exhibiting mitotic defects. They demonstrate that supplementing a good nitrogen source in the medium can rescue the mitotic defects in these mutants. Notably, this rescue occurs independently of addressing lipid composition defects or altering the expression of lipid metabolism genes. Furthermore, the study implicates TORC1 activity as a key player in integrating nitrogen availability for the effective execution of mitosis. Despite well-controlled and meticulously executed experiments, the overall study lacks comprehensiveness and appears to add to the list of existing reports without offering mechanistic insights into the unexplained impact that the TOR pathway (or nitrogen source) has on mitosis.

      Major Comments:

      1. The discussed link between Tor and mitosis is not a novel finding. An yet unexplained link between mitosis and the tor pathway has been previously reported by Yanagida lab and several years later by the Hauf Lab. Recent reports from the Hauf lab suggest that the relation of Tor to mitotic fidelity could be associated with the translational sensitivity of mitotic proteins to tor pathway or more directly to translational response to nitrogen availability for growth. Therefore, based on these leads it would be informative to see if the authors could expand on this idea and explore more on the mechanistic aspect of how nitrogen availability which feeds into tor functionality can influence mitotic progression.

      Based on the results presented here, it is reasonable to assume that in the lipid metabolism mutants which are rescued on nitrogen supplementation, TORC1 would be rendered inactivate as these cells are apparently nitrogen starved. TORC1 inactivation is known to downregulate translation and could impact the levels of critical mitotic genes. Therefore, it warrants the testing of this possibility. 2. TORC1 is known to restrain mitotic progression by opposing securin-separase and TORC2 to aid G2 to M transition by regulating the timing of Cdc2 de-phosphorylation. Earlier studies have seen rescue of mitotic defects in securin and separase by tor2 mutants (TORC1). However, here the rescue is executed by increasing the activity of TORC1 or impairing the TORC2 pathway by mutations in tor1. It might be good to present this result in context of previous reports and discuss how mitotic defects exhibited by lipid metabolism defects differ from those of mutants in core mitotic pathway such as separase and securin. The current discussion section does not explicitly explain this difference.

      Significance

      The inquiry central to the present study, namely the investigation into the impact of the TOR pathway on the proficient execution of mitosis, holds significant scientific relevance. Unraveling the mechanisms through which TOR enhances mitotic fidelity has the potential to enhance current drug interventions and pave the way for the development of informed and efficient therapeutic strategies, particularly in cancer.However, in the current form, the study lacks mechanistic insights and does not add much to the already known literature as I have detailed above in my comment.

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      Referee #2

      Evidence, reproducibility and clarity

      Zemlianski et al. present an analysis of the interaction between various mitotic phenotypes and Tor1-dependent nitrogen signaling in fission yeast. They make two interesting observations. First, the mitotic disruption caused by defects in lipid metabolism are not due to direct effects of such defects on mitotic mechanisms because the mitotic phenotypes can be suppressed by nitrogen supplementation without resolving the lipid metabolism defects. Second, the effects of nitrogen supplementation are due to nitrogen's effect on TOR signaling, not to the direct effect of the nutrient, because TOR mutants have similar effects. The work is straight forward, appears to be well done, and the conclusions are well supported by the data.

      The one part of the manuscript that I do not understand is the effect of rapamycin on mitotic fidelity. The presented genetics suggest that nitrogen increases mitotic fidelity by activating TORC1. However, rapamycin inhibits TORC1, yet also increases mitotic fidelity. The authors need to state and address this apparent contradiction much more directly than they currently do (unless I badly misunderstand something, and then they need only explain it to me).

      Referees cross-commenting

      I agree with the comments of the other reviews. They all seem reasonable and addressable by the authors.

      Significance

      The significance of the work is limited by the lack of mechanistic insight or even a plausible hypothesis as to how TOR-dependent nitrogen signaling is affecting mitotic fidelity. In something of an understatement, the authors note that "the exact mechanism of the nitrogen-mediated rescue of mitotic fidelity remains to be characterised in detail". Until that mechanism can be at least suggested, these observation do not provide much biological insight into the question of mitotic regulation.

      The work will be of interest to workers specifically involved in the regulation of mitotic fidelity in yeast, but, until more mechanistic insight can be generated, not much beyond that group.

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      Referee #1

      Evidence, reproducibility and clarity

      The authors show that mitotic fidelity can be improved by a good nitrogen source. Such 'rescue' applies to a range of unrelated mitotic mutants in fission yeast (S.pombe). Rescue appears not to be achieved by restoring lipid metabolism. Instead they argue for an indirect mechanism of suppression, and find that the TOR signalling network is involved. The paper is well written and the data clearly presented.

      Major comments:

      • most claims and conclusions are supported by the data. The catastrophic mitosis (Fig 1A) should be better described in this manuscript, rather than referring the reader to Ref. 18.
      • 'rescue' is often used in figure legends and text (eg. Fig 5-7 titles). Is this the most appropriate word? In most cases this rescue is partial. Perhaps 'suppresses' is more appropriate?
      • The authors write in the results: "taken together the ammonium-mediated rescue of mitotic defects.........seems to operate early in the cell cycle, prior to anaphase." Can they be more precise here? If cell cycle checkpoints were activated, to lengthen G2 or early M, this may not always help reduce chromosome mis-segregation. The authors have previously shown that combining a sac mutation with cbf11 did not rescue mitotic defects (ref 18). Have the authors tested these double mutants to see if the prolonged mitosis observed in cbf11 is shortened? Have other checkpoints been tested, apart from the sac?
      • "Figure 7. The TOR network is critical for the ammonium-mediated rescue of Δcbf11 mitotic defects." The data shows that inhibiting the TOR network (rapamycin) has a similar impact to ammonia. These are not additive, and it is argued that both rapamycin and ammonium must affect the same pathway. However, they do not test the impact of nitrogen sources in the genetic tor mutant backgrounds. Where is the mechanistic evidence that tor signalling is required for the ammonium-mediated rescue?
      • Optional: can the authors support their interpretation by providing some biochemical evidence that the tor signalling pathway is active in relevant conditions. For example, is tor signalling reduced when a good nitrogen source is added? Is tor signalling enhanced in a cbf11 mutant?

      Minor comments:

      • Methods: how was the area of nuclear cross-section measured (for Figure 2)?
      • I question whether the statistical t-tests used are always appropriate. In some experiments (eg. Fig2 and 4C) should ANOVA be performed? I am no expert in this, but the authors should get advice.

      Significance

      The data presented will be of interest to those studying cell division, lipid homeostasis and TOR signalling networks. However, in my opinion the mechanistic link with TOR signalling (Fig 7) should be strengthened.

      I am an expert on mitotic regulation and chromosome segregation in yeast.

  2. Apr 2024
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      Reply to the reviewers

      __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ Summary In this manuscript the authors address the largely unexplored role of micro RNAs (miRNAS) in Drosophila melanogaster brain development, in particular in neural stem cell lineages. The authors for the first time adapt the Ago protein Affinity Purification by Peptides (AGO-APP) technology for Drosophila. They show that this technique works efficiently in neural stem cell lineages and identify several cell type specific active miRNAs. Through a series of bioinformatic analysis the authors identify candidate mRNA targets for these miRNAs. The authors then functionally analyse the role of some of the identified miRNAs, focusing on miRNAs significantly over-represented in neuroblasts.

      By overexpressing Mir-1, the authors demonstrate that this miRNA effectively targets the UTR of Prospero, resulting in the overproliferation of neuroblasts. In a parallel experiment, overexpression of Mir-9c causes neuroblast differentiation defects, similar to the phenotype caused by nerfin-1 mutants, a previously validated target. Loss of function analyses show that knock down of single miRNAs has little functional effects in neuroblast size, showing that the individual effect caused by miRNAs knock down is likely compensated. In contrast, a sponge against a selected group of miRNAs leads to a reduction in poxn positive neuroblasts. Overall these results validate the approach and support the theory that miRNAs cooperate in functional modules during stem cell differentiation.

      We thank Reviewer 1 for its overall positive review. We are grateful for the useful suggestions and we believe the additional experiments we have performed and added strongly improve the quality of the study and will hopefully satisfy the reviewer's concerns.

      Comments

      Title: As the authors do not really explore exit from neural stem cell state this should be altered. The authors do not assess for the levels of any temporal genes, nor other markers of neural stem cell state exit (e.g. nuclear Pros).

      We now have further evidence that the identified microRNA module preserves neuroblasts, in particular in the optic lobe. We have modified the title accordingly: "In vivo AGO-APP identifies a module of microRNAs cooperatively preserving neural progenitors"

      The observed effects, with the available experiments, rather say that neural stem cell state is not maintained in general, not being clear what mechanistically happens to these cells expressing Cluster 2 sponges. The described phenotype caused by the expression of sponges against individual miRNAs also rather shows a blockage in differentiation.

      -The miRNAs analysed were found in Ago-APP to be predominantly active in neuroblasts, but was there any phenotypes of OE or KD in neurons or glial cells?

      Since the analyzed miRNAs were either not or poorly expressed in neurons or glia overall, it seemed less essential to investigate potential phenotypes in these cells. However, we did mis-expressed miR-cluster1sponge and miR-cluster2sponge in neurons and in glial cells (using elav-GAL4 and Repo-GAL4, respectively) throughout development, and did not observe any major impact on viability. All pupae were able to hatch.

      In addition, we show now that mis-expression of the miR-cluster2sponge (that induces strong phenotypes in neuroblasts) specifically in the wing pouch throughout development did not lead to any phenotype in the adult (e.g. wing size (tissue growth), patterning defects (cell differentiation)) (Fig6K,L). Importantly, this experiment rules out unspecific effects of the sponge construct on cell fitness, and highlight the tissue-specificity of the phenotype.

      • The authors obtained a phenotype when using a sponge against Cluster 2 in poxn neuroblasts. Is this specific for these 6 neuroblasts? What happens if this sponge is expressed with a pan-neuroblast driver in central brain/VNC/optic lobe? These experiments should be included as they would show if these are conserved effects for all neuroblasts.

      We already showed in Fig.4B of the first version of the manuscript (using a flip-out approach in clones) that miR-cluster1sponge or miR-cluster2sponge expression leads to an overall reduction in the neuroblast size in the VNC and CB.

      We have now added four more experiments, all suggesting that these sponges specifically affect type I neuroblasts:

      • using the pan-neuroblast driver nab-GAL4, we show that neuroblasts in the VNC and CB expressing these sponges are significantly smaller in late L3. Also, their number is reduced, indicated that some neuroblasts are eliminated (Fig.4C-G).
      • Using pox-GAL4 (already in first version) and eagle-GAL4, we show that different subset of type I neuroblasts in the VNC exhibit different sensitivities to the sponges (from light/medium - neuroblast shrinkage, to high - neuroblast elimination) (Fig.4H-J, S6C-E)
      • using the dpnOL-GAL4 driver, that is specific and strongly active in medulla neuroblasts in the optic lobe, we demonstrate that both, miR-cluster1sponge and miR-cluster2sponge, induce neuroblast shrinking. In addition, we find that the width of the medulla neuroblast stripe is strongly reduced when using the miR-cluster2sponge, providing further evidence for precocious neuroblast elimination (6C,D). Importantly, this leads to a smaller medulla in late L3 (Fig 6F), implying that in these conditions, medulla neuroblasts produce fewer neuronal progeny. Because medulla neuroblasts generate GMCs that undergo a single division, they are also considered as type I neuroblasts
      • using a worniu-GAL4, ase-GAL80 driver, that is specifically active in type II neuroblasts, we show that expression of miR-cluster1sponge and miR-cluster2sponge does not affect neuroblast size and the number of intermediate progenitors (Fig 6H-J). Together, these additional experiments in different types of neuroblasts and in non-neural tissue (the wing pouch, see above) demonstrate a type I neuroblast-specific effect. Our new results also imply that the microRNA module is active in most, if not all type I neuroblasts. In contrast, it is not present or not affecting differentiation genes in type II neuroblasts. Importantly, in Type II lineages, intermediate progenitors produced by neuroblasts undergo themselves a few rounds of divisions before differentiating, unlike GMCs that give rise to two differentiated progeny after a single division. Therefore, the dynamics of differentiation is different in the two lineages, involving a distinct sequential expression of differentiation factors, and possibly different miRNAs.

      The authors do different analyses in different brain regions, making also a hard to conclude if all brain regions behave the same way. As authors show that some miRNAs are only expressed in sub-sets of cells, this becomes particularly relevant.

      The new set of experiments in different types of type I neuroblasts and in type II neuroblasts, presented above, addresses the points on the specificity of the microRNA module.

      Could sponge of cluster 1 cause a phenotype if it had been expressed in other neuroblast lineages?

      Yes, it can. See our new experiments discussed above.

      __ __In addition, a discussion of the results obtained from sponge 1 should be included and put in context with miRNA function, technical limitations, levels/cell, targets, pitfalls of analyses, sponges, etc.

      We have more carefully acknowledge that sponge mediated knock-down is not very efficient and dose-dependent. We also clarified that other approaches will be required in the future to rigorously assess the specificity of each miRNA/mRNA interaction as well as their cooperativity.

      For example: "In contrast to genetic miR-1KO (Fig. 3O), we found that sponge mediated knock-down of this miRNA, or of other individual miRNAs in the module, had never a significant effect on neuroblast size (Fig. 4B), likely because the inhibition induced by sponges is incomplete. However, expression of either multi-sponge 1 or multi-sponge 2 significantly reduced neuroblast size in a dose dependent manner - two copies of the transgene exacerbate the phenotype (Fig. 4B)."

      We also state at the end of the discussion: "In the future, the combination of Ago-APP with complementary genetic strategies will be required to rigorously assess the specificity of each miRNA/mRNA interaction as well as their cooperativity."

      It would also be interesting to further explore the phenotypes caused by Mir-1 sp expression - are there any milder lineage defects?

      We observed an increase in Prospero expression and a decrease of the neuroblast size in miR-1null mutant neuroblast clones (Fig.3L-O). These phenotypes are not observed when miR-1sponge is mis-expressed. This is probably due to the fact that miR-1sponge expression leads to only a partial knock -down of miR-1. Moreover, we have added data about the expression of miR-1sponge in medulla neuroblasts in the optic lobe, showing an absence of obvious phenotype when assessing neuroblast size and neuroblast maintenance. This contrasts with expression of miR-cluster1sponge and miR-cluster2sponge (Fig. 4F,G). This new data is in line with our hypothesis that the knockdown of miRNAs of a common module synergize/cooperate to produce the phenotype expected from the deregulation of their common target mRNAs.

      Any defects in other brain regions/lineages, like in type 2 neuroblasts that usually do not express Pros?

      As suggested by the reviewer, and discussed above, we tested expression of miR-cluster1sponge and miR-cluster2sponge in type-II neuroblasts using the worniu-GAL4, asense-GAL80 driver (Neumüller et al., 2011). Interestingly, in contrast to type I neuroblasts in VNC, CB and OL regions, we did not observe neuroblast shrinking or changes in INP numbers. This suggests that either the self-renewing state is more robust in Type II than in Type I neuroblasts, or that that the uncovered miRNA module is more specific to type I neuroblasts than to type II. We have added and discussed these important data in Fig 6H-J in the revised version.

      Ago-APP identifies cell type specific miRNAs in larval neurogenesis section: - "...29oC... allows Gal4-dependent expression (Fig.1B,C)" - this description of Gal80ts/Gal4 works is not correct, expression is not prevented.

      Gal80 directly binds to Gal4 carboxy terminus and prevents Gal4-mediated transcriptional activation.

      We have tried to clarify this point in the revised version.

      "Thus, when x-GAL4, tub-GAL80ts, UAS-T6B animals are maintained at 18{degree sign}C (restrictive temperature), GAL80 binds to Gal4 and inhibits its activity. *Switching to 29{degree sign}C (permissive temperature) for 24 hours inactivates GAL80, allowing for GAL4-mediated transcriptional activation of UAS-T6B" *

      • Fig S1 - nab-Gal4 also drives expression in GMCs and neurons, rephrase text. Is nab-Gal4 expressed in optic lobe, VNC and central brain neuroblasts?

      nab-GAL4 drives UAS-T6B expression in neuroblasts (in the VNC and in the CB), but also at lower levels in the medulla neuroblasts of the OL.

      We now describe this expression more precisely in the text and in Fig.S1C:

      "nab-GAL4 was used for T6B expression in all neuroblasts. However, because GAL4 is inherited by neuroblast progeny, T6B will also be present in GMCs and a few immature neurons (Fig.S1A,C)24. Of note, nab-GAL4 is highly expressed in the neuroblasts of the ventral nerve cord (VNC) and of the central brain (CB), and weaker in the neuroblasts of the optic lobe (OL) (Fig. S1C)".

      • "20 late larval CNS" - mention the exact stage

      We mention now the precise stage: the wandering stage.

      • Providing a more detailed and interpretive description of Figures 1D and 1E would greatly enhance their clarity. Currently, the descriptions of these pannels resemble typical figure legends.

      We now provide a more detailed description of the data, emphasizing that they are consistent with previous studies on specific miRNAs.

      • Fig. 1F,G,H - It is not clear why the authors sometimes use the optic lobe, other ventral nerve cord as both regions have both neuroblasts, neurons and glia. Are the drivers used for Ago-APP not expressed in all brain regions?

      We now document the activity of the GAL4 drivers used for AGO-APP throughout the entire larval central nervous system in Fig.S1B-D. We also show images of the entire larval central nervous system for the different reporter lines (Fig S1E-K) and focus on regions of interest in the main Fig 1F-M with quantitative measurement of reporter gene expression.

      • Show "data not shown" for 1H.

      It is now shown in Fig. 1M'.

      • Fig. 1F, G, H - Please quantify intensity levels in the different cell types to facilitate comparison with Ago-APP graphs. Include in figure legend what is "cpm".

      Quantification of intensity levels is now represented in Fig. 1F,I and L. Cpm means "counts per millions". We added this in the figure legend.

      A regulatory module controlling neuroblast-to-neuron transition section: - Fig. 2C - A more detailed explanation in text is required in addition to what is mentioned in the figure legend. Including a brief summary/conclusion of the results would be helpful. If possible, add in X-axis 1, 2, 3.

      We clarified this point in the text:

      "We used the Targetscan algorithm1 to determine the predicted target genes of each neuroblast-enriched miRNA. Next, we investigated the correlation between the identified miRNAs and the presence of their targets, based on independently generated mRNA expression data44.

      *This analysis showed that neuroblast-enriched miRNAs predominantly target mRNAs that are normally highly expressed in neurons (Fig. 2C), consistent with a differentiation inhibiting function." *

      • Figure S2B - as mentioned in the text elav is expressed from the neuroblast, although this is not represented in the figure.

      I In this scheme, we depict the expression of proteins, not the presence of mRNAs. elav mRNA is indeed present at low levels in neuroblasts but the protein is absent from both neuroblasts and GMCs (as shown by all our immunostainings against Elav). This fact strongly suggests post-transcriptional repression of elav mRNA (possibly by miRNAs). This likely explains why the elav-GAL4 is also active in neuroblasts. It also suggests some post-transcriptional mechanisms to silence elav in the neuroblasts/GMCs (miRNAs?)

      It is hard to tell what are young vs maturing neurons in the cartoon, pls add a label/legend.

      We added new labels in Fig S2B to uncouple neuronal maturation from temporal identity. We hope it is clearer now.

      • Fig.3I - please shown a control brain. The merge images are not easy to see. I think it would be nicer to change the figures to be color-blind friendly.

      We added the control brain in Fig 3I for VNC clones, and Fig S3A for OL clones.

      We also changed all the figures to be color-blind friendly.

      • Fig. 3K,L - why is this now done in the VNC?

      We now focus on the VNC in the main Figure 3 (Fig.3I,J,K,L,N), and show similar phenotypes in the OL in the Supplemental Figure S3 (Fig.S3A-C).

      • Are there any lineage defects when Mir-1 sp is expressed?

      See previous comment on miR-1sponge.

      • Based on which parameters/variables of the predicted targets was the Hierarchical clustering done? A brief explanation would help the interpretation of the results and of the choice of the clusters that were further analysed.

      Hierarchical clustering is now explained in the "Bioinformatics analysis" section of the Material & Methods section with an additional matrix available in Table S1.

      • "revealed the presence of three main groups" - this should be rephrased as this "grouping" was done arbitrarily by the authors and not by hclust. Hclust is set to merge individual clusters/sub-trees up to 1. Furthermore, a more detailed explanation that supported this decision of choosing this 3 large clusters should be included.

      See previous question.

      • Fig. 4B, S4B - please include in legend how were these clones generated. S4B - scale bars missing.

      We included the missing information and added the missing scale bars.

      • Fig. 4H - was the ratio of UAS/Gal4 kept in both experimental conditions? Increasing the number of UAS/Gal4 leads to weaker expression of UAS and thus could lead to a weaker phenotype. Including in legends genotype details would help.

      This is a very good point as the number of copies of the UAS and/or GAL4 can influence transgene expression and consequently the phenotype observed. We indeed kept the ratio of UAS/GAL4 in both experimental conditions. The exact genotypes for the experiments are:

      Hs-FLP/+; act>stop>Gal4, UAS-GFP/+; UAS-RFP/UAS-miR-1

      Hs-FLP/+; act>stop>Gal4, UAS-GFP/UAS-cluster2sp; UAS-miR-1/+.

      To address this important issue in the manuscript, we added a table (Table S3) listing the precise genotypes for each experiment.

      Minor - Abstract: "a defined group of miRNAs that are predicted to redundantly target all..." This is only predicted, not experimentally shown, this should be modified accordingly.

      Although the request here is not clear to us, we made a few minor changes to the abstract that we hope will satisfy the reviewer.

      • Intro: "Elav, an RNA binding protein, is expressed as soon as post-mitotic neurons..." - Elav is expressed already in neuroblasts, as also mentioned by the authors in the result section. Correct, add references.

      elav is indeed already transcribed in neuroblasts and GMCs. However, the protein is absent in the two cell types (as shown by all our immunostainings), and only present in neurons. Thus, there is a level of post-transcriptional regulation that prevents elav mRNA translation in neuroblasts and GMCs (likely at least partly mediated by miRNAs). This also explains why in elav-GAL4; UAS-T6B brains T6B is expressed in neuroblasts and GMCs, as the GAL4 mRNA transgene is not submitted to the same post-transcriptional regulation.

      • Last paragraph of Intro (Bioinformatic analyses...) - it is not easy to understand the content of this paragraph. Rewrite to improve clarity.

      The paragraph has been rewritten for more clarity with the addition of Table S1

      • All legends: Please mention which developmental stage is being analysed in each panel (i.e. wandering 3IL, hours After Larval Hatching, hours After Puparium Formation, or other), in which brain region the analyses/images are being done.

      The CNS regions are now systematically annotated in the figures. All experiments have been done in wandering L3 (except for the new Fig.6 K,L, where the experiment is done in the adult wing). We now systematically mention in the text and legend the developmental stage at which the experiment is performed.

      Please include more detailed information about the genetics in figure legends.

      We added Table S3 that describes the exact genotype of all crosses done in this study.

      • Please include brief explanation of the genetics of miR-10KOGal4 line.

      This is now also explained in the new Table S3.

      • Why are miRNAs sometimes referred as (e.g.) "miR-1" and others "miR-1-3p"?

      The miRNA found enriched (and thus active) in the neuroblast is the miR-1-3p strand. The UAS-miR-1-sponge has been designed to be complementary to the miR-1-3p strand, and is then referred as miR-1-3psp in the text and figure legend. The miR-1 null clones have been made using the miR-1KO allele, which inactivates the entire locus and therefore both, the miR-1-3p and miR-1-5p strands. This is referred to as miR-1KO or miR-1 in the text. Finally, constructions used to mis-expressed miR-1 and other miRNAs are made with the pre-miRNA, meaning that both strands of the miRNA are mis-expressed. This is then referred as miR-1 in the text.

      • Fig. 3I-M - stage of the animal? 3M - in which brain region is this?

      We have systematically mentioned the brain region on panels on all figures.

      • Fig. 3N - can actual sizes be additionally shown, or at least averages mentioned in text?

      Average sizes are indicated in the legend of new Fig. 4F.

      • If non differentially expressed miRNAs, or miRNA with other expression patterns, had been analysed to determine their targets in the sub-set of genes expressed in neuroblasts (from the transcriptome) would different targets been found? Meaning, how specific are these binding patterns for the selected miRNA?

      This is an interesting and important point. To answer, we added a new analysis (Fig.S2C), where the total number of target sites in the 3'UTR of the pro-differentiation/temporal network genes are shown for different categories of miRNAs: neuroblast-enriched miRNAs (analysed in this study), neuron-enriched miRNAs, glia-enriched miRNAs, and random miRNAs not expressed in the brain. This analysis shows that neuroblast-enriched miRNAs exhibits a higher level of promiscuity with the iconic pro-differentiation/temporal genes than other identified or random miRNAs, arguing for functional relevance.

      **Referees cross-commenting**

      *think this study is very interesting as it optimizes a novel technique in Drosophila for the investigation of cell-specific active miRNAs, and it globally addresses the role of miRNAs in neural stem cell lineages. Although the authors do not explore deeply the biological effect of these miRNAs in neural lineages, I think that the technical contribution and the identification of some miRNA targets is relevant on its own. The authors use Prospero as an example, which is very interesting, as this gene is required to be lowly expressed in Neuroblasts and then upregulated during differentiation. Which the authors propose can be regulated by miRNAs, identifying a novel player in this differentiation mechanism. I do not feel the authors need to perform additional experiments to corroborate their findings, as they are well supported by the experiments presented. I do agree that the authors did not explore deeply the biological effect in neural lineages, and the claims regarding premature terminal differentiation, nerfin, etc need to be toned down accordingly.

      * Reviewer #1 (Significance (Required)):

      This study is both a technical and conceptual advance. It is very interesting as it optimizes a novel technique in Drosophila for the investigation of cell-specific active miRNAs, and it globally addresses the role of miRNAs in neural stem cell lineages. However, the text, especially in the results section, could benefit from increased detail to enhance the comprehension of the experiments, results, and conclusions. Given that the functional analyses were not conducted at a very detailed level, there exist certain instances of over-interpretation, which could be easily addressed either by revising the text or by incorporating additional experiments, as elaborated upon below. This manuscript will be interesting for research fields interested in stem cell differentiation, brain development, micro RNAs, both for Drosophilists and scientists working with other animal models. I am an expert in Drosophila brain development.

      __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Summary MicroRNAs (miRNAs) have a well-established role in fine-tuning gene expression. Because the mechanisms by which miRNAs recognize specific target transcripts are poorly understood, their functionally relevant targets in the physiological context are mostly poorly defined. Studies in vertebrates have suggested that miRNAs play a prominent role in regulating cell type specification during brain development. Insight into miRNA regulation of target selection will improve our understanding of neural development. Cell type-specific gene expression patterns and functions in the neural stem cell (neuroblast) lineage in the fly larval brain are well characterized. The fly genome is compact, and gene redundancy including miRNAs is significantly less than vertebrates. For these reasons, the authors chose to investigate how miRNAs regulate cell-state transitions by first establishing a comprehensive miRNA expression profile for major cell types in the fly larval brain. They combined the AGO-APP strategy and the GAL4-UAS inducible expression system to pull-down cell type-specific miRNAs from fly larval brain. The authors focused on miRNAs that are enriched in neuroblasts and examine how multi-miRNA modules regulate the maintenance of an undifferentiated state in neuroblasts. The cell type-specific inducible AGO-APP system introduced in this study is innovative and allows for systematic identification of miRNAs that most standard RNA-sequencing techniques missed in previously published datasets. The technological note sets high promise for this study, but the findings appear tame. It is my opinion that there are a number of shortcomings that can improve the rigor of this study. For example, strategies used to determine spatial expression patterns of miRNAs as well as to validate miRNA target genes are indirect with high likelihood of caveats. The choices of candidate target genes to assess the function of miRNAs in the cell state transition appear counterintuitive.

      We thank the reviewer for qualifying our study as "technologically excellent" and for emphasizing the "innovative character of AGO-APP" and the potential of such studies to "be hugely significant to the general audience".

      We are aware that there could be ways to more rigorously and systematically investigate the interactions between miRNAs and their targets and assess their cooperativity. Beyond in vitro luciferase assays (an approach we have used in this study), this would ideally involve multiple new transgenic assays, with point mutations in various miRNA sites in the 3'UTR of predicted target genes as proposed by Reviewer 2. Also, measuring the direct effect of miRNA knockdown on its target is notoriously difficult as it can be modest (and only be revealed through the cooperative action with other miRNAs, as proposed in this study), and sometimes not detected by measuring mRNA levels (e.g. by transcriptomic approaches or FISH).

      One of our aims in the future is to develop such non-trivial approaches, which will take a considerable amount of time and work. At this stage we believe that it would go beyond the scope of the present study which aims at illustrating how introducing a new technology for miRNA isolation (AGO-APP) can help to reassess important questions on miRNA biology and function (e.g. miRNA cooperation within in the context of developmental transitions). We discuss this point now in the last paragraph of the discussion in the revised version.

      Our unbiased AGO-APP results reveal a group of neuroblast enriched miRNAs that are predicted to target multiple times pro-differentiation genes (prospero, elav, nerfin-1, brat) while not targeting stemness genes such as miranda, worniu, inscuteable, deadpan, grainyhead. Mutation in pro-differentiation genes are known to either promote neuroblast tumors (prospero, nerfin-1, brat ) (https://doi.org/10.1016/j.cell.2006.01.03; 10.1101/gad.250282.114) or perturb neuronal differentiation (elav) (https://doi.org/10.1002/neu.480240604). On the other hand, mis-expression of these genes in neuroblasts often promotes shrinkage, precocious differentiation and /or cell cycle-exit (10.1016/j.cell.2008.03.034 ; 10.7554/eLife.03363 ; 10.1101/gad.250282.114). Therefore, bioinformatic prediction and previous studies made it likely that GOF of the neuroblast-enriched miRNAs would lead to neuroblast expansion or differentiation defects, and that LOF would lead to neuroblast shrinkage, cell cycle exit or differentiation. All these predictions are experimentally validated in our study. To reinforce our data, we have performed a number of additional experiments that are described below.

      Furthermore, the authors provided no rationale as to why they chose cell types that are not in the brain (such as wing cells and cells in the optic lobe) to assess the phenotypic effect of manipulating miRNAs.

      All our analysis were done either in the different types of neuroblasts found in the central nervous system (CNS) composed of the ventral nerve cord (VNC) (equivalent to vertebrate spinal cord) and brain (comprising the central brain (CB) and the optic lobes (OL) (10.1016/j.neuron.2013.12.017) - not to be confused with eye imaginal discs that produce the retina but do not contain neuroblasts. We tested the role of the neuroblast-enriched miRNAs in all neuroblasts of the CNS based on the pan-neuroblast activity of the nab-GAL4 driver used for the AGO-APP experiment. We then focused on different types of neuroblasts using lineage specific GAL4 drivers (poxn-GAL4, eagle-GAL4, dpnOL-GAL4, type II-GAL4). This is shown in the entirely revisited last paragraph of the results (Fig 4, 5, 6, S6 and S7). These experiments demonstrate that sponges simultaneously targeting several miRNAs of the module only affect type I neuroblasts but not type II neuroblasts.

      To investigate whether miR-1 directly regulates prospero mRNA in vivo, we used a tissue where prospero is not normally expressed (the wing pouch of the wing imaginal disc in late l3 larvae), allowing us to test how over-expressing miR-1 post-transcriptionally affects versions of prospero mRNAs that either possess or not its endogenous 3'UTR. The obtained results are consistent with in vitro luciferase assays, and miR-1 gain-of function in neuroblasts and GMCs, supporting the hypothesis that prospero mRNA is a direct target of miR-1 via its 3'UTR. We have clarified these points in the revised version of the manuscript.

      Using solely a reduced cell size as the functional readout for "precocious differentiation" is not rigorous and should be complemented with additional measures.

      Reduced neuroblast size always precedes neuroblast differentiation and has been widely used as functional readout of precocious differentiation (this is more clearly emphasized and referenced in the revised version). We have now also observed this phenotype in the neuroblasts of the optic lobe (Fig 6), together with precocious "plunging" of old neuroblasts in the deep layer of the medulla (Fig S7G), another sign of differentiation. These experiments show that the shrinkage phenotype is robust to all type I neuroblasts (medulla neuroblasts of the optic lobe can also be considered as type I neuroblasts because they generate GMCs that undergo a single division).

      Moreover, opposite to precocious differentiation induced by the simultaneous knockdown of multiple miRNAs of the neuroblast module, we now show that mis-expression of many of the miRNAs of the module prevents proper neuronal differentiation (miR-1, miR-9, miR-92a, miR-8) (Fig S5). Taken together, these experiments strongly suggest that the miRNAs of the module have the ability to block neuronal differentiation and that they represent a functional module in type I neuroblasts.

      Major concern: 1. The authors should use a direct method to confirm the expression pattern of identified miRNAs such as miRNA scope (ACD) in the whole mount brain instead of indirect methods such as reporters.

      Such techniques are not trivial and do not represent a standard in Drosophila. Instead, the reporter genes we have used in our study have been already validated in other studies to reflect the expression of particular miRNAs in different tissues. We thus have taken advantages of these available lines to correlate expression patterns as reflected by transgenics with our AGO-APP experiment. All reporter lines tested quantitatively support the AGO-APP data as now shown in the revised Fig 1F,I,L.

      The entire figure 3 aims to provide evidence to support that prospero mRNA is a direct target of miR-1-3p. These convoluted experiments with significant caveats should be replaced with mutating the endogenous miR-1-3p binding sites in the 3'UTR of the prospero reading frame, and demonstrate that the endogenous prospero transcript level is increased by sm-FISH. The authors could also use this novel allele to assess the phenotypic effect of "unregulated prospero" in the larval brain.

      It would indeed be an interesting experiment to perform to show that miR-1 directly regulates pros RNA in vivo. However, our miR-1 mutant clones suggests that miR-1 on its own has only a small contribution to prospero mRNA regulation during the neuroblast-to-neuron transition. This could be due to the low physiological levels of miR-1-3p in neuroblasts and to the fact that several miRNAs of the module may act partly redundantly and collaboratively to maintain the correct level of prospero mRNA. Thus, in this case, it is well possible that changes in the endogenous prospero mRNA transcript may not be significant and detected by smFISH, unless more miRNA sites are mutated. Such an experiment would involve the generation of several new transgenic lines using the CRISPR technology, which represents a long-term project.

      Again, these approaches are powerful and we agree that they would represent a more rigorous assessment of miRNA cooperation. But we feel that it goes beyond the scope of this article, as mentioned above.

      The effect of overexpressing mir-1 on the prospero transgene with its 3'UTR vs without 3'UTR cannot easily compared since the UTR might be regulated by other regulatory mechanisms in addition to mir-1.

      To minimize the potential effect of other regulators, we only compare conditions where the only difference is the presence or absence of miR-1. We do not directly compare levels of Prospero with its 3'UTR vs without 3'UTR. However, there is indeed still the possibility that miR-1 overexpression would change the expression of a protein that regulates prospero mRNA via its 3'UTR.

      Considering this we have tuned-down our conclusion concerning this part in the revised version of the manuscript and now used the sentence:

      "These experiments performed in two different cellular contexts strongly suggest that prospero mRNA is a direct target of miR-1-3p."

      How could the author use evidence-based strategy to demonstrate that massive amplification of Mira-expressing cells induced by overexpressing mir-1 in the optic lobe is indeed due to mis-regulation of prospero instead of mimicking the prospero-mutant phenotype?

      First, we noted that miR-1 overexpression in neuroblast clones causes neuroblast amplification in all regions of the CNS (not only in the optic lobe) at the expense of neuronal differentiation. This is now shown in Fig 3 and S3.

      Second, multiple chemical or genome-wide RNAi screens have been performed (Gould lab, Chia lab, Knoblich lab, etc) to identify genes whose downregulation causes efficient neuroblast amplification (10.1186/1471-2156-7-33 ; 10.1016/j.stem.2011.02.022). In VNC type I neuroblasts, only inactivation of prospero or miranda can lead to efficient neuroblast amplification in late larvae, generating tumour-like structures devoid of neurons. We find that while Miranda is highly expressed in neuroblast clones overexpressing miR-1 (Fig 3J), Prospero is completely absent, suggesting that it is efficiently silenced by miR-1 overexpression, and therefore responsible for the observed phenotype. This new result is now added in Fig.S3D. It is very unlikely that the down-regulation of another gene is responsible for this phenotype. However, we cannot exclude that other genes are deregulated that contribute to this phenotype in addition to prospero knockdown.


      Similarly, what is the evidence that the phenotype associated with mir-9a knockout is due to mis-regulation of nerfin-1?

      In contrast to prosperoKD clones that are devoid of neurons, nerfin-1 mutant clones are known to be composed of a mix of neuroblasts and neurons (Fig S4E,G) (10.1101/gad.250282.114 ). When over-expressing miR-9 in neuroblast clones in the VNC, we observed a strong downregulation of nerfin-1 (Fig S4A, C) showing that nerfin-1 is a likely target of miR-9. However, downregulation is not complete which could explain why we do not see neuroblast amplification in the VNC (Fig 4F). Together with the significant up-regulation of nerfin-1 upon miR-9sponge expression, and the results of our luciferase assays, these data are consistent with nerfin-1 being a direct target of miR-9. Finally, the fact that overexpression of miR-9 in the optic lobes triggers phenotypes very similar to loss of function of nerfin-1 (but different from loss of function of prospero which is upstream of Nerfin-1 in epistatic tests) suggests that down-regulation of nerfin-1 is at least partially responsible for the phenotype (Fig S4D,E).

      Again, we cannot exclude that other deregulated targets contribute to the phenotype.

      Most of look-alike mutant phenotypes presented by the authors appear to occur in the OL. Is there any reason why cells in the visual center, which is not a part of the brain, appears to be more suspectable to loss of function of miRNAs? This is particularly important when manipulating the same miRNAs appear to have very subtle effects on VNC neuroblasts.

      Optic lobes (OL) are a part of the brain (10.1016/j.neuron.2013.12.017). Indeed, each OL constitutes a large region located on both sides of the central brain that integrates signals from retinal photoreceptors coming from the retina in the eyes. Moreover, medulla neuroblasts in the OL can be considered as type I neuroblasts because they generate GMCs that undergo a single division, in contrast to intermediate progenitors (INPs) produced by type II neuroblasts.

      In the original version of our manuscript, we mainly showed gain-of-function in the OL , as for some of the miRNAs the phenotypes were more striking than elsewhere. We have now more systematically tested our gain-of-function and loss-of-function in both the VNC (type-I neuroblasts) (Fig 3, 4, 5, S3, S4, S6) and in the OL (medulla neuroblasts) (Fig 6, S4, S5, S7).

      Results in the VNC are presented generally in the main figures, while results in the OL are presented mainly in supplemental figures; but phenotypes obtained in both parts are now clearly described in the text of the revised version.

      How do the authors know that multi-sponge 2 expression leads to loss of stemness potential in neuroblasts? Any additional evidence that supports precocious differentiation but not death or cell cycle exit?

      This is indeed an important point which we have investigated further in the new version. We now show that inhibiting apoptosis partially rescues neuroblast elimination but not shrinkage when miR-cluster2sponge is expressed in the poxn lineage in the VNC (Fig.4L,M). This shows that VNC neuroblast can disappear by apoptosis upon miR-cluster2sponge, but that shrinkage precedes apoptosis. We also show that optic lobe neuroblasts also shrink upon miR-cluster2sponge and are precociously eliminated as indicated by the thinner neuroblast stripe, by a mechanism independent of apoptosis (Fig 6C,D, S7F). Indeed, the neuroblast stripe in the optic lobe remains free of anti-activated caspase 1 (Dcp1), a widely used label of apoptotic cells, upon miR-cluster2sponge (Fig S7F). Finally, we also show precocious "plunging" of the old OL neuroblasts deep in the medulla, another sign of precocious differentiation (Fig S7G).

      Therefore, these experiments reinforce the conclusion that the neuroblast-enriched miRNA module is involved in neuroblast maintenance and that down-regulation of this module leads to the progressive loss of the neuroblast state.

      Lastly, we show that miR-cluster2sponge has no effect on type II neuroblasts or wing imaginal discs arguing for a specific type I neuroblast effect (including VNC, CB and medulla neuroblasts).

      Again, how do the authors know that mir-1 overexpression efficiently silenced prospero mRNA in neuroblasts and GMCs in Fig. 4F?

      This relevant question is addressed in our response to questions 2 and 3.

      Have the authors considered other targets to better assess the function of these miRNAs enriched in neuroblasts. For example, could these miRNAs function to dampen the expression of genes that are required for maintaining these cells in an undifferentiated state? Several studies using the neuroblast model suggest that the expression of these genes needs to be downregulated at the transcriptional and post-transcriptional levels. Perhaps, these miRNAs might target these "stemness" transcripts instead of "differentiation" transcripts. Is there evidence for or against this possibility?

      This is definitely a good point that we have now discussed in the revised version. We found that neuroblast identity genes (e.g. Mira, Dpn, Insc, etc) are not targeted by the miRNA module. However, the module of miRNA in late L3 neuroblasts also appears to target the early temporal genes (Chinmo, Imp), that are strongly oncogenic and stemness promoting. These need to be silenced in late L3 to ensure that neuroblasts stop dividing during metamorphosis ( 10.7554/eLife.13463). Therefore, there is indeed a strong possibility that the miRNA module we have identified in late L3 both maintains stemness by inhibiting differentiation genes and dampens stemness by silencing early temporal genes ensuring timely elimination in pupal stage. We are actively working on the regulation of temporal genes by microRNAs along development and will describe this in details in another study.

      This point was clarified in the discussion as followed:

      "In this context it is interesting to note that, in addition to differentiation factors, the early temporal factors Chinmo and Imp are predicted to be highly targeted by the neuroblast-enriched miRNA module. Given the strong oncogenic potential of these genes30*, it possible that the microRNA module not only protects neuroblasts against precocious differentiation but also protects against uncontrolled self-renewal. Therefore, in principle the same miRNA module could control neuroblast activity through the control of both self-renewal and differentiation, two seemingly opposing biological activities." *

      Minor point 1. There are a number of mis-leading statements throughout the manuscript. -In the abstract, the authors indicated "isolate actively inhibiting miRNAs from different neural cell populations in the larval Drosophila central nervous system". For example, the expression patterns of Nub-Gal4 an Elav-Gal4 drivers appear to be partially overlapping in multiple cell types and might be active in the visual center (optic lobe). If true, it was unclear to me what neural cell types were actually used in their analyses and how they could confidently indicate that cell types in the central nervous system were used in their study. Aren't there more specific Gal4 drivers or more sophisticated genetic tools available to increase the purity of cell types? If not, the alternative could be a much more precise secondary screening step to directly determine where these miRNAs are actually detected instead of relying on indirect readouts of where they might be expressed.

      The expression patterns with additional figures are now more clearly described in the main text and in Fig.S1C,D.

      We are in the process of using other GAL4 drivers that target more specific populations of neurons. But this is beyond the scope of this first study and will be published later.

      -The statement "GMCs lacking Prospero, Nerfin-1 or Brat fail to differentiate and reacquire a neuroblast identity" is very problematic. Nerfin-1 does not appear to be expressed in GMCs according to Fig. S2B. Furthermore, Froldi et al., 2015 suggested that Nerfin-1 appears to prevent activated Notch from reverting neurons to ectopic neuroblasts.

      Indeed, Nerfin-1 is not expressed in GMCs but in immature neurons to stabilize neuronal identity and prevent reversion as shown by Froldi et al. and other studies (DOI: 10.1101/gad.250282.114 ; https://doi.org/10.1242/dev.141341). We have now clarified this point in the introduction: "This process involves the sequential activity of key cell fate determinants such as the transcription factor Prospero and the RNA-binding protein Brat in the GMCs followed by the transcription factor Nerfin-1 and the RNA-binding protein Elav in the maturing neurons20-23. GMCs lacking Prospero, or immature post-mitotic progeny lacking Nerfin-1, fail to initiate or maintain differentiation respectively, and progressively reacquire a neuroblast identity, leading to neuroblast amplification 21,23-25."

      -The statement on page 6 "Strikingly, the group of genes ... contained all iconic genes known to induce neuron differentiation after neuroblast asymmetric division, including nerfin-1, prospero, elav and brat" is problematic. Again, Nerfin-1 probably functions to maintain a neuronal state rather to induce differentiation. Is there evidence that Elav induces neuron differentiation after neuroblast asymmetric division? Brat seems to downregulate Notch signaling in neuroblast progeny rather than instructing neuron differentiation. Furthermore, previous studies suggested that loss of brat function does not affect identity of GMCs and their symmetric division to generate neurons. A similar statement is used at the end of this same paragraph to reiterates mis-leading messages.

      Prospero and Nerfin-1 are sequentially expressed in maturing neurons. Nerfin-1 shares many similar targets as Prospero. It has been proposed that Nerfin-1 prolonged the action of Prospero, allowing stabilisation/maintenance of the differentiated neuronal state (10.1101/gad.250282.114 ; 10.1016/j.celrep.2018.10.038)

      Brat is also involved in the sequence of events needed to produce neurons upon neuroblast asymmetric division. However, the mode of action of Brat in GMCs from type-I neuroblasts and in INPs from type-II neuroblasts is unclear. It was shown that Brat is an RNA-binding protein that has multiple targets. For example, it can bind and silence Myc, Zelda and Deadpan, and promote neuroblast-to-INP differentiation. It may also inhibit Notch signaling which is required for neuroblast-to-INP differentiation (https://doi.org/10.1016/j.devcel.2006.01.017; 10.1016/j.devcel.2008.03.004 ; https://doi.org/10.15252/embr.201744188; https://doi.org/10.1158/0008-5472.CAN-15-2299)

      We have clarified the difference between Type I and Type II neuroblasts in the introduction: "A sparse subset of neuroblasts (Type II) generate intermediate progenitors (INPs) that can undergo a few more asymmetric divisions, allowing for larger lineages to be produced. The neuroblast-to-neuron process in Type II lineages involves a slightly different sequential expression of differentiation factors21,24."

      We have also added a new reference describing that neuronal differentiation and maintenance are severely affected upon elav loss of function:

      Yao, K.-M., Samson, M.-L., Reeves, R. & White, K. Gene elav of Drosophila melanogaster: A prototype for neuronal-specific RNA binding protein gene family that is conserved in flies and humans. J. Neurobiol. 24, 723-739 (1993).

      **Referees cross-commenting**

      My main concern about data in this study remains direct vs. indirect effects of manipulating miRNA functions and the corresponding phenotype in various cell types in flies. The authors focused most of their effort on using genes that promote GMC differentiation in order to establish the role of neuroblast-specific miRNAs. Most of the experiments were not rigorously performed to the level that eliminates obvious caveats and suggests their interpretation is the most likely possibility. It is a technologically excellent study but lacks in-depth analyses in biological effects.

      Reviewer #2 (Significance (Required)):

      I believe there is a strong general interest in better appreciating how miRNAs regulate precise gene expression. Deriving some sort of rules such as the specificity of target selection or the efficiency of downregulating gene expression will be hugely significant to the general audience

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      Referee #2

      Evidence, reproducibility and clarity

      Summary

      MicroRNAs (miRNAs) have a well-established role in fine-tuning gene expression. Because the mechanisms by which miRNAs recognize specific target transcripts are poorly understood, their functionally relevant targets in the physiological context are mostly poorly defined. Studies in vertebrates have suggested that miRNAs play a prominent role in regulating cell type specification during brain development. Insight into miRNA regulation of target selection will improve our understanding of neural development. Cell type-specific gene expression patterns and functions in the neural stem cell (neuroblast) lineage in the fly larval brain are well characterized. The fly genome is compact, and gene redundancy including miRNAs is significantly less than vertebrates. For these reasons, the authors chose to investigate how miRNAs regulate cell-state transitions by first establishing a comprehensive miRNA expression profile for major cell types in the fly larval brain. They combined the AGO-APP strategy and the GAL4-UAS inducible expression system to pull-down cell type-specific miRNAs from fly larval brain. The authors focused on miRNAs that are enriched in neuroblasts and examine how multi-miRNA modules regulate the maintenance of an undifferentiated state in neuroblasts.

      The cell type-specific inducible AGO-APP system introduced in this study is innovative and allows for systematic identification of miRNAs that most standard RNA-sequencing techniques missed in previously published datasets. The technological note sets high promise for this study, but the findings appear tame. It is my opinion that there are a number of shortcomings that can improve the rigor of this study. For example, strategies used to determine spatial expression patterns of miRNAs as well as to validate miRNA target genes are indirect with high likelihood of caveats. The choices of candidate target genes to assess the function of miRNAs in the cell state transition appear counterintuitive. Furthermore, the authors provided no rationale as to why they chose cell types that are not in the brain (such as wing cells and cells in the optic lobe) to assess the phenotypic effect of manipulating miRNAs. Using solely a reduced cell size as the functional readout for "precocious differentiation" is not rigorous and should be complemented with additional measures.

      Major concern:

      1. The authors should use a direct method to confirm the expression pattern of identified miRNAs such as miRNA scope (ACD) in the whole mount brain instead of indirect methods such as reporters.
      2. The entire figure 3 aims to provide evidence to support that prospero mRNA is a direct target of miR-1-3p. These convoluted experiments with significant caveats should be replaced with mutating the endogenous miR-1-3p binding sites in the 3'UTR of the prospero reading frame, and demonstrate that the endogenous prospero transcript level is increased by sm-FISH. The authors could also use this novel allele to assess the phenotypic effect of "unregulated prospero" in the larval brain. The effect of overexpressing mir-1 on the prospero transgene with its 3'UTR vs without 3'UTR cannot easily compared since the UTR might be regulated by other regulatory mechanisms in addition to mir-1.
      3. How could the author use evidence-based strategy to demonstrate that massive amplification of Mira-expressing cells induced by overexpressing mir-1 in the optic lobe is indeed due to mis-regulation of prospero instead of mimicking the prospero-mutant phenotype? Similarly, what is the evidence that the phenotype associated with mir-9a knockout is due to mis-regulation of nerfin-1?
      4. Most of look-alike mutant phenotypes presented by the authors appear to occur in the OL. Is there any reason why cells in the visual center, which is not a part of the brain, appears to be more suspectable to loss of function of miRNAs? This is particularly important when manipulating the same miRNAs appear to have very subtle effects on VNC neuroblasts.
      5. How do the authors know that multi-sponge 2 expression leads to loss of stemness potential in neuroblasts? Any additional evidence that supports precocious differentiation but not death or cell cycle exit?
      6. Again, how do the authors know that mir-1 overexpression efficiently silenced prospero mRNA in neuroblasts and GMCs in Fig. 4F?
      7. Have the authors considered other targets to better assess the function of these miRNAs enriched in neuroblasts. For example, could these miRNAs function to dampen the expression of genes that are required for maintaining these cells in an undifferentiated state? Several studies using the neuroblast model suggest that the expression of these genes needs to be downregulated at the transcriptional and post-transcriptional levels. Perhaps, these miRNAs might target these "stemness" transcripts instead of "differentiation" transcripts. Is there evidence for or against this possibility?

      Minor point

      1. There are a number of mis-leading statements throughout the manuscript. -In the abstract, the authors indicated "isolate actively inhibiting miRNAs from different neural cell populations in the larval Drosophila central nervous system". For example, the expression patterns of Nub-Gal4 an Elav-Gal4 drivers appear to be partially overlapping in multiple cell types and might be active in the visual center (optic lobe). If true, it was unclear to me what neural cell types were actually used in their analyses and how they could confidently indicate that cell types in the central nervous system were used in their study. Aren't there more specific Gal4 drivers or more sophisticated genetic tools available to increase the purity of cell types? If not, the alternative could be a much more precise secondary screening step to directly determine where these miRNAs are actually detected instead of relying on indirect readouts of where they might be expressed. -The statement "GMCs lacking Prospero, Nerfin-1 or Brat fail to differentiate and reacquire a neuroblast identity" is very problematic. Nerfin-1 does not appear to be expressed in GMCs according to Fig. S2B. Furthermore, Froldi et al., 2015 suggested that Nerfin-1 appears to prevent activated Notch from reverting neurons to ectopic neuroblasts. -The statement on page 6 "Strikingly, the group of genes ... contained all iconic genes known to induce neuron differentiation after neuroblast asymmetric division, including nerfin-1, prospero, elav and brat" is problematic. Again, Nerfin-1 probably functions to maintain a neuronal state rather to induce differentiation. Is there evidence that Elav induces neuron differentiation after neuroblast asymmetric division? Brat seems to downregulate Notch signaling in neuroblast progeny rather than instructing neuron differentiation. Furthermore, previous studies suggested that loss of brat function does not affect identity of GMCs and their symmetric division to generate neurons. A similar statement is used at the end of this same paragraph to reiterates mis-leading messages.

      Referees cross-commenting

      My main concern about data in this study remains direct vs. indirect effects of manipulating miRNA functions and the corresponding phenotype in various cell types in flies. The authors focused most of their effort on using genes that promote GMC differentiation in order to establish the role of neuroblast-specific miRNAs. Most of the experiments were not rigorously performed to the level that eliminates obvious caveats and suggests their interpretation is the most likely possibility. It is a technologically excellent study but lacks in-depth analyses in biological effects.

      Significance

      I believe there is a strong general interest in better appreciating how miRNAs regulate precise gene expression. Deriving some sort of rules such as the specificity of target selection or the efficiency of downregulating gene expression will be hugely significant to the general audience

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      Referee #1

      Evidence, reproducibility and clarity

      Summary

      In this manuscript the authors address the largely unexplored role of micro RNAs (miRNAS) in Drosophila melanogaster brain development, in particular in neural stem cell lineages. The authors for the first time adapt the Ago protein Affinity Purification by Peptides (AGO-APP) technology for Drosophila. They show that this technique works efficiently in neural stem cell lineages and identify several cell type specific active miRNAs. Through a series of bioinformatic analysis the authors identify candidate mRNA targets for these miRNAs. The authors then functionally analyse the role of some of the identified miRNAs, focusing on miRNAs significantly over-represented in neuroblasts.

      By overexpressing Mir-1, the authors demonstrate that this miRNA effectively targets the UTR of Prospero, resulting in the overproliferation of neuroblasts. In a parallel experiment, overexpression of Mir-9c causes neuroblast differentiation defects, similar to the phenotype caused by nerfin-1 mutants, a previously validated target. Loss of function analyses show that knock down of single miRNAs has little functional effects in neuroblast size, showing that the individual effect caused by miRNAs knock down is likely compensated. In contrast, a sponge against a selected group of miRNAs leads to a reduction in poxn positive neuroblasts. Overall these results validate the approach and support the theory that miRNAs cooperate in functional modules during stem cell differentiation.

      Comments

      Title: As the authors do not really explore exit from neural stem cell state this should be altered. The authors do not assess for the levels of any temporal genes, nor other markers of neural stem cell state exit (e.g. nuclear Pros). The observed effects, with the available experiments, rather say that neural stem cell state is not maintained in general, not being clear what mechanistically happens to these cells expressing Cluster 2 sponges. The described phenotype caused by the expression of sponges against individual miRNAs also rather shows a blockage in differentiation.

      • The miRNAs analysed were found in Ago-APP to be predominantly active in neuroblasts, but was there any phenotypes of OE or KD in neurons or glial cells?
      • The authors obtained a phenotype when using a sponge against Cluster 2 in poxn neuroblasts. Is this specific for these 6 neuroblasts? What happens if this sponge is expressed with a pan-neuroblast driver in central brain/VNC/optic lobe? These experiments should be included as they would show if these are conserved effects for all neuroblasts. The authors do different analyses in different brain regions, making also a hard to conclude if all brain regions behave the same way. As authors show that some miRNAs are only expressed in sub-sets of cells, this becomes particularly relevant. Could sponge of cluster 1 cause a phenotype if it had been expressed in other neuroblast lineages? In addition, a discussion of the results obtained from sponge 1 should be included and put in context with miRNA function, technical limitations, levels/cell, targets, pitfalls of analyses, sponges, etc. It would also be interesting to further explore the phenotypes caused by Mir-1 sp expression - are there any milder lineage defects? Any defects in other brain regions/lineages, like in type 2 neuroblasts that usually do not express Pros?

      Ago-APP identifies cell type specific miRNAs in larval neurogenesis section: - "...29oC... allows Gal4-dependent expression (Fig.1B,C)" - this description of Gal80ts/Gal4 works is not correct, expression is not prevented. - Fig S1 - nab-Gal4 also drives expression in GMCs and neurons, rephrase text. Is nab-Gal4 expressed in optic lobe, VNC and central brain neuroblasts? - "20 late larval CNS" - mention the exact stage - Providing a more detailed and interpretive description of Figures 1D and 1E would greatly enhance their clarity. Currently, the descriptions of these pannels resemble typical figure legends. - Fig. 1F,G,H - It is not clear why the authors sometimes use the optic lobe, other ventral nerve cord as both regions have both neuroblasts, neurons and glia. Are the drivers used for Ago-APP not expressed in all brain regions? - Show "data not shown" for 1H. - Fig. 1F, G, H - Please quantify intensity levels in the different cell types to facilitate comparison with Ago-APP graphs. Include in figure legend what is "cpm".

      A regulatory module controlling neuroblast-to-neuron transition section: - Fig. 2C - A more detailed explanation in text is required in addition to what is mentioned in the figure legend. Including a brief summary/conclusion of the results would be helpful. If possible, add in X-axis 1, 2, 3. - Figure S2B - as mentioned in the text elav is expressed from the neuroblast, although this is not represented in the figure. It is hard to tell what are young vs maturing neurons in the cartoon, pls add a label/legend. - Fig.3I - please shown a control brain. The merge images are not easy to see. I think it would be nicer to change the figures to be color-blind friendly. - Fig. 3K,L - why is this now done in the VNC? - Are there any lineage defects when Mir-1 sp is expressed? - Based on which parameters/variables of the predicted targets was the Hierarchical clustering done? A brief explanation would help the interpretation of the results and of the choice of the clusters that were further analysed. - "revealed the presence of three main groups" - this should be rephrased as this "grouping" was done arbitrarily by the authors and not by hclust. Hclust is set to merge individual clusters/sub-trees up to 1. Furthermore, a more detailed explanation that supported this decision of choosing this 3 large clusters should be included. - Fig. 4B, S4B - please include in legend how were these clones generated. S4B - scale bars missing. - Fig. 4H - was the ratio of UAS/Gal4 kept in both experimental conditions? Increasing the number of UAS/Gal4 leads to weaker expression of UAS and thus could lead to a weaker phenotype. Including in legends genotype details would help.

      Minor

      • Abstract: "a defined group of miRNAs that are predicted to redundantly target all..." This is only predicted, not experimentally shown, this should be modified accordingly.
      • Intro: "Elav, an RNA binding protein, is expressed as soon as post-mitotic neurons..." - Elav is expressed already in neuroblasts, as also mentioned by the authors in the result section. Correct, add references.
      • Last paragraph of Intro (Bioinformatic analyses...) - it is not easy to understand the content of this paragraph. Rewrite to improve clarity.
      • All legends: Please mention which developmental stage is being analysed in each panel (i.e. wandering 3IL, hours After Larval Hatching, hours After Puparium Formation, or other), in which brain region the analyses/images are being done. Please include more detailed information about the genetics in figure legends.
      • Please include brief explanation of the genetics of miR-10KOGal4 line.
      • Why are miRNAs sometimes referred as (e.g.) "miR-1" and others "miR-1-3p"?
      • Fig. 3I-M - stage of the animal? 3M - in which brain region is this?
      • Fig. 3N - can actual sizes be additionally shown, or at least averages mentioned in text?
      • If non differentially expressed miRNAs, or miRNA with other expression patterns, had been analysed to determine their targets in the sub-set of genes expressed in neuroblasts (from the transcriptome) would different targets been found? Meaning, how specific are these binding patterns for the selected miRNA?

      Referees cross-commenting

      I think this study is very interesting as it optimizes a novel technique in Drosophila for the investigation of cell-specific active miRNAs, and it globally addresses the role of miRNAs in neural stem cell lineages. Although the authors do not explore deeply the biological effect of these miRNAs in neural lineages, I think that the technical contribution and the identification of some miRNA targets is relevant on its own. The authors use Prospero as an example, which is very interesting, as this gene is required to be lowly expressed in Neuroblasts and then upregulated during differentiation. Which the authors propose can be regulated by miRNAs, identifying a novel player in this differentiation mechanism.

      I do not feel the authors need to perform additional experiments to corroborate their findings, as they are well supported by the experiments presented.

      I do agree that the authors did not explore deeply the biological effect in neural lineages, and the claims regarding premature terminal differentiation, nerfin, etc need to be toned down accordingly.

      Significance

      This study is both a technical and conceptual advance. It is very interesting as it optimizes a novel technique in Drosophila for the investigation of cell-specific active miRNAs, and it globally addresses the role of miRNAs in neural stem cell lineages. However, the text, especially in the results section, could benefit from increased detail to enhance the comprehension of the experiments, results, and conclusions. Given that the functional analyses were not conducted at a very detailed level, there exist certain instances of over-interpretation, which could be easily addressed either by revising the text or by incorporating additional experiments, as elaborated upon below.

      This manuscript will be interesting for research fields interested in stem cell differentiation, brain development, micro RNAs, both for Drosophilists and scientists working with other animal models. I am an expert in Drosophila brain development.

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      Reply to the reviewers

      1. General Statements__: The manuscript entitled "__Dual antiviral mechanisms of Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue viruses with insights into Dengue methyltransferase-CAPE crystal structure" is the first report of broad spectrum alphavirus and flavivirus inhibitors with dual roles that efficiently inhibit virus replication by diminishing the levels of polyamines in the host cells as well as inhibit the enzymatic activity of the virus-specific methyltransferase (MTases). Chikungunya virus (CHIKV) and Dengue virus (DENV) are re-emerging alpha- and flaviviruses respectively. Until now, no antivirals are commercially available to combat these two viral infections. This study delves into the antiviral mechanisms of Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) against DENV and CHIKV. Treatment of Vero cells with these compounds resulted in polyamine depletion. However, adding exogenous polyamines did not completely rescue the virus, suggesting alternative antiviral mechanisms. Interestingly, these compounds exhibited anti-MTase activity against purified viral MTases of CHIKV and DENV. The crystal structure of the DENV 3 MTase in complex with CAPE revealed its binding site within the GTP-binding region of DENV MTase. This study presents the novel dual inhibition mechanism of HC and CAPE, offering promising prospects for developing broad-spectrum antivirals.

      2. Point-by-point description of the revisions

      We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to the improvement of the manuscript. We believe that the thoughtful critiques and suggestions have significantly enhanced the overall quality of our work. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

      Reviewer 1: -

      Comment 1: My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question..

      I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section

      • Response: We appreciate the reviewer's constructive feedback for their insightful remarks on the potential extensive influence of polyamine depletion on host cell metabolism. We acknowledge the critical role polyamines play in genome folding and stability, and their depletion could indeed disrupt cellular homeostasis. In response to this valuable feedback, we conducted a comprehensive literature review. This literature review uncovered studies investigating the targeting of the polyamine biosynthetic pathway as a potential therapeutic strategy for combating various infections and diseases. Additionally, DFMO , a drug that targets polyamine biosynthetic pathway enzyme is an FDA-approved drug for African sleeping sickness and high-risk neuroblastoma (Bouteille & Dumas, 2003; Nazir et al., 2024) indicating that despite the critical role of polyamines in cellular metabolic homeostasis, the host polyamine pathway can also be successfully targeted for antiviral drug discovery. As recommended, we have added this information in the revised manuscript. * Additionally, ribavirin, an FDA-approved antiviral agent, employs various mechanisms to inhibit viral replication, including the reduction of polyamine levels (Tate et al., 2019). Furthermore, we have also examined the protocols available in the literature for CAPE, HC, and DFMO treatment. Most of these studies have employed MTT assay, as illustrated in the research conducted by Arisan et al. 2012 and Shen et al. 2013 (Arisan et al., 2012; Shen et al., 2013). Notably, Aljabr et al.,2016 also employed the MTT assay for viability testing, underscoring its relevance (Aljabr et al., 2016). Similarly, our manuscript employed the MTT assay at various compound concentrations to ensure the utilization of non-cytotoxic concentrations for antiviral activity testing. *

      As per reviewer's recommendation, we have discussed the potential adverse effects of polyamine depletion on cellular processes in the revised manuscript's discussion section.

      *Line no.s 513 – 523 of the revised manuscript have the revised text as per the suggestion. *

      Reviewer 2:-

      Comment 1:- Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with a site involved in binding of RNA

      Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

      • *Response :-We appreciate the reviewer's positive feedback of on the crystal structure and as pointed out towards data quality and analysis, we have tried and made significant improvements, including enhancing data representation and providing detailed protocols in the supplementary materials where necessary. Additionally, we have addressed key technical details that were previously missing and ensured that all experiments are described adequately. We acknowledge the need for clearer writing and have now mentioned clearly that independent experiments have been carried out in the study. We have made suggested revisions to the revised manuscript. *

        Comment 2:- Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery

      • Response:-We thank the reviewer for pointing this out. We have modified the text as follows: lines 64-65 "Viral genomes lack protein synthesis machinery, and the ability to hijack the host cell's resources for replication is crucial for all viruses". to lines 65-67 "Viral particles lack essential protein synthesis machinery. Consequently, viruses rely on the host cell's resources to replicate effectively."

        Comment 3:- line 137 flavonoids play a role in reducing the levels of nsP1 in CHIKV - what can this possibly mean? Are shown to reduce the level of nsP1 in CHIKV-infected cells?

      • Response: We appreciate the reviewer for bringing this to our attention, and we acknowledge that it was due to a writing issue in English. This has now been rectified. A dose-dependent reduction of the CHIKV E2, nsP1, and nsP3 proteins was observed upon treatment with baicalein and fisetin. This finding would suggest that baicalein and fisetin might inhibit the production of CHIKV protein, especially the proteins involved in the negative-strand synthesis and part of the replicase unit (Lani et al., 2016). To account for this suggestion, we have modified the text in the revised manuscript to (line 145-147): " Moreover, flavonoids treatment has demonstrated the dose-dependent decrease in CHIKV titer due to reduced levels of CHIKV viral proteins, including nsP1*. *

      __Comment 4 :-__line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, and inserted between the NheI and XhoI restriction sites... …..It should be impossible as one cannot insert RNA into bacterial plasmid DNA.

      • Response:- We thank the reviewer for pointing this out. line 250-251 – "RNA was isolated from the infected cells' supernatant……..". This has been changed to line 267-271 " RNA was isolated from the supernatant of the cells infected with DENV 3, and used for cDNA preparation, cloning of the MTase gene fragment into the pET28c (+) vector using NheI and XhoI restriction sites."

        __Comment 5 :-__Missing parts. Examples

      the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies, but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)

      • Response:- We thank the reviewer for the comments. In response to the reviewer's concern regarding the source of the nsP1 protein from CHIKV, we would like to clarify that the recombinant protein was expressed and purified from E. coli Rossetta cells in our laboratory. We acknowledge the importance of this point and apologize for any oversight in not explicitly stating it in the manuscript. In response to the reviewer's suggestion, we have incorporated a detailed expression and purification protocol into the manuscript supplementary methodology (line number 1068-1091).
      • Response:- Alphaviruses share a high degree of sequence similarity (>80%), particularly within the nsP1 protein, with conserved active site residues (Supplementary Figure 2). Several studies investigating nsP1 proteins from alphaviruses, including Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, have successfully employed E. coli Rosetta cells for protein expression, followed by enzyme activity assays (Abdelnabi et al., 2020; Li et al., 2015; Tomar et al., 2011). Our laboratory is working on this protein for more than a decade and have conducted extensive assays on the activity of nsP1 protein purified from bacterial expression system. Our results are reproducible. These studies have been published in reputed peer reviewed research articles, including (Kaur et al., 2018; Mudgal et al., 2020). Additionally, similar assays have been demonstrated in the study by Bullard-Feibelman et al., 2016. We trust that this clarification resolves the reviewer's concern, and we are delighted to address any further inquiries.

        Comment 6:- Figure lacks quality (and figure legends are unclear) Examples:

      • it is impossible to understand what exactly is shown in Figure 1J

      • important information is missing, for example, it is not clear what were concentrations of antiviral compounds for panels 1F and 1I

      • Response :- We thank the reviewer for the constructive comments that has helped us to improve the revised manuscript. We have revised Figure 1J and as suggested we have updated the legends accordingly. Similar revisions have been made in the revised manuscript to the TLC protocol and results to ensure clarity. We thank the reviwer for pointing out the missing information regarding the concentrations of the antiviral compounds used in panels 1F and 1I. As per your suggestion, we added the antiviral compounds concentrations for these experiments in figure legends.

      Comment 7:- 4. wrong data - line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023 - line 476 refers to family alphaviridae. This does not exist, family is Togaviridae

      • Response:- We appreciate the reviewer for bringing this to our attention. We have accordingly revised the sentences for accuracy. "Although human viruses belong to several viral families, Alphaviridae and Flaviviridae are the most significant burden on public health" changed to line number 505-506 "Although human viruses belong to several viral families, Togaviridae and Flaviviridae impose one of the most significant burdens on public health"
      • *

      Line no.. 478 “ Neither commercially available drugs nor vaccines are available for these viruses.” Changed to line number 508 to 509 “Although FDA-approved vaccines for Dengue and Chikungunya viruses are available, no antiviral therapies have been approved against these viral infections.”

      Comment 8: ____5. unjustified conclusions. Example

      • authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)
      • Response:-Following the reviewer's recommendation, we have included Salmonid alphavirus, an aquatic virus, and Eilat virus, an insect-specific virus, in our comparison along with other human-infecting alphaviruses. Additionally, for flaviviruses, we have incorporated Palm Creek virus, an insect-specific virus, and Wenzhou shark flavivirus, an aquatic virus. As suggested, the relevant modifications have been done to the MSA protocol, results, and figure legends.

        Comment 9:- 6. Insufficient analysis of data. In some cases, there is a significant discrepancy between the results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies.

      (CE and ELISA-based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided one can assume that it is due to experimental errors.

      • Response:- We thank the reviewers for their valuable comments. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * Our explanation: Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, it is only able to detect SAH concentrations above ~ 300 µM. A previously validated CE-based assay for Chikungunya virus (CHIKV) nsP1 by Mudgal et al.,2020 addresses this limitation. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. However, *CAPE is observed to inhibit DENV at ~2.5 micro, supporting that viral inhibition not only is due to MTase inhibition but through other mechanism i.e. host cells polyamine depletion.

      • *

      • Therefore, this presents one plausible explanation, although we cannot currently dismiss the possibility of other mechanisms that could also contribute to viral inhibition by CAPE.*

      The established ELISA assay of nsP1 utilizes an indirect detection method, which exhibits higher sensitivity. Additionally, previously published studies on alphaviral nsP1 inhibitors also report nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020).Therefore, differing substrate concentrations and CE-based assay limitations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays. Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.

      In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response curve IC50 format to a bar chart for enhanced clarity. This bar chart effectively conveys the key finding of a dose-dependent decrease in activity observed for both HC and CAPE.

      Similarly, we again tried to reoptimize the MTase CE-based assay by reducing the GTP concentration in enzyme reaction from 4 mM to 0.3 mM. This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. Furthermore, our approach with CE based assay is centered around detecting inhibition rather than conducting quantitative analyses.

      • *

      The discrepancy in the in vitro vs the enzyme test tube assay could be attributed to HC and CAPE's multifaceted mechanism of action when used in vitro (i.e polyamine depletion and anti methyltransferase activity). However, only methyltransferase inhibition has been assessed in enzymatic assay. Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends for clarifications. Additionally, the results have been appropriately discussed in the discussion section.

      • *

      Comment 10 :-6. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

      > Response: We appreciate the reviewer's suggestion regarding the discussion section; we have incorporated a comprehensive discussion in the revised manuscript.

      3rd reviewer :-

      The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

      Comment 1:-

      __Major points:- __ While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition is not precise enough in the case of CHIK nsp1 and too weak in the case of DENV NS5 (detailed below).

      Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

      The discussion section requires more elaborate scientific justification than simply re-reporting the results.

      • Response:- We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to in the improvement of our manuscript. We believe that the thoughtful critiques and suggestions have substantially improved the overall quality of our work. The changes made in the revised manuscript are highlighted in red. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

        Comment 2:-

      It would be best to organize the ms as follows: - Crystal structure of DENV MTase in complex with CAPE - Building of a model of nsp1 by superimposition with NS5 MTase - Modeling compound binding - Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

      • Response :- We appreciate the reviewer's suggestion regarding the manuscript organization. We understand the value of presenting the data in a logical flow. For this study, our initial investigations focused on the polyamine depletion ability of HC and CAPE, followed by antiviral activity assays. Based on the preliminary data from cell-based polyamine depletion assay and antiviral assays, the identified molecules were used for in silico investigations, followed by biochemical and biophysical validation. the crystal structure studies were performed to gain a deeper understanding of the inhibition mechanism. Therefore, we believe this flow, approach and the current structure have merit and is request to be considered.

        Comment 3:- Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

      • If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

      Figure 5 is problematic.

      • When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points.

      • Panel F and G: A reduction of 25 % at the highest inhibitor concentration is a strong indication that there is no effect.

      • Response:- We sincerely thank the reviewers for their valuable comments and insights regarding the discrepancies observed in our data. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, typically only detecting SAH concentrations exceeding ~300 µM. *

      *A previously validated CE-based assay for Chikungunya virus (CHIKV) nsp1 by Rajat et al. addresses this limitation and has been mentioned in the revised manuscript with the reference. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. The established ELISA assay utilizes an indirect detection method and exhibits higher sensitivity. Also, previous studies on alphaviral nsP1 inhibitors have also reported nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020). *

      Hence, differing substrate concentrations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays. Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.

      • *In response to the reviewer's suggestion to test compounds at lower dilutions, we acknowledge that we are currently unable to perform an assay for lower dilutions as recommended due to time constraints and limited availability (screen shot below) of "MABE419 Sigma-Aldrich (Merk), Anti-m3G-cap, m7G-cap Antibody, clone H-20 antibody" used as the primary antibody (Kaur et al., 2018). Our attempts to procure this antibody from Sigma were unsuccessful.For India it shows limted availability and the vendor has given the estimated shipment time of more than 7 weeks. As per reviewers suggestion and the current limitations in the IC50 data, we have revised the graphical representation from a non-linear regression format (which estimates IC50) to a bar chart format. In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response IC50 format to a bar chart for clarity. This bar chart effectively conveys the key finding of inhibitory activity observed for both HC and CAPE.

      We tried to reoptimize the Dengue virus MTase CE-based assay by reducing the GTP concentration from 4 mM to 0.3 mM. This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. The CE-based assay for HC and CAPE data clearly indicates inhibition above >50%. Our approach with this assay is centered around detecting inhibition rather than conducting quantitative analyses. Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends. Additionally, the results have been appropriately discussed in the discussion section.

      Comment 4- Please describe more panel D in the legend.

      • Response :-We sincerely appreciate your suggestion and wish to express our gratitude. We have revised figure legend 6 D from. Line no. 791 "The CE based HC and CAPE Methyltransferase inhibition activity assay CHIKV nsP1" changed to line no. 884 to 886 "CE-based nsP1 MTase activity inhibition assay as described previously by Mudgal et al. 2020". HC and CAPE compounds were tested at a concentration of 200 µM and CAPE 1000 µM respectively.

        Minor Points/Comments/ Suggestions:

      Comment 1:-

      In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

      Response:- Thank you for your suggestion; we have revised the text accordingly. Line no. 58 "It is estimated that ~100-400 million DENV infections occur annually" changed to line no. 58 to 61 "It is estimated that annually ~100-400 million DENV infections occur worldwide. The Philippines and Vietnam are among the most affected countries. Moreover, dengue is endemic in India, Indonesia, Myanmar, Sri Lanka, and Thailand (Bhatt et al., 2013; Lobo et al., 2011, National Center for Vector Borne Diseases Control Report 2022 (NCVBDC)."

      __Comment 2:- __B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

      Response :- Thank you for your feedback; brief introduction of alphaviruses have been added.

        • 4 (line 92) Alphaviruses belonging to the Togaviridae family include viruses such as Chikungunya, Eastern equine encephalitis, Venezuelan equine encephalitis, etc.*
      1. *

      Comment 3:- C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3.

      Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

      • Response:-Thank you for your valuable input. Dengue serotypes have been introduced, and we have meticulously reviewed and rectified all inconsistencies regarding their nomenclature. Line no. 120 to 123 "Flaviviruses are classified within the Flaviviridae family and encompass viruses like Dengue, Zika, Japanese encephalitis, etc. Dengue virus consists of four distinct antigenic types: DENV 1, DENV 2, DENV 3, and DENV 4. DENV 2 has been India's most prevalent serotype for the past 50 years, however serotypes 3 and 4 have also appeared in some recent epidemics (Kalita et al., 2021)."

        Comment 4:- D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicate the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

      • Response:-Line 100 changed from "Cellular enzyme capping mechanisms usually involve the methylation of guanosine triphosphate (GTP) after transferring it to the 5' end of the RNA. However, the molecular mechanism of viral mRNA capping in alphaviruses is distinct." To line no. 102 to 108 "Cellular enzymes use conventional capping mechanisms, usually where GTP is first transferred to RNA's 5' end, followed by its methylation. On the other hand, viral capping in the case of alphaviruses is unconventional, where GTP is first methylated, followed by the guanyltion of viral RNAs. Furthermore, Cap 0 alphaviruses feature monomethylation at the N7 position of the guanosine nucleotide, while Cap 1 in flaviviruses has additional methylation at both the N7 and 2'O positions."

        Comment 5:-

      • Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

      • Response :- We have addressed the reviewer's insightful suggestion regarding the citation and included the references accordingly.

        Comment 6:- F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

      • Response :-We sincerely appreciate the reviewer's input. We have incorporated the suggested changes as follows : In line 126, we changed "Flaviviridae" to "Flaviviridae".

      In line 476 (Discussion section), we corrected "alphaviridae" to "Togaviridae".

      We ensured consistency in the formatting of taxonomic names throughout the manuscript.

      Comment 7:-

      1. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.
      • Response :-The reviewer's feedback has been invaluable, and we've acted upon it accordingly. In response to the suggestion, we've made it clear in the manuscript to refer to the supplementary experimental procedures for detailed protocols where appropriate. Additionally, we've listed certain protocols exclusively in the supplementary material to enhance clarity and avoid repetition.

        Comment 8:-

      • M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

      • Response :-We appreciate the reviewer's attention to detail. At the time of usage, further dilutions were prepared in 2% DMEM media. Additionally, individual polyamines (putrescine, spermidine, and spermine) stocks were diluted in sterile tissue culture-grade water from Alfa-Aeser, USA, and used as indicated. As such, we have revised the sentence to enhance clarity. Line number 173 to 175 "At the time of usage, further dilutions were prepared in culture media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in water and used as designated." changed to this "At the time of usage, further dilutions were prepared in 2 % DMEM media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in sterile tissue culture grade water and used as designated."

      • *

      Comment 9:-

      1. M&M, line 274: What is CE? Please expand the term before using the abbreviation.

      2. Response :- Thank you for bringing that to our attention. CE mentioned in line 294 stands for Capillary electrophoresis__.__

        Comment 10:-

      line 306. Ref. 53: This is not a reference.

      • Response :-Thank you for bringing this to our attention. We understand that reference 53 does not correspond to a valid source. We acknowledge this and want to clarify that due to the unavailability of the proper reference, we included this reference. We have now changed the reference to the Crysalis Pro software.

        Comment 11:-

      • Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B.

      • Response :-Thank you for bringing this to our attention. We apologize for any confusion caused by including Fig. 1C, especially since the data it presents overlaps with that of Fig. 1B. To ensure clarity, we have made modifications accordingly. Figures (A) and (C) depict the viability of Vero cells measured by an MTT assay after a total incubation of 134 hours. This protocol involved a 12-hour pre-treatment with either HC (A) or CAPE (C), followed by additional incubation steps as detailed in the legend. In contrast, figure (B) shows the cell viability of Vero cells treated with CAPE only, measured after a total incubation of 38 hours.

      • To avoid further confusion figure legend has been changed from "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment and 24 hr post-treatment and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment and 24 hr post-treatment. " to "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment followed by a 2-hour incubation with maintenance media, 24 hr post-treatment, and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment, followed by a 2-hour incubation with maintenance media and 24 hr post-treatment."

        Comment 12:-

      Fig. 1G and H are not referred to in the result text.

      • Response :-Thank you for pointing out the oversight regarding Fig. 1G and H not being referred to in the results text. We have added following statement Results p.1 Line no. 354 "Likewise, HC and CAPE treatment to Vero cells has shown a decrease in viral titer DENV-infected cells in a dose-dependent manner (Figure 1 G-H)."
      • *

      Comment 12:-

      Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned?

      • Response:-*Thank you for bringing this to our attention. We acknowledge this mistake regarding the mentioned concentrations at lines 342 and 343. RT-PCR was conducted for CHIKV using concentrations of 200 µM for HC, 25 µM for CAPE, and 1000 µM for DFMO. Similarly, for DENV, RT-PCR was performed with concentrations of 200 µM for HC, 2.5 µM for CAPE, and 1000 µM for DFMO. To avoid further confusion, Figure legends were revised and line no. 846 to 848 "(1F) RT-PCR for CHIKV with HC 200 µM, CAPE 25 µM, DFMO 1000 µM concentration (1I) RT-PCR for DENV with HC 200 uM, CAPE 2.5 uM and DFMO 1000 µM" *
      • *

      Comment 13:-

      qRT-PCR data is not very clear. Please consider elaborating on some details. Why were the statistics only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident.

      • Response:- We regret that the clarity of the qRT-PCR data was not satisfactory. We acknowledge your feedback and understand the importance of elaborating on certain details. The statistics were performed for all treatment groups, including HC, CAPE, and DFMO. However, the representation in the graph was adjusted by replacing the "top square bracket" with a "line" to avoid confusion. The y-axis of the graph depicts the log10 fold change in target gene expression relative to a designated virus control (VC). A value of ~ -2 on this axis corresponds to a significant downregulation, reflecting a 97-fold decrease in expression compared to the VC. A comparable graphical depiction is also evident in the work by Mudgal et al. (2022).

        Comment 14:-

      Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed'

      • Response :-Done as suggested. We have revised the sentence in question and similar ones accordingly. "In CHIKV nsP1, SAM is formed by residues Gly65, Ser66, Ala67, Pro83, Arg85, Ser86, Asp89, Thr137, Asp138…" changed to line no. 393 "In CHIKV nsP1, SAM binding site is lined by,….."

        Comment 15:-

      Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results.

      • Response:- Thank you for bringing these points to our attention. Done as suggested. We have simplified the presentation of the TLC results to enhance clarity and revised the methodology, results, figure, and legend accordingly. Also, we have quantified the TLC results. * -*Polyamine determination by Thin-layer chromatography (TLC)

      -Vero cells were treated with HC, CAPE and DFMO, as mentioned in the antiviral assay protocol. Similarly, HC-treated cells were collected after 12, 24, and 36 hr of treatment." Revised to " Vero cells were treated with CAPE (25 µM), HC (200 µM), and DFMO (1000 µM) for 36 hr …… Further, TLC images were quantified utilizing ImageJ software." *Figure legend 1:- (J) depicts the effect of polyamines level after treating with HC (200 µM) and CAPE (25 µM). Polyamine level of Vero treated cells at 12, 24, and 36 hr for HC and pre (12 hr) and post-treatment (24 h) for CAPE and DEMO, using untreated cells as a cell control (CC) for both of the conditions. 0.1 μM putrescine (put), spermine (spm), and spermidine (spd) as a positive control marker. changed to *

      "(J) the chromatographic analysis of polyamine levels in Vero cells after 36 hr treatment with (from left) CAPE (25 µM), HC (200 µM), DFMO(1000 µM), and cell control (CC), 0.1 μM putrescine (Put), spermine (Spm), and spermidine (Spd) as a positive control marker. "

      Results: Line no. 351 "Polyamine levels in cells treated with CAPE were significantly lower as compared to DFMO treatment (Figure 1J). Meanwhile, HC showed a reduction in polyamine levels with the initial 12 hr treatment; later, polyamine levels elevated gradually with time."

      Revised to line no. 371 to 373"After treatment with CAPE, HC, and DFMO to Vero cells, overall residual polyamine levels are 28.33%, 29.67 %, and 46 %, respectively, compared to cell control."

      Comment 16:-

      Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be

      'Panels D-G depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'

      • Response:-Thank you for the suggestion. We have updated the figure legend to ensure clarity based on your recommendation. (D,E,G,H) depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'.

        Comment 17:-

      Line 755: DFMO is wrongly written as 'DEMO'

      • Response:- Thank you for bringing that to our attention. We have corrected the typo, changing Line 845 'DEMO' to 'DFMO' as appropriate.

        Comment 18:-

      Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

      • Response:- We thank reviewer for your input. As per the suggestion we have elaborated the results on IFA. The qualitative application of IFA was chosen because of the absence of dedicated paid software/hardware for image quantification on the Thermofisher EVOS platform, thereby impeding our quantification efforts.

        Comment 19:-

      Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here.

      • Response:- Thank you for the feedback. Line no. 377:We have updated the line to specify the time as “ after 2 h of virus infection," and we have also revised this in the methodology section for clarity.

        Comment 20:-

      Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM?

      What will be the effect on increasing concentrations?

      Why were all these three polyamines added together?

      What is the effect of addition of individual polyamine in the rescue of viral titer?

      Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added simultaneously?

      Response:- We thank the reviewer, for raising these insightful questions. We performed an Exogenous polyamine addition assay as per Mounce et al. 2016 to maintain consistency with established practices and the research focus. The concentration of 1 µM biogenic polyamines (Putrescine, Spermidine, and Spermine) was chosen based on the findings of Mounce et al. (2016), where viral titers were restored to levels comparable to non-treated conditions at this concentration (Mounce, Cesaro, et al., 2016; Mounce, Poirier, et al., 2016)*. Furthermore, increasing the concentration of these polyamines did not yield significant additional effects on viral titer rescue, as observed in their study. *

      The potential influence of pre-treating cells with the biogenic polyamines (putrescine, spermidine, spermine) prior to viral infection, compared to simultaneous addition with the compound in question, is an interesting point. While Mounce et al. (2016) suggest this order may not significantly impact the rescue effect (Mounce, Poirier, et al., 2016)*. Further investigations are warranted to address this question definitively within the context of our specific experimental design. *

      Comment 20:-

      It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

      • Response:-Thank you for your insightful suggestion. We have addressed these results in detail in the discussion section of the manuscript. We conducted an Exogenous Polyamine Addition Assay following the methodology outlined by Mounce et al. (2016) to adhere to established procedures and align with our research objectives. Treatment with DFMO in the presence of exogenous polyamines, as well as treatment with DFMO followed by polyamine addition, led to the rescue of virus titers, as indicated by Mounce et al. (2016). Therefore, according to the data, the timing of exogenous polyamine addition may not be a significant factor. In our manuscript, the timing of polyamine and compound addition was consistent across all treatments (HC, CAPE, and DFMO).

        Comment 21:-

      Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID.

      • Response:-Thank you for bringing this to our attention. We have updated the figure legend in Supplementary Figure 2 to include complete information, indicating the virus names corresponding to the Accession numbers and GenBank IDs.

        Comment 22:-

      Line 392: '2 dimensions' ?

      • Response:-Thank you for bringing this to our attention. As suggested, we have made the change, replacing "2 dimensions" with "2D" for clarity.

        Comment 23:-

      Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

      • Response:- We appreciate the reviewer's feedback. We have expanded upon these results in more detail in the discussion section. Discussion p.4 line no. 512 "Biophysical interactions by TFS indicate distinct red shift for nsP1 and NS5 MTase, with each compound displaying specific affinities toward the target proteins." revised to line no. 551 to 557 "The binding affinities of SAM and GTP with CHIKV nsP1 and DENV NS5 MTase were investigated and used as a reference to compare with HC and CAPE. HC has a high binding affinity for both enzymes, as evidenced by the Kd values. Conversely, CAPE demonstrates a more selective binding profile, exhibiting a significantly stronger affinity towards nsP1 than NS5 MTase. Significantly, both HC and CAPE have demonstrated a dose-dependent red shift, indicating structural changes upon interaction (Figure 5 and Supplimentary figure 5)."
      • *

      Comment 25 Result 4. Fig. 6A and 6E: The text does not report this result (SDS-PAGE). Fig. 6

      • Response We appreciate the reviewer for bringing this to our attention. As per suggestion, we have incorporated the SDS-PAGE results in Fig. 6 in the text.line no. 467 to 468 "Single band at ~ 56 and ~ 32 kDa was observed in 12% SDS-PAGE for purified nsP1 and NS5 MTase, respectively ( Figure 6A and 6E)."

        Comment 24:-

      Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

      • Response:- Thank you for your intriguing question. We appreciate the reviewer's interest. We opted not to conduct enzymatic assays (inhibition assays) with DFMO, as it is a known analog of ornithine, a well-established inhibitor of the polyamine pathway (ornithine decarboxylase inhibitor). This decision was made as it was deemed outside the scope of our study.

        Comment 25:-

      Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

      • Response:- We thank the reviewer for their meticulous attention to detail. As per your observation, we have carefully reviewed the manuscript and made the necessary corrections, including changing "ml" to "mL", "µl" to "µL", and "E. coli" to " coli" (line no.. 1042). Additionally, we have standardized the usage of "titre" to "titer" across multiple figures. __References: __

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      Terui, Y., Yoshida, T., Sakamoto, A., Saito, D., Oshima, T., Kawazoe, M., Yokoyama, S., Igarashi, K., & Kashiwagi, K. (2018). Polyamines protect nucleic acids against depurination. The International Journal of Biochemistry & Cell Biology, 99, 147–153. https://doi.org/10.1016/J.BIOCEL.2018.04.008

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      Referee #3

      Evidence, reproducibility and clarity

      The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

      Major points

      While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition are not precise enough in the case of CHIK nsp1, and too weak in the case of DENV NS5 (detailed below).

      Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

      The discussion section requires more elaborate scientific justification rather than simply re-reporting the results.

      Specific major remarks:

      It would be best to organize the ms as follows: - Crystal structure of DENV MTase in complex with CAPE - Building of a model of nsp1 by superimposition with NS5 MTase - Modeling compound binding - Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature. - If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

      Figure 5 is problematic. - When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points. - Please describe more panel D in the legend. - Panel F and G: A reduction of 25 % at the highest concentration of inhibitor is a strong indication that there is no effect.

      Minor Points/Comments/ Suggestions:

      A. In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

      B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

      C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3. Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

      D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicates the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

      E. Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

      F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

      G. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.

      H. M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

      I. M&M, line 274: What is CE? Please expand the term before using the abbreviation.

      J. line 306. Ref. 53: This is not a reference.

      K. Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B. Fig. 1G and H are not referred to in the result text. Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned? qRT-PCR data is not very clear. Please consider elaborating on some details. Why the statistics were only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident. Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed' Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results. Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be 'Panels D-G depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'. Line 755: DFMO is wrongly written as 'DEMO' Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

      Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here. Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM? What will be the effect on increasing concentrations? Why were all these three polyamines added together? What is the effect of addition of individual polyamine in the rescue of viral titer? Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added at the same time? It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

      Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID. Line 392: '2 dimensions' ?

      Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

      Result 4. Fig. 6A and 6E: This result (SDS-PAGE) is not reported in the text. Fig. 6

      Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

      Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

      Significance

      This a body of work that is very interesting and has good potential, however it lacks the correct demonstration of the additive effect of MTase inhibition to polyamine depletion.

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      Referee #2

      Evidence, reproducibility and clarity

      Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with site involved in binding of RNA

      Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

      There are several points that need to be addressed, so here I provide some examples:

      1. Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery line 137 flavonoids play role in reducing of the levels of nsP1 in CHIKV - what this can possibly mean? Are shown to reduce level of nsP1 in CHIKV infected cells? line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, an inserted between the NheI and XhoI restriction sites... It should be impossible as one cannot insert RNA into bacterial plasmid DNA
      2. Missing parts. Examples
        • the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)
        • description of experiment shown on Figure 4 is missing
      3. Figure lacks quality (and figure legends are unclear) Examples:
        • it is impossible to understand what exactly is shown on Figure 1J
        • important information is missing, for example it is not clear what were concentrations of antiviral compounds for panels 1F and 1I
      4. wrong data
        • line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023
        • line 476 refers to family alphaviridae. This does not exist, family is Togaviridae
      5. unjustified conclusions. Example
        • authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)
      6. Insufficient analysis of data. Some cases there is significant discrepancy between results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies (CE and ELISA based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided on can assume that it is due to experimental errors.
      7. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

      The reviewer is sorry for not being able to provide more specific and useful comments and suggestions. To my opinion, manuscript should have been better prepared before submitting for review. Multiple mistakes, discrepancies and lack of clarity makes it difficult (for me nearly impossible) to focus on scientific value of study and provide constructive comments

      Significance

      It is difficult to assess the significance of the studys findings as the data presented and writing lacks sufficient quality and depth. While some experiments that can be understood (crystal structure, some antiviral assays) show potentially interesting scientific findings, the manuscript needs a major overhaul before it can be considered relevant for the scientific community.

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      Referee #1

      Evidence, reproducibility and clarity

      In this manuscript, the authors use structural and functional approaches to investigate the potential anti-DENV and anti-CHIKV activity of HC and CAPE, two naturally occurring compounds. They find that these compounds reduce cellular polyamine levels and specifically inhibit the viral methyltransferase (MTase) activity. Hence, the authors propose that HC and CAPE have anti-viral potential against DENV and CHIKV, which have been implicated in severe disease in humans.

      Overall, this is a straightforward investigation and is quite suitable for publication as a "first report" on the anti-MTase activity of these compounds. The data support the conclusions. This will be of interest to researchers in the anti-virals field. A strength of this investigation is the multi-faceted approach to get to the target of these compounds, i.e., the viral MTase enzymes. This is commendable.

      My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question.

      I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section.

      Significance

      Strengths- multi-faceted approach

      Target audience- researchers interested in anti-virals

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      Reply to the reviewers

      We would like to express our gratitude to the reviewers for their comments, which helped us to improve the quality of our manuscript. Below are the responses to each comment. We hope that these responses will satisfy the reviewers.

      Reviewer #1

      Evidence, reproducibility and clarity

      Summary: The nonsense-mediated mRNA decay (NMD) is and RNA quality pathway that eliminates mRNAs containing premature termination codons. Its mechanism has been studied for several decades but despite enormous progress we still don't have a satisfactory model that would explain most of the published observations. In particular, the mechanism has been proposed to differ substantially between yeast and metazoa. Yeast Nmd4 protein was previously shown to be involved in NMD, to interact with UPF1 and exhibit similarities with metazoan SMG6 and SMG5/7, that are normally believed to be specific for metazoan NMD (Dehecq et al., EMBO J, 2018). Barbarin-Bocahu et al now describe the crystal structure of the complex between the yeast UPF1 RNA helicase and Nmd4. Importantly, the authors show that interaction is required for NMD activity and increases the ATPase activity of UPF1. Barbarin-Bocahu et al equally show that this interaction and its role in NMD is conserved in the human UPF1-SMG6 complex, thus providing additional novel evidence for universal conservation of the NMD mechanism in eukaryotes. The manuscript carefully combines biochemistry, biophysics with functional in vivo studies. In my opinion, all the experiments are very well executed, generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe could strengthen the manuscript and enhance our confidence in the findings.

      We are grateful for the useful suggestions that have enabled us to improve our manuscript.

      Major comments:*

      *Page 7 - "Since the D1353A mutation completely abolishes the enzymatic activity of SMG6 (34), this strongly suggests that the PIN domain of Nmd4 is not endowed with endonucleolytic activity. " Could/was the endonucleolytic activity of NMD4 be tested?

      We agree with this important point. Our statement is based on previous site directed mutagenesis experiments on the PIN domain of human SMG6 (Galvan et al; 2006; EMBO Journal; PMID : 17053788 / Eberle et al; 2008; Nat. Struct. Mol. Biol.; PMID : 19060897), which showed that D1353 is the critical residue of SMG6 active site involved in the endonuclease enzymatic activity. Given that in yeast Nmd4 proteins, the corresponding residue is hydrophobic (Leu112 in S. cerevisiae Nmd4 and Phe114 in Kluyveromyces lactis Nmd4) and therefore cannot participate directly in catalysis, we assume that yeast Nmd4 proteins have no endonucleolytic activity.

      Furthermore, despite decades of research in this field, no endonucleolytic activity has been described as being involved in the NMD pathway of S. cerevisiae (the model system in which the NMD mechanism was discovered in the 1970's), whereas it has been well characterized in the NMD pathway of metazoans for more than twenty years (Gatfield and Izaurralde; Nature; 2004; PMID : 15175755 / Huntzinger et al; RNA; 2008; PMID : 18974281 / Eberle et al; Nat. Struct. Mol. Biol.; 2009; PMID : 19060897 / Lykke-Andersen et al; Genes Dev.; 2014; PMID : 25403180). Our attempts to demonstrate an endonucleolytic activity of purified Nmd4 in vitro were not successful. This negative result could be due to many reasons, including loss of enzymatic activity in the tested buffer, the absence of an important cofactor or the choice of the tested RNA. For these reasons, we prefer not to include this type of negative result in the current manuscript.

      We hope that, on the basis of the above informations, the reviewer will agree that further substantial efforts to demonstrate a hypothetical endonucleolytic activity of Nmd4 are unlikely to be fruitful. Moreover, we believe that even if yeast Nmd4 turns out to behave as an endonuclease, this fact does not change the main message of the manuscript.

      Page 10 - The two proteins bind RNA with reasonable affinity. The complex binds polyU RNA with Kd of 0.44 μM . The authors suggest, based on structure superpositions, that RNA fragments bound to the PIN domain and Upf1-HD have opposite orientations. But since they have the complex ready to crystallize, did they attempt to determine the structure with of the complex with RNA? The complex is quite small (~100 kDa with RNA) but it could be even visible by cryo-EM. I don't insist that such a structure needs to be included but it might perhaps be easy to do and would surely strengthen the story. If it is too difficult, it could at least be mentioned that it was tried?

      We agree that it would be interesting to determine the crystal structure of the complex with a short RNA fragment. Unfortunately, despite extensive efforts, we could not obtain crystals of the complex in the presence of RNA. This is probably due to the large movements of the RecA2 and 1B domains relative to the RecA1 domain observed in former studies upon RNA binding to Upf1. We have mentioned that we tried to crystallize this complex in the absence or the presence of a short oligonucleotide in our revised manuscript.

      As far as single-particle cryo-EM is concerned, we are aware that recent advances in this field should make it possible to determine the structure of the Nmd4-Upf1-RNA complex, but we do not yet have the necessary expertise in this technique. Despite the interesting information that such a structure could provide, we therefore consider that this would require a very significant investment and that it is beyond the scope of this manuscript.

      I think it is important to demonstrate that the structure-based mutants don't significantly impact the overall structure of the proteins (e.g. glycine residues are mutated within helices). At least gel filtration profiles with gels of the WT and mutated proteins should be shown in SI.

      Thank you very much for highlighting this point. We fully agree that it is important to demonstrate that the Upf1 and Nmd4 mutants used in the in vitro experiments (pull-down and ATPase assays) are not affected in their overall folding. As suggested by the reviewer, we have included gel filtration chromatograms for WT and mutant proteins (Figures S2A for Upf1-HD proteins and S2B for His6-ZZ-Nmd4 proteins). These chromatograms clearly show that the different mutants behave very similarly to the WT proteins during purification, demonstrating that the overall structures of the mutants are very similar to those of the wild-type proteins. We have also included the Coomassie blue stained SDS-PAGE analysis of the proteins present in the main peak to show the purity of the final proteins.

      Perhaps the main finding of this manuscript is the conservation of the UPF1-Nmd4 interaction in human UPF1-SMG6. But the interaction is only demonstrated by co-IP with ectopically expressed human proteins in human cells that contain all the other human proteins as well. It would probably be more convincing to demonstrated the interaction in pull-downs with purified proteins as done for the yeast complex.

      Thank you for highlighting what we consider to be one of the most interesting findings presented in our manuscript. We agree that pull-down experiments using pure protein fragments expressed in E. coli would have been ideal to further confirm our co-IP results and to validate that mutations do not affect the overall structure of SMG6. Unfortunately, despite considerable efforts, we were unable to express sufficient quantities of the SMG6-[207-580] fragment or shorter versions as soluble proteins in E. coli. Indeed, Elena Conti's laboratory had the same experience according to a statement in a paper on SMG6 (Chakrabarti et al; 2014 Nucleic Acids Research; PMID: 25013172), indicating that this region protein is very difficult to work with. As we have not yet set up protein over-expression techniques in human cells or baculovirus-infected insect cells in our laboratory, we have not been able to try these expression systems to express these SMG6 domains. These are the reasons why we decided to demonstrate this interaction by co-IP experiment using ectopically expressed tagged proteins in human cells and all appropriate controls.

      In addition, using purified proteins would enable testing whether the mutations in SMG6 don't affect the overall structure of the mutants compared to the WT.

      We agree that this is an important issue. Several bioinformatics tools, including AlphaFold2 (identifier: AF-Q86US8-F1), predict that the human SMG6-[207-580] fragment is largely unstructured (see panel A of figure below). Furthermore, the pLDDT values or confidence scores for this region in the AlphaFold2 model are very low (below 50), indicating that the structure of this region is poorly predicted (see panel B of figure below). Therefore, biophysical techniques to assess that the overall structure of this fragment is not affected by the introduced mutations are very limited. However, we did not observe reduced levels of SMG6 mutants compared with WT in human cells expressing these variants (Fig. 4B and S4), so we believe that these mutants behave similarly to the wild-type fragment, as is often postulated by scientists for in cellulo studies. Furthermore, if these mutants drastically affect the overall structure of SMG6, we would expect NMD to be strongly affected, resulting in a notable accumulation of NMD RNA substrates in our in cellulo experiments when the effect of the double mutant (M2) is compared to that of the SMG6 WT protein (Fig. 4C). This was not the case. On the basis of all these elements, we assume that the overall structure of the SMG6 protein is not affected by these mutations.

      Figure for reviewing purpose : Model of the three-dimensional structure of human SMG6 protein generated by AlphaFold2.

      A. Model of human SMG6 protein (green) with the region 207-580 used in our study colored in red.

      B. Model of human SMG6 protein (green) colored according to the pLDDT values. Orange : pLDDT 90.

      Since the detected similarity to Nmd4 is only in a region covering residues 440-470, why is the tested construct much larger (207-580) including extra, large disordered regions.

      For in cellulo studies, it has previously been shown that the SMG6-[207-580] fragment is expressed as a stable protein in human cells and is responsible for the phospho-independent interaction between UPF1 and SMG6 (Chakrabarti et al; 2014; Nucleic Acids Research; PMID: 25013172). As our aim was not to reduce this SMG6 region to a shorter peptide but to conduct an amino acid-level analysis by site-directed mutagenesis, we decided to perform our experiments using the same SMG6 domain as Conti's laboratory and to mutate conserved residues on this fragment.

      Finally, the most convincing way to show and characterize the human UPF1-SMG6 interaction would be an X-ray structure. It might be feasible to crystallize human UPF1 HD domain with a SMG6 peptide. Or at least an Alphafold model could be included? I had a quick try just with the Colabfold and using the HD domain and the SMG6 peptide, Alphafold can predict convincingly the binding of the region around W456 and in some models even around R448. I think that this would strengthen the conclusions in this part of the manuscript.

      We agree that determination of the crystal structure of human UPF1 HD linked to this region of SMG6 protein interaction would have further supported our conclusions on the conservation of UPF1-Nmd4 interaction in human UPF1-SMG6. However, due to the SMG6 expression problems mentioned above, we were unable to reconstitute the human complex in vitro, which precluded crystallization assays.

      Based on this suggestion, we generated a model of human UPF1-HD bound to the 421-480 region of human SMG6 using AlphaFold2 Colabfold. Of the various models proposed (25 in total), most are very similar and show that the side chains of R448 and W456 of SMG6 bind to regions of human UPF1 corresponding to the region of the yeast protein that interacts with R210 and W216 of Nmd4. This model is consistent with our hypothesis and we have decided to include it in the revised manuscript as suggested (Fig. EV6). We thank the reviewer for this constructive comment.

      We have added the following text to mention this model : « Based on this observation, we generated a model of the complex between human UPF1-HD and the region 421-480 of SMG6 using AlphaFold2 software (1,2). In this model, the SMG6 fragment binds to the same region of UPF1-HD as the Nmd4 « arm » (Fig. EV6). In particular, the R448 and W456 side chains of SMG6 match almost perfectly with R210 and W216 side chains of S. cerevisiae Nmd4, suggesting that this conserved region from SMG6 is involved in the interaction between the SMG6 and UPF1-HD proteins. »

      Does the SMG6 addition also increases the ATPase activity of UPF1?

      This is a very good point and we agree that the results of such an experiment may have further supported our conclusions about the conservation of the Upf1-Nmd4 interaction in human UPF1-SMG6. Unfortunately, due to the SMG6 protein expression problems mentioned above, we could not perform these in vitro experiments.

      Minor comments: Examples of electron density omit maps of the key interaction interfaces should be shown in Supplementary Information for the reader to be able to judge the crystallography data quality.

      Following this suggestion, we have added two panels showing electron density omit maps of residues at the interface in Fig. S1. We hope that this will convince the reader of the quality of our crystallographic data. We have also added the following sentence to the main text : « The overall quality of the electron density map allowed us to unambiguously identify the residues of the two proteins involved in the formation of the complex (Fig. S1A-B). »

      I suggest to add the Kd values to ITC panels for clarity in main and EV figures.

      We have taken this suggestion into account for figures 2A and EV5.

      On page 10: What experiment is this referring to : "This is in agreement with our ITC experiments (carried out in the absence of a non-hydrolyzable ATP analog), which revealed no major synergistic effect between the two proteins for RNA binding." Results in EV4A? Or some other not shown data? The results in EV4A do show an increase in RNA binding when both proteins are in a complex.

      Thank you for your comment. We realize that this sentence was not clear. We refer to the ITC data for the interaction of Upf1-HD, Nmd4 or the complex with RNA (Fig. EV5A). These data show a 2.3-fold increase in the affinity of Upf1 for RNA in the presence of Nmd4, which we consider to be a notable effect but not a major one. Based on the second reviewer's comments that our comparison between Nob1 and the PIN domain of Nmd4 is not convincing, we have decided to delete this speculative section, which did not address an important point in our current study. We will address this point using more direct and sophisticated methods in future work.

      On page 16, "organsms" should be" organisms"

      Typo corrected.

      In certain figure legends the panel labels (A,B,C..) are missing (e.g. Fig 3, EV1, EV5).

      We apologize for this problem ,which was due to a conversion problem when preparing the PDF file of the submitted article. This problem has now been corrected.

      The PIN domain structure was solved only to determine the structure of the complex? I only found it mentioned in the methods and no other mention of this structure in the main text. Maybe one sentence could be added to the results to explain why this structure was solved and how it compares to the complex structure.

      We agree that we forgot to explain why we solved the structure of the PIN domain of Nmd4. The point was to help in the determination of the structure of the complex. We have added the following sentence to the main text to explain this point: « We also determined the 1.8 Å resolution crystal structure of the PIN domain of Nmd4 (residues 1 to 167) to help us determine the structure of the Nmd4/Upf1-HD complex. As this structure is virtually identical to the structure of the PIN domain of Nmd4 in the complex (rmsd of 0.5 Å over 163 C𝛼 atoms between the two structures), we will only describe the structure of this domain in the Upf1-Nmd4 complex. »

      Significance

      This is a important study, providing detailed insight into the function on Nmd4, SMG6 and UPF1 NMD. The results also point towards a conserved mechanism on NMD between yeast and human. I would like to highlight the quality of the experiments. This study will be of great interest to people working on NMD but also more broadly to scientists working on RNA, helicases and structural biologists.

      We are very grateful for the reviewer's comments about the broad interest and overall quality of our work.

      Reviewer #2

      Evidence, reproducibility and clarity

      In this study, the authors solved the crystal structure of the UPF1 helicase domain in complex with Nmd4. Through the structure and biochemical studies, they uncovered a region responsible for Nmd4 binding to UPF1, also important for their function in NMD. In the end, the authors also extended their findings to the human SMG6, proposing a conserved mechanism for Nmd4 and SMG6.

      The mechanism of UPF1 functioning during NMD is a long-existing question. For decades, people have been trying to find out the roles of all the NMD factors during this process. This study visualized the first direct connection between UPF1 and the putative SMG6 homolog, Nmd4. Undoubtedly, it will aid our understanding of how the whole process works.

      One of the limitations of this study is the conservation between Nmd4 and SMG6. Although they both have a PIN domain, Nmd4 is inactive while SMG6 is active. During NMD, SMG6 is thought to work to cut the mRNA, thus promoting the degradation of the non-functional mRNA. Therefore, Nmd4 and SMG6 may only share a similar binding mode with UPF1, however, they do not share similar functions. This study might only apply to yeast study.

      We respectfully disagree with this comment. The role of SMG6 in NMD cannot be attributed solely to the endonuclease activity of the SMG6 PIN domain alone. Indeed, recruitment of the SMG6 PIN domain alone to an mRNA is not sufficient to destabilize it (Nicholson et al; 2014; Nucleic Acids Research; PMID: 25053839). This clearly indicates that other regions of SMG6 are critical for NMD. In our manuscript, we unveil the conservation of the Upf1-Nmd4 interaction in human UPF1-SMG6 (and probably more generally in metazoans) and show that this interaction plays a role in the optimal removal of NMD substrates. We strongly believe that our results are not only applicable to the study of yeast, but will fuel future studies in human cells aimed at describing the mechanistic details of the human NMD pathway.

      comments: the study write in a very clear way, and most of the experiments are clear and sound. I do not have any major comments. I only have a few minor comments, listed below:

      We are very grateful for the reviewer's comments about the overall quality of our manuscript and of the experimental work.

      1:The authors also solved the PIN domain of the SMG6. This is a result worth showing in the main figure.

      In our study, we did not solve the structure of the human SMG6 PIN domain. This was done by Dr. Conti's group in 2006 (Galvan et al; 2006; EMBO Journal; PMID : 17053788). This is the reason why we do not include this in the main figure. However, we have solved the crystal structure of Nmd4 PIN domain alone to help us determine the structure of the complex. Since it is very similar to the structure of the Nmd4 PIN domain in the complex with Upf1, we do not describe this structure in details. Following up the suggestion from another reviewer, we have included the following sentence mentioning that we have also determined the structure of Nmd4 PIN domain in the main text : « We also determined the 1.8 Å resolution crystal structure of the PIN domain of Nmd4 (residues 1 to 167) to help us determine the structure of the Nmd4/Upf1-HD complex. As this structure is virtually identical to the structure of the PIN domain of Nmd4 in the complex (rmsd of 0.5 Å over 163 C𝛼 atoms between the two structures), we will only describe the structure of this domain in the Upf1-Nmd4 complex. »

      2:It would be easier to read if the authors could add all the binding constants directly into the ITC panels.

      We have taken this suggestion into account for figures 2A and EV5.

      3:I am confused with His6-ZZ. Is ZZ a protein tag?

      The ZZ protein is a tag consisting of a tandem of the Z-domain from Staphylococcus aureus protein A. This domain binds to the Fc region of IgG and has been shown to improve expression levels and stability of recombinant proteins. In our case, it proved crucial to obtain mg amounts of the yeast Nmd4 protein and to enhance considerably its stability. We have added the following sentence in the « Materials and methods » section of the manuscript : « The ZZ-tag consists in a tandem of the Z-domain from Staphylococcus aureus protein A and was used as an enhancer of protein expression and stability. »

      4:The comparison between Nob1 and the PIN domain of Nmd4 is not convincing for me. Since the PIN domain is not required for the binding between Nmd4 and UPF1, the conformation of the PIN domain could be a result of the crystal packing. Thus, it is still possible that Nmd4 and UPF1 bind to the same RNA. To this end, I challenge the conclusion the authors have made on the mRNA binding part.

      We agree with your comment. Since this comparison is purely speculative and is not a major focus of our study, we decided to remove this section. We will address this point using more direct and sophisticated methods in future work aimed at elucidating this aspect.

      5: "Showing that Nmd4 stabilizes Upf1-HD on RNA in the absence of ATP and that Upf1 is the main RNA binding factor in the Nmd4/Upf1-HD complex." As mentioned above, I don't think one can make the conclusion UPF1 is the main RNA binding factor; there shouldn't be a main and minor. Meanwhile, what will happen if you add ATP in? Or AMPPNP? Or ADP?

      We agree with your comment that our current data do not allow to conclude precisely about the role of Upf1 as major RNA binding factor. We have replaced this sentence by the following one : « Whether this increase in affinity is due to a synergistic effect between both proteins or to an allosteric stimulation of one partner on the RNA binding property of the second partner remains to be clarified. ».

      Regarding the role of the nucleotides on RNA binding properties of the Upf1 helicase domain or the complex, we faced precipitation problems when mixing high concentrations Upf1 and nucleotides for ITC experiments, making difficult to determine Kd values for the interaction between Upf1 and RNA in the presence of nucleotides. However, in a previous study (Dehecq et al; 2018; EMBO J; PMID : 30275269), we observed that AMPPNP did not affect the amount of Nmd4 and Upf1-HD co-precipitated by an RNA oligonucleotide, indicating that nucleotide does not significantly affect the interaction of the complex with RNA.

      6: "But also that a physical interaction between Upf1-HD and the PIN domain exists in vitro, although we were unable to detect it using our various interaction assays." This also confused me, since one cannot detect the interaction in any assay, how could you be so confident there is a physical interaction? Have you tested assays which are good for weak binding?

      We understand that this sentence may be confusing. The tests we have used to determine whether there is a physical interaction between the PIN domain of Nmd4 and Upf1-HD are ITC and pull-down. These are excellent methods for detecting stable interactions with dissociation constants (Kd) in the nanomolar to tens of micromolar range. These two methods did not indicate any direct interaction between the PIN domain of Nmd4 and Upf1-HD. However, we observed that the PIN domain of Nmd4 stimulates the ATPase activity of Upf1-HD to the same extent as the « arm » of Nmd4. This is an indirect indication that the Nmd4 PIN may interact with Upf1-HD, otherwise a stimulatory effect would not be expected. Our radioactivity-based ATPase assay is very sensitive, allowing the detection of a stimulatory effect due to a transient interaction between the PIN domain of Nmd4 and Upf1-HD, which, as indicated above, could not be detected with the interaction assays used. We would also like to point out that in our ATPase conditions, Upf1-HD (0.156 µM) is incubated with a 20-fold molar excess (3.12 µM) of its partners (Nmd4-FL, Nmd4 « arm » or Nmd4 PIN). Such an excess cannot be used in our interaction tests. This could explain the stimulatory effect detected for the PIN domain of Nmd4 in our ATPase assay.

      We have clarified this section by adding the following sentences: « We were unable to detect such an interaction using our different interaction assays (pull-down and ITC), which are optimal for studying interactions with dissociation constants (Kd) in the nanoM to tens of microM range. We therefore assume that a transient low-affinity interaction (high Kd value not detected by our binding assays) exists between Upf1-HD and PIN Nmd4 and can only be detected by highly sensitive assays such as our radioactivity-based ATPase assay, which was performed with a 20-fold molar excess of PIN Nmd4 domain over Upf1-HD. »

      7: Figure 4B should be done in the context of the full length of SMG6 and UPF1.

      **Referees cross-commenting**

      *This session contains comments from both Rev1 and Rev2*

      Rev1:

      There seems to be a contradiction in comments on Figure 4B. I agree with Reviewer 2 that using FL proteins will be informative to see whether the FL proteins indeed interact (or not in the case of the mutants).

      If one wants to use this experiment to map the interacting regions, then I think that the UPF1 HD domain and the short conserved region of SMG6 should be used. The long fragment SMG6 207-580 is not ideal for either. The short constructs would be more suited for a pull-down experiments (like done for the yeast proteins).

      Rev2

      Response to reviewer #1, It is necessary to use the full-length protein (FL protein) to map the interface unless they have pre-existing information to support mapping down to short fragments.

      In addition, performing further structural work would be beyond the scope of this study. Given the additional time and effort required, I do not recommend doing so for this study.

      Rev1:

      As I said, I agree with using the FL proteins. The pre-existing information supporting the mapping comes from sequence alignments with the yeast structure and the mutagenesis. This is further confirmed by Alphafold modeling which in my opinion should be included. As I mentioned in my review, I don't insist on further structural work

      Thank you very much for this comment and the discussions between reviewers, which show that we didn't explain our experimental strategy clearly. Human UPF1 has been shown to interact with SMG6 in both phospho-dependent and phospho-independent modes. In our manuscript, we focus on characterizing the phospho-independent interaction. For this reason, we cannot perform this experiment using the full-length version of SMG6 and UPF1, otherwise the effects of our point mutants on the UPF1-SMG6 interaction could be masked by the phospho-dependent interaction occurring between domain 14.3.3 of SMG6 and the C-terminus of Upf1. To circumvent this problem, we were inspired by former in cellulo studies, which have shown that the SMG6-[207-580] fragment is expressed as a stable protein in human cells and is responsible for the phospho-independent interaction between UPF1 and SMG6 (Chakrabarti et al; 2014; Nucleic Acids Research; PMID: 25013172). Similarly, the helicase domain of UPF1 was found to be sufficient for this phospho-independent interaction with human SMG6 (Nicholson et al; 2014; Nucleic Acids Research; PMID: 25053839). These are the reasons why we decided to use this protein domains in our in cellulo studies to test the effect of our point mutants on the interaction. As indicated above in an answer to one comment to reviewer #1, as our aim was not to reduce this SMG6 region to a shorter peptide but to conduct an amino acid-level analysis by site-directed mutagenesis, this is also why we decided to perform our experiments using the same SMG6 domain as Conti's laboratory and to mutate conserved residues on this fragment. We have also included the AlphaFold2 model of the complex between human UPF1 and SMG6 in our revised version.

      To clarify this point, we have amended the relevant section as follows: « To determine whether this motif might be involved in the interaction between SMG6 and UPF1-HD proteins, we ectopically expressed the region comprising residues 207-580 of human SMG6 fused to a C-terminal HA tag (SMG6-[207-580]-HA) and human UPF1-HD (residues 295-921 fused to a C-terminal Flag tag; UPF1-HD-Flag) in human HEK293T cells, as these regions have previously been shown to be responsible for the phosphorylation-independent interaction between these two proteins. Compared to the full-length UPF1 and SMG6 proteins, these constructs also preclude our findings of any interference from the phosphorylation-dependent interaction occurring between the C-terminus of UPF1 and the 14-3-3 domain of SMG6. »

      8: "The NMD mechanism not only targets mRNAs but also small nucleolar RNAs (snoRNAs) and long noncoding RNAs (lncRNAs) harboring bona fide stop codons but in a specific context such as short upstream open reading frame (uORF), long 3'-UTRs, low translational efficiency or exon-exon junction located downstream of a stop codon." "First, for mRNAs with long 3'-UTRs, the 3'-faux UTR model posits that a long 3 spatial distance between a stop codon and the mRNA poly(A) tail destabilizes NMD substrates by preventing the interaction between the eRF1-eRF3 translation termination complex bound to the A- site of a ribosome recognizing a stop codon and the poly(A)-binding protein (Pab1 or PABP in S. cerevisiae and human, respectively)." These are difficult to read.

      Thank you for this suggestion to improve the clarity of our manuscript. We have tried to make these sentences easier to read as follow:

      « The NMD mechanism also targets mRNAs, small nucleolar RNAs (snoRNAs) and long noncoding RNAs (lncRNAs) carrying normal stop codons located in a specific context (short upstream open reading frame or uORF, long 3'-UTRs, low translational efficiency or exon-exon junction located downstream of a stop codon (3-11)). »

      « The first model, the 3'-faux UTR model posits that for mRNAs with long 3'-UTRs, a long spatial distance between a stop codon and the mRNA poly(A) tail destabilizes NMD substrates. Indeed, it would prevent the physical interaction between the eRF1-eRF3 translation termination complex recognizing a stop codon in the A-site of the ribosome and the poly(A)-binding protein (Pab1 or PABP in S. cerevisiae and human, respectively) bound to the 3' poly(A) tail (12-14). »

      9: please add the Ramachandran plot values.

      Thank you for pointing out this omission. These values have been included in Table EV1.

      __Significance __

      NMD is one of the major topics in the field of gene translational regulation research. this study will be of interest to a broad audience. i am an expert in the structure study in translation. However, I have limited experience in the in vivo study of NMD substrates.

      We are very grateful for the reviewer's comments about the broad interest and the overall quality of our work.

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      Referee #2

      Evidence, reproducibility and clarity

      In this study, the authors solved the crystal structure of the UPF1 helicase domain in complex with Nmd4. Through the structure and biochemical studies, they uncovered a region responsible for Nmd4 binding to UPF1, also important for their function in NMD. In the end, the authors also extended their findings to the human SMG6, proposing a conserved mechanism for Nmd4 and SMG6.

      The mechanism of UPF1 functioning during NMD is a long-existing question. For decades, people have been trying to find out the roles of all the NMD factors during this process. This study visualized the first direct connection between UPF1 and the putative SMG6 homolog, Nmd4. Undoubtedly, it will aid our understanding of how the whole process works.

      One of the limitations of this study is the conservation between Nmd4 and SMG6. Although they both have a PIN domain, Nmd4 is inactive while SMG6 is active. During NMD, SMG6 is thought to work to cut the mRNA, thus promoting the degradation of the non-functional mRNA. Therefore, Nmd4 and SMG6 may only share a similar binding mode with UPF1, however, they do not share similar functions. This study might only apply to yeast study.

      comments: the study write in a very clear way, and most of the experiments are clear and sound. I do not have any major comments. I only have a few minor comments, listed below:

      1:The authors also solved the PIN domain of the SMG6. This is a result worth showing in the main figure.

      2:It would be easier to read if the authors could add all the binding constants directly into the ITC panels.

      3:I am confused with His6-ZZ. Is ZZ a protein tag?

      4:The comparison between Nob1 and the PIN domain of Nmd4 is not convincing for me. Since the PIN domain is not required for the binding between Nmd4 and UPF1, the conformation of the PIN domain could be a result of the crystal packing. Thus, it is still possible that Nmd4 and UPF1 bind to the same RNA. To this end, I challenge the conclusion the authors have made on the mRNA binding part.

      5: "Showing that Nmd4 stabilizes Upf1-HD on RNA in the absence of ATP and that Upf1 is the main RNA binding factor in the Nmd4/Upf1-HD complex." As mentioned above, I don't think one can make the conclusion UPF1 is the main RNA binding factor; there shouldn't be a main and minor. Meanwhile, what will happen if you add ATP in? Or AMPPNP? Or ADP?

      6: "But also that a physical interaction between Upf1-HD and the PIN domain exists in vitro, although we were unable to detect it using our various interaction assays." This also confused me, since one cannot detect the interaction in any assay, how could you be so confident there is a physical interaction? Have you tested assays which are good for weak binding?

      7: Figure 4B should be done in the context of the full length of SMG6 and UPF1.

      8: "The NMD mechanism not only targets mRNAs but also small nucleolar RNAs (snoRNAs) and long noncoding RNAs (lncRNAs) harboring bona fide stop codons but in a specific context such as short upstream open reading frame (uORF), long 3'-UTRs, low translational efficiency or exon-exon junction located downstream of a stop codon." "First, for mRNAs with long 3'-UTRs, the 3'-faux UTR model posits that a long 3 spatial distance between a stop codon and the mRNA poly(A) tail destabilizes NMD substrates by preventing the interaction between the eRF1-eRF3 translation termination complex bound to the A- site of a ribosome recognizing a stop codon and the poly(A)-binding protein (Pab1 or PABP in S. cerevisiae and human, respectively)." These are difficult to read.

      9: please add the Ramachandran plot values.

      Significance

      NMD is one of the major topics in the field of gene translational regulation research. this study will be of interest to a broad audience. i am an expert in the structure study in translation. However, I have limited experience in the in vivo study of NMD substrates.

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

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      Summary:

      The nonsense-mediated mRNA decay (NMD) is and RNA quality pathway that eliminates mRNAs containing premature termination codons. Its mechanism has been studied for several decades but despite enormous progress we still don't have a satisfactory model that would explain most of the published observations. In particular, the mechanism has been proposed to differ substantially between yeast and metazoa. Yeast Nmd4 protein was previously shown to be involved in NMD, to interact with UPF1 and exhibit similarities with metazoan SMG6 and SMG5/7, that are normally believed to be specific for metazoan NMD (Dehecq et al., EMBO J, 2018). Barbarin-Bocahu et al now describe the crystal structure of the complex between the yeast UPF1 RNA helicase and Nmd4. Importantly, the authors show that interaction is required for NMD activity and increases the ATPase activity of UPF1. Barbarin-Bocahu et al equally show that this interaction and its role in NMD is conserved in the human UPF1-SMG6 complex, thus providing additional novel evidence for universal conservation of the NMD mechanism in eukaryotes. The manuscript carefully combines biochemistry, biophysics with functional in vivo studies. In my opinion, all the experiments are very well executed, generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe could strengthen the manuscript and enhance our confidence in the findings.

      Major comments:

      Page 7 - "Since the D1353A mutation completely abolishes the enzymatic activity of SMG6 (34), this strongly suggests that the PIN domain of Nmd4 is not endowed with endonucleolytic activity. " Could/was the endonucleolytic activity of NMD4 be tested?

      Page 10 - The two proteins bind RNA with reasonable affinity. The complex binds polyU RNA with Kd of 0.44 μM . The authors suggest, based on structure superpositions, that RNA fragments bound to the PIN domain and Upf1-HD have opposite orientations. But since they have the complex ready to crystallize, did they attempt to determine the structure with of the complex with RNA? The complex is quite small (~100 kDa with RNA) but it could be even visible by cryo-EM. I don't insist that such a structure needs to be included but it might perhaps be easy to do and would surely strengthen the story. If it is too difficult, it could at least be mentioned that it was tried?

      I think it is important to demonstrate that the structure-based mutants don't significantly impact the overall structure of the proteins (e.g. glycine residues are mutated within helices). At least gel filtration profiles with gels of the WT and mutated proteins should be shown in SI.

      Perhaps the main finding of this manuscript is the conservation of the UPF1-Nmd4 interaction in human UPF1-SMG6. But the interaction is only demonstrated by co-IP with ectopically expressed human proteins in human cells that contain all the other human proteins as well. It would probably be more convincing to demonstrated the interaction in pull-downs with purified proteins as done for the yeast complex. In addition, using purified proteins would enable testing whether the mutations in SMG6 don't affect the overall structure of the mutants compared to the WT. Since the detected similarity to Nmd4 is only in a region covering residues 440-470, why is the tested construct much larger (207-580) including extra, large disordered regions. Finally, the most convincing way to show and characterize the human UPF1-SMG6 interaction would be and X-ray structure. It might be feasible to crystallize human UPF1 HD domain with a SMG6 peptide. Or at least an Alphafold model could be included? I had a quick try just with the Colabfold and using the HD domain and the SMG6 peptide, Alphafold can predict convincingly the binding of the region around W456 and in some models even around R448. I think that this would strengthen the conclusions in this part of the manuscript.

      Does the SMG6 addition also increases the ATPase activity of UPF1?

      Minor comments:

      Examples of electron density omit maps of the key interaction interfaces should be shown in Supplementary Information for the reader to be able to judge the crystallography data quality.

      I suggest to add the Kd values to ITC panels for clarity in main and EV figures.

      On page 10: What experiment is this referring to : "This is in agreement with our ITC experiments (carried out in the absence of a non-hydrolyzable ATP analog), which revealed no major synergistic effect between the two proteins for RNA binding." Results in EV4A? Or some other not shown data? The results in EV4A do show an increase in RNA binding when both proteins are in a complex.

      On page 16, "organsms" should be" organisms"

      In certain figure legends the panel labels (A,B,C..) are missing (e.g. Fig 3, EV1, EV5).

      The PIN domain structure was solved only to determine the structure of the complex? I only found it mentioned in the methods and no other mention of this structure in the main text. Maybe one sentence could be added to the results to explain why this structure was solved and how it compares to the complex structure.

      Referees cross-commenting

      This session contains comments from both Rev1 and Rev2

      Rev1:

      There seems to be a contradiction in comments on Figure 4B. I agree with Reviewer 2 that using FL proteins will be informative to see whether the FL proteins indeed interact (or not in the case of the mutants). If one wants to use this experiment to map the interacting regions, then I think that the UPF1 HD domain and the short conserved region of SMG6 should be used. The long fragment SMG6 207-580 is not ideal for either. The short constructs would be more suited for a pull-down experiments (like done for the yeast proteins).

      Rev2

      Response to reviewer #1, It is necessary to use the full-length protein (FL protein) to map the interface unless they have pre-existing information to support mapping down to short fragments. In addition, performing further structural work would be beyond the scope of this study. Given the additional time and effort required, I do not recommend doing so for this study.

      Rev1:

      As I said, I agree with using the FL proteins. The pre-existing information supporting the mapping comes from sequence alignments with the yeast structure and the mutagenesis. This is further confirmed by Alphafold modeling which in my opinion should be included. As I mentioned in my review, I don't insist on further structural work

      Significance

      This is a important study, providing detailed insight into the function on Nmd4, SMG6 and UPF1 NMD. The results also point towards a conserved mechanism on NMD between yeast and human. I would like to highlight the quality of the experiments. This study will be of great interest to people working on NMD but also more broadly to scientist working on RNA, helicases and structural biologists.

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      Learn more at Review Commons


      Reply to the reviewers

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

      In the present manuscript, the authors analyzed diel oscillations in the brain and olfactory organs' transcriptome of Aedes aegypti and Anopheles culicifacies. The analysis of their RNAseq results showed an effect of time of day on the expression of detoxification genes involved in oxidoreductase and monooxygenase activity. Next, they investigated the effect of time of day on the olfactory sensitivity of Ae. aegypti and An. gambiae and identified the role of CYP450 in odor detection in these species using RNAi. In the last part of the study, they used RNAi to knock down the expression of one of the serine protease genes and observed a reduction in olfactory sensitivity. Overall, the experiments are well-designed and mostly robust (see comment regarding the sample size and data analysis of the EAG experiments) but do not always support the claims of the authors. For example, since no experiments were conducted under constant conditions, the circadian (i.e., driven by the internal clocks) effects are not being quantified here. In addition, knocking down the expression of a gene showing daily variations in its expression and observing an effect on olfactory sensitivity is not sufficient to show its role in the daily olfactory rhythms. Knowledge gaps are not well supported by the literature, and overstatements are made throughout the manuscript. Our detailed comments are listed below.

      We sincerely thank the reviewer for their time and consideration, and appreciate the thorough review of our manuscript. Their insightful comments have greatly enriched our work. We also apologies for instances of overinterpreting the data. Your feedback has helped us recognize areas where clarity and caution are needed, and we are committed to addressing these concerns in our revisions. Thank you for your valuable input and guidance.

      Major comments

      Introduction

      1. Several statements made in the introduction are misleading and suggest that authors are trying to exaggerate the impact of their work. For example, "Furthermore, different species of mosquitoes exhibit plasticity and distinct rhythms in their daily activity pattern, including locomotion, feeding, mating, blood-feeding, and oviposition, facilitating their adaptation into separate time-niches (7, 8), but the underlying molecular mechanism for the heterogenous temporal activity remains to be explored." is not accurate since daily rhythms in mosquitoes' transcriptomes, behavior, and olfactory sensitivity have been the object of several publications. Even though some of them are listed later in the introduction, they contradict the claim made about the knowledge gap. See:

      Rund, S. S., Gentile, J. E., & Duffield, G. E. (2013). Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC genomics, 14(1), 1-19

      Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., & Duffield, G. E. (2011). Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences, 108(32), E421-E430

      Rund, S. S., Bonar, N. A., Champion, M. M., Ghazi, J. P., Houk, C. M., Leming, M. T., ... & Duffield, G. E. (2013). Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Scientific reports, 3(1), 2494

      Rund, S. S., Lee, S. J., Bush, B. R., & Duffield, G. E. (2012). Strain-and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. Journal of insect physiology, 58(12), 1609-1619

      Leming, M. T., Rund, S. S., Behura, S. K., Duffield, G. E., & O'Tousa, J. E. (2014). A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti. BMC genomics, 15(1), 1-9

      Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

      Rivas, G. B., Teles-de-Freitas, R., Pavan, M. G., Lima, J. B., Peixoto, A. A., & Bruno, R. V. (2018). Effects of light and temperature on daily activity and clock gene expression in two mosquito disease vectors. Journal of Biological Rhythms, 33(3), 272-288

      Response: We apologies for this oversight. In the revised manuscript, we have added these references and made changes to the text as suggested by the reviewer.

      The knowledge gap brought up in the next paragraph of the introduction doesn't reflect the questions asked by the experiments: "But, how the pacemaker differentially influences peripheral clock activity present in the olfactory system and modulates olfactory sensitivity has not been studied in detail." Specifically, the control of peripheral clocks by the central pacemaker has not been evaluated here.

      Response: This statement has been modified in the revised manuscript.

      "In vertebrates and invertebrates, it is well documented that circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" should also cite work on cockroaches and kissing bugs:

      Lubinski, A. J., & Page, T. L. (2016). The optic lobes regulate circadian rhythms of olfactory learning and memory in the cockroach. Journal of Biological Rhythms, 31(2), 161-169

      Page, T. L. (2009). Circadian regulation of olfaction and olfactory learning in the cockroach Leucophaea maderae. Sleep and Biological Rhythms, 7, 152-161

      Vinauger, C., & Lazzari, C. R. (2015). Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus. Journal of Experimental Biology, 218(19), 3110-3117

      Response: These references have been added in the revised manuscript as suggested by the reviewer.

      The sentence: "Previous studies showed that synaptic plasticity and memory are significantly influenced by the strength and number of synaptic connections (43, 44)." should be nuanced as the role of neuropeptides such as dopamine has also been showed to influence learning and memory in mosquitoes:

      Vinauger, C., Lahondère, C., Wolff, G. H., Locke, L. T., Liaw, J. E., Parrish, J. Z., ... & Riffell, J. A. (2018). Modulation of host learning in Aedes aegypti mosquitoes. Current Biology, 28(3), 333-344 Wolff, G. H., Lahondère, C., Vinauger, C., Rylance, E., & Riffell, J. A. (2023). Neuromodulation and differential learning across mosquito species. Proceedings of the Royal Society B, 290(1990), 20222118

      Response: We agree with the reviewer. We have modified this statement and added the references in the revised manuscript.

      Overall, the paragraph dealing with the idea that "circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" is very confusing. This paragraph discusses mechanisms of learning-induced plasticity but seems to ignore the simplest (most parsimonious) explanations for the circadian regulation of learning (e.g., time-dependent expression of genes involved in memory consolidation). In addition, the sentence quoted above is circumvoluted to simply say that training at different times of the day affects memory acquisition and consolidation. Although the authors did look at one gene involved in neural function, learning, memory, or circadian effects were not analysed in this study. Please reconsider the relevance of the paragraph.

      Response: We have modified this paragraph as per the suggestions of the reviewer in the revised manuscript.

      The sentence: "But, how the brain of mosquitoes entrains circadian inputs and modulates transcriptional responses that consequently contribute to remodel plastic memory, is unknown." should be rephrased. First, it should be "entrains TO circadian inputs", and second, it suggests that the study will be investigating circadian modulation of learning and memory, which is not the case. Furthermore, the term "remodel plastic memory" is unclear and doesn't seem to relate to any specific cellular or neural processes.

      Response: This statement has been removed from the revised manuscript.

      Given the differences in mosquito chronobiology observed even between strains, why perform the RNAi and EAGs on a different species of Anopheles than the one used for the RNAseq (or vice versa)?

      Response: We agree with the reviewer that there are differences in mosquito chronobiology between different strains and therefore species variation may be challenging for data interpretation. Considering the strict nocturnal behavioral pattern of An. culicifacies and dirurnal behavior of Aedes aegypti, we performed RNA-Seq study with these respective species. However, 1) due to unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available), 2) challenges in rearing and adaptation of An. culicifacies in a new environment/laboratory, 3) to validate the proof-of-concept of CYP450 function in odorant detection and olfactory sensitivity, we opt for the current collaborative study. We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (68). For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates, therefore, the arguments for species difference can be overruled.

      S. S. C. Rund, J. E. Gentile, G. E. Duffield, Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC Genomics. 14 (2013), doi:10.1186/1471-2164-14-218. S. S. C. Rund, T. Y. Hou, S. M. Ward, F. H. Collins, G. E. Duffield, Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 108 (2011), doi:10.1073/pnas.1100584108. S. S. C. Rund, N. A. Bonar, M. M. Champion, J. P. Ghazi, C. M. Houk, M. T. Leming, Z. Syed, G. E. Duffield, Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Sci. Rep. 3, 2494 (2013).

      Results

      1. "As reported earlier, a significant upregulation of period and timeless during ZT12-ZT18 was observed in both species (Figure 1C)." Please provide effect size and summary statistics.

      Response: The statistics are provided in the Figure S2 in the revised manuscript.

      "Next, the distribution of peak transcriptional changes in both An. culicifacies and Ae. aegypti was assessed through differential gene-expression analysis. Noticeably, An. culicifacies showed a higher abundance of differentially expressed olfactory genes (Figure 1D)" Please provide effect size and summary statistics.

      Response: The statistics are provided in the Table 1 in the revised manuscript.

      "Taken together, the data suggests that the nocturnal An. culicifacies may possess a more stringent circadian molecular rhythm in peripheral olfactory and brain tissues." What do the authors mean by "stringent"? At this point, this should be stated as a working hypothesis, as the statement is not backed up by the data. It is possible that the fewer differentially expressed genes of Aedes aegypti are more central to regulatory networks and cascade into more "stringent" rhythmic control of activities and rhythms.

      Response: We thank the reviewer for this suggestion. We have modified this statement as suggested by the reviewer.

      The section title: "Circadian cycle differentially and predominantly expresses olfaction-associated detoxification genes in Anopheles and Aedes" doesn't make sense. The expression of genes can be modulated by circadian rhythms, but cycles don't express genes. Please rephrase. In addition, this whole section deals with "circadian rhythms" while no experiment has been conducted under constant conditions. The observed daily variations are therefore diel rhythms until their persistence under constant conditions is established.

      Response: We agree with the reviewer and changed the statement accordingly.

      "The downregulated genes of Ae. aegypti did not show any functional categories probably due to the limited transcriptional change." Could the authors explain if this is actually the phenomenon or due to a lack of temporal resolution in the study design (i.e., 4 time points)?

      Response: We do not agree with the reviewer’s comments about the lack of temporal resolution in the current study. The functional categories of differentially expressed genes are deduced by gene set enrichment analysis, which identify the classes of genes that are overrepresented in a large set of genes. The statistical significance value is dependent on the abundance of query and background genes. In our experiments, as the number of queries (i.e. number of downregulated genes) is limited, the enrichment tool, i.e. shinyGo didn’t able to show significant enrichment of downregulated genes with FDR cut-off 0.05 and top 10 pathways were selected. Though we have selected 4 time points, previous study by Rund et al. (BMC Genomics 2013) also showed that compared to Aed. aegypti, An. gambiae possess higher number of rhythmic genes (2.6 fold higher). Therefore, it can be stated that the data that we received is not due to the pitfalls of study design, but probably the physiological difference between Anopheles and Aedes mosquitoes.

      "a GO-enrichment analysis was unable to track any change in the response-to-stimulus or odorant binding category of genes (including OBPs, CSPs, and olfactory receptors)." This finding doesn't corroborate the statements made previously and doesn't align with previously published studies. Is it due to pitfalls in the study design?

      Response: The functional categories of differentially expressed genes are deduced by gene set enrichment analysis, which identify the classes of genes that are overrepresented in a large set of genes. The statistical significance value is dependent on the abundance of query and background genes. Though, differential expression analysis revealed a significant upregulation of a subset of CSPs (~ 5-fold) and OBP6 (~3.3-fold) transcripts in An. culicifacies mosquitoes during ZT12, as the number of queries (i.e. number of chemosensory genes) is limited (i.e. 3), the enrichment tool, i.e. shinyGo didn’t able to show significant enrichment of these categories of genes when FDR cut-off 0.05 and top 10 pathways were selected.

      Moreover, we do not agree with the reviewer regarding the comment on pitfalls of study design because our previous experiments with An. culicifacies according to diel rhythm, considering more extended time points, also revealed similar expression pattern of chemosensory genes (Das De et.al., 2018).

      "In contrast, three different clusters of OBP genes in Ae. aegypti showed a time-of-day dependent distinct peak in expression starting from ZT0-ZT12 (Figure 2F)." Please provide summary statistics.

      Response: Please find the table for summary statistics in the supplemental file 1.

      "In the case of An. gambiae, the amplitudes of odor-evoked responses were significantly influenced by the doses of all the odorants tested (repeated measure ANOVA, p {less than or equal to} 2e-16) (Figure S4B)." Did the authors use a positive control for the EAGs? How did the authors normalize the responses across the two species? Given the way the data is presented, how were the data normalized to allow inter-species comparisons? In addition, It is highly unlikely that all the mosquito preps used in the EAG assay responded to all the odors tested. If that was the case, then the dataset includes missing data for certain odors and time points. We believe the authors have ensured there are at least a certain number of responses per odor and time point combinations. If this is true, repeated measures ANOVA is not suited for analyzing this data because this statistical technique requires all repeated measures within and across preps without missing values. Also, the authors need to correct the summary statistics for multiple comparisons within this framework to avoid inflating type-I errors. Has this been done?

      Response: In our study involving An. gambiae, we observed significant influences of odorant doses on the amplitudes of odor-evoked responses (repeated measure ANOVA, p ≤ 2e-16) (Figure S4B). It's important to note that we did not employ a separate positive control for the electroantennogram (EAG) assays, as the compounds utilized in our research are already known to be EAG active in at least one of the mosquito species under investigation (mentioned in supplementary file 3).

      Our primary objective for performing EAG studies is to correlate the diel-rhythmic molecular data with the diel-rhythmic electroantennographic response in nocturnal and diurnal mosquitoes. To address the normalization of responses across the two species, we opted to control for dose and time rather than normalizing using one of the EAG active compounds. Further, the EAG responses were measured in relation to solvent control. In our experimental design, we utilized different batches of mosquitoes from the same cohort to test each odorant at various time points. EAG responses were acquired using the same mosquito across different dilutions for a single odor or volatile compound, rather than across time points. Hence, we didn’t end up with missing values.

      For individual species analysis, we performed repeated measures ANOVA for each compound's EAG response, considering dose and time as variables. This enabled not only enabled us select compounds which where ‘Time’ or its interaction terms were found to be significant. Subsequently, for compounds showing significance, we conducted a basic one-way ANOVA using only time as a variable, segregating the data by each individual dose. Post-hoc Tukey tests were then carried out to compare between time points. When comparing between species, we generated a dataset by combining both species and adding species as a variable as well. Repeated measures ANOVA for each compound's EAG response, considering species, dose, and time as variables, was applied. This enabled us select compounds which where ‘Time’ or its interaction terms were found to be significant. For significant compounds, a two-way ANOVA was performed using time and species as variables. Data were segregated by each individual dose, and post-hoc Tukey tests were employed to compare between time points. It's worth mentioning that our analysis aims to account for repeated measures within and across preparations. Additionally, we have implemented post-hoc Tukey tests to correct for multiple comparisons within this framework, ensuring that we avoid inflating type-I errors in our statistical interpretations.

      "Ae. aegypti was found to be most sensitive to all the odorants (4-methylphenol, β-ocimine, E2-nonenal, benzaldehyde, nonanal, and 3-octanol) during ZT18-20 except sulcatone (Figure 3C - 3H)." Although some of these chemicals are associated with plants and Ae. aegypti is suspected to sugar feed at night, how do the authors explain that the peak olfactory sensitivity occurs at night for compounds such as nonanal? It would be interesting to discuss how these results compare to previous studies such as:

      Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

      Response: The possible explanations have been added in the revised MS.

      "Additionally, our principal components analysis also illustrates that most loadings of relative EAG responses are higher towards the Anopheles observations (Figure S4C)." The meaning of this sentence is unclear? Please clarify.

      Response: Considering the limited clarity of the statement we have removed it from the revised manuscript.

      "Taken together these data indicate that An. gambiae may exhibit higher antennal sensitivity to at least five different odorants tested, as compared to Ae. aegypti." As mentioned above, how did the authors normalized across species to allow comparisons? If not normalized, how do you ensure that higher response magnitudes correlate with higher olfactory sensitivity, given potential differences in the morphology or size differences between the two species? Furthermore, An. gambiae has been exclusively used in the EAG assay. Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable. This, however, is a major caveat in the experiment design, given previous studies (indicated below) have reported species-specific variations in olfactory sensitivity. In its present form, the EAG data from An. gambiae is not a piece of appropriate evidence that the authors could use to complement or substantiate the findings from other aspects of this study on An. culicifacies.

      Wheelwright, M., Whittle, C. R., & Riabinina, O. (2021). Olfactory systems across mosquito species. Cell and Tissue Research, 383(1), 75-90. Wooding, M., Naudé, Y., Rohwer, E., & Bouwer, M. (2020). Controlling mosquitoes with semiochemicals: a review. Parasites & Vectors, 13, 1-20.

      iii. Gupta, A., Singh, S. S., Mittal, A. M., Singh, P., Goyal, S., Kannan, K. R., ... & Gupta, N. (2022). Mosquito Olfactory Response Ensemble enables pattern discovery by curating a behavioral and electrophysiological response database. Iscience, 25(3).

      Response: The data is normalized as described above in the point 15. Also, it is technical limitation that we had to use multiple species of the mosquito for this study (please refer to the point 7).

      The reviewer’s statement “Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable” is not true, as we never assume similar olfactory sensitivity between An. culicifacies and An. gambiae. We only consider nocturnal activity for both the mosquito species. Moreover, we are aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (no other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (68). For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Furthermore, we would like to emphasize that the primary focus of the current manuscript is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validated this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

      "Similar to An. gambiae, a comparatively high amplitude response was also observed in An. stephensi (Figure S4D)." This is interesting but what would be even more relevant to the present study is to discuss how the time-dependent responses compare between the two Anopheles species.

      Response: We agree that it will be interesting to compare time-dependent response between the two Anopheles species. However, it is not our primary interest and objectives, and is beyond the scope of the current manuscript. Thus, we remove the data from the revised MS.

      The paragraph titled "Daily temporal modulation of neuronal serine protease impacts mosquito's olfactory sensitivity" is confusing because the authors move on to test the effect of knocking down a serine protease gene (found to be differentially expressed throughout the day) on olfactory sensitivity. While this is interesting in and of itself, the link between the role of this gene in learning-induced plasticity, the circadian modulation of "brain functions" and olfactory sensitivity is 1) unclear and 2) not explicitly tested. We agree with the authors that what has been tested is "the effect of neuronal serine protease on circadian-dependent olfactory responses," but the two paragraphs leading to it seem to be extrapolating functional links that have yet to be determined. In this context, their conclusions that "Our finding highlights that daily temporal modulation of neuronal serine-protease may have important functions in the maintenance of brain homeostasis and olfactory odor responses." is misleading because although they used the hypothetical "may", the link between the temporal modulation of one serine protease gene and the maintenance of brain homeostasis is not explicitly tested here.

      Response: Though, we strongly believe that neuronal serine protease are involved in remodelling of extracellular matrix and the maintenance of brain homeostasis, the limitation of experimental validation by neuroimaging (out of the scope of the current manuscript), restricting us to draw the conclusion. Therefore, we have modified our conclusions based on the available data as suggested by the reviewer.

      Discussion

      1. The first sentence of the discussion: "In this study, we provide initial evidence that the daily rhythmic change in the olfactory sensitivity of mosquitoes is tuned with the temporal modulation of molecular factors involved in the initial biochemical process of odor detection i.e., peri-receptor events" is not true since studies from Rund and Duffield previously revealed the daily modulation of OBP gene expression. It also contradicts the next sentence: "The findings of circadian-dependent elevation of xenobiotic metabolizing enzymes in the olfactory system of both Ae. aegypti and An. culicifacies are consistent with previous literature (26, 31), and we postulate that these proteins may contribute to the regulation of odorant detection in mosquitoes."

      Response: This statement is modified in the revised manuscript.

      The use of "circadian" in the discussion of the results is also misleading as only diel rhythms were evaluated in the present study.

      Response: This is changed in the revised manuscript.

      "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58)." This is not really what these references show.

      Response: The statement and the references have been changed in the revised manuscript.

      "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58), it can be hypothesized that detection of any specific signal in such a noisy environment, mosquitoes may have evolved a sophisticated mechanism for rapid (i) odor mobilization and (ii) odorant clearance, to prevent anosmia (24)." One could argue that this is a requirement for all insects, regardless of the size of their olfactory repertoire.

      Response: We agree with the reviewer and modified the text accordingly.

      "Taken together, we hypothesize that circadian-dependent activation of the peri-receptor events may modulate olfactory sensitivity and are key for the onset of peak navigation time in each mosquito species." This is not entirely accurate since spontaneous locomotor activity rhythms are also observed in the absence of olfactory stimulation. While "navigation" does imply olfactory-guided behaviors, "peak navigation time" appears to be driven by other processes. See, for example, all studies testing mosquito activity rhythms in locomotor activity monitors. Response: Considering the concern of the reviewer, we have modified the text.

      "Due to technical limitations, and considering the substantial data on the circadian-dependent molecular rhythmicity" please clarify what the technical limitations were. Is this something that prevented the authors specifically, or something tied to mosquito biology and would prevent anybody from doing it? Also, why couldn't the transcriptomic analysis be performed on An. gambiae?

      Response: As previously mentioned, primarily, unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available) is the major challenge for us to proof our hypothesis. Secondly, transportation of An. culicifacies was not possible due to Govt. regulations and also adaptation and establishment of the colony of An. culicifacies take long time as it is not easily adapted (Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. Trans R Soc Trop Med Hyg. 1999;93:573–577) in a new environment/laboratory. Thirdly, An. culicifacies colony was not available at our collaborative laboratory. These are the major technical limitations.

      Therefore, to validate the hypothesis of CYP450 function in odorant detection and olfactory sensitivity, we opt for the current collaborative study. We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (68). For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Performing another RNA-Seq study with An. gambiae would not be possible for the current MS. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

      "In contrast to An. gambiae, the time-dose interactions had a higher significant impact on the antennal sensitivity of Ae. aegypti. An. gambiae showed a conserved pattern in the daily rhythm of olfactory sensitivity, peaking at ZT1-3 and ZT18-20." These two sentences are very confusing. Doesn't it simply mean that the co-variation is not linear or not the same across odors? In addition, what does it mean for a pattern to be more conserved? How can one conclude about the "conserved" nature of a pattern by looking at time-dependent variations in dose-response curves?

      Response: This section of discussion is re-written in the revised version of the manuscript.

      "Together these data, we interpret that mosquito's olfactory sensitivity possibly does not follow a fixed temporal trait" is unclear and suggests that the authors are discussing global versus odor-specific rhythms. Please rephrase.

      Response: This section of discussion is re-written in the revised version of the manuscript.

      "Moreover, we hypothesize that under standard insectary conditions, mosquitoes may not need to exhibit foraging flight activity either for nectar or blood, and during the time course, it may minimize their olfactory rhythm, which is obligately required for wild mosquitoes." This hypothesis is not supported by the results of the study and contradicts work by others (Rund et al., Eilerts et al., Gentile et., etc).

      Response: This section of discussion is re-written in the revised version of the manuscript.

      The same comment applies to "Therefore, it is reasonable to think that the mosquitoes used for EAG studies may have adapted well under insectary settings and, hence carry weak olfactory rhythm." as this statement is not supported by results of the present study or comparisons of the results to previous studies based on field-caught mosquitoes. Although it is an interesting question to ask in the future, it should be stated as a future research avenue rather than a working hypothesis that results from the present study.

      This section of discussion is re-written in the revised version of the manuscript.

      "Aedes aegypti displayed a peak in antennal sensitivity at ZT18-20 to the higher concentrations of plant and vertebrate host-associated odorants tested. Given the time-of-day dependent multiple peaks (at ZT6-8 and ZT18-20 for benzaldehyde and at ZT12-14 and ZT18-20 for nonanal) in antennal sensitivity to different odorants, our data supports the previous observation of bimodal activity pattern of Ae. aegypti (50)." Rephrase by saying that results are "aligned with the previous observations of bimodal activity". Olfactory rhythms don't "support" the activity patterns because olfactory processes and spontaneous locomotor activity are independent processes.

      Response: We have made these changes in the revised manuscript as per the suggestions of the reviewer.

      "our preliminary data indicate that Anopheles spp. may possess comparatively higher olfactory sensitivity to a substantial number of odorants as compared to Aedes spp." Consider removing this sentence unless the way the data has been normalized to allow for comparisons between species is clarified.

      Response: This statement is removed from the revised manuscript.

      In "A significant decrease in odorant sensitivity for all the volatile odors tested in the CYP450-silenced Ae. aegypti," please change "silenced" to "reduced" because RNAi doesn't silence (i.e. knockout) gene expression.

      Response: It has been modified as per the suggestions of the reviewer.

      The title "Neuronal serine protease consolidates brain function and olfactory detection" is extremely misleading. Do the authors refer to memory consolidation, which has not been tested here? What is brain function consolidation??

      Response: We agree with the reviewer. The title has been modified in the revised manuscript.

      The reference used in "Despite their tiny brain size, mosquitoes, like other insects, have an incredible power to process and memorize circadian-guided olfactory information (7)." is not appropriate. Also, "circadian-guided" is unclear. Consider replacing it with "circadian-gated".

      Response: It has been modified as per the suggestions of the reviewer.

      What is the "the homeostatic process of the brain"?

      Response: The process of maintaining a stable state can be defined as homeostasis. Here, the statement "the homeostatic process of the brain" is used to convey that after the active host-seeking/olfaction phase of mosquitoes during which the co-ordinated and integrated functions of both olfactory and neuronal system is required for crucial decision-making events, brain may undergo a homeostatic process (comes down from excitatory state to stable state) during the resting period. However, in view of reviewer’s concern we have modified the statement.

      "the temporal oscillation of the sleep-wake cycle of any organism is managed by the encoding of experience during wake, and consolidation of synaptic change during inactive (sleep) phases, respectively (70)." By experience, do the authors refer to learning? This seems out of topic as this process has not been evaluated here.

      Response: It has been modified as per the suggestions of the reviewer.

      "We speculate that after the commencement of the active phase (ZT6-ZT12), the serine peptidase family of proteins in the brain of Ae. aegypti mosquitoes may play an important function in consolidating brain actions (after ZT12) and aid circadian-dependent memory formation." The value of this statement is unclear. Circadian-dependent memory formation is not being evaluated here, and the results from the present study do not directly support this speculation, also because other processes involved in memory formation are not evaluated here. This seems at odds with the literature on learning and memory.

      Response: We have modified these statements in the revised manuscript and mentioned it as future research hypothesis.

      "Subsequent work on electrophysiological and neuro-imaging studies are needed to demonstrate the role of neuronal-serine proteases in the reorganization of perisynaptic structure." Sure. But the link between "the role of neuronal-serine proteases in the reorganization of perisynaptic structure" and rhythms in olfactory sensitivity is unclear.

      Response: It has been modified as per the suggestions of the reviewer.

      As a general comment, EAGs seem inappropriate to evaluate the effect of the central-brain processing in the regulation of peripheral olfactory processes. This is a critical comment that needs to be considered by the authors and clarified in the manuscript. If rhythms of central brain processes are important for olfactory-guided behaviors, these should be evaluated at the level of the central brain or via behavioral metrics. The effect of the RNAi knockdowns on peripheral sensitivity is interesting, but its link with central processes is unclear and doesn't support the speculations made by the authors about learning and memory.

      Response: We agree with the reviewer that EAG study is not enough/appropriate to comment on the effect of central-brain processing in the regulation of olfactory processes. Further validation by either neuroimaging or behavioral studies are needed to make any conclusion. We clearly mention in the manuscript that our data indirectly indicating this function of serine protease and further confirmatory studies are needed to prove this hypothesis.

      Methods

      1. No explanations are provided for how the EAG data are normalized to allow comparisons between species.

      Response: Please refer to the response of the point no. 15 of the reviewer 1.

      Figures 42. Figure 1: The daily rhythm depicted in A, are not representative of the actual profiles. See: Benoit, J. B., & Vinauger, C. (2022). Chapter 32: Chronobiology of blood-feeding arthropods: influences on their role as disease vectors. In Sensory ecology of disease vectors (pp. 815-849). Wageningen Academic Publishers. Or any other paper on mosquito activity rhythms.

      Response: Considering the reviewer’s concern we have revised the figure.

      Figure 3 and 4: The EAG results are plotted twice. This is redundant and misleading as it makes the reader think there is more data than actually presented.

      Response: Considering the reviewer’s comment we shifted figure 4 into the supplemental file.

      Figure 5: Please clarify the sample size for each panel. In C - F, what would be used as a reference? In other words, what is a Relative EAG Response of 1? And if it is "relative", are the units really mV? In E and F, it would be great to show how the Ethanol control compares to the no solvent condition. This could be placed in supplementary materials.

      Response: The sample size was mentioned in the figure legends. However, for the reviewer’s clarification, the odor response was tested with 40 individual mosquitoes of control and dsrRNA-treated groups. Therefore, sample size N=40 for Fig. 5C.

      Respective solvent control (hexane solvent) used as a reference to calculate the relative EAG response for both the dsrLacZ and dsrCYP450 group. As it is relative EAG amplitude we have removed the unit in the revised MS.

      Figures 5 and 6, given the dispersion in the EAG data, the treatments where N=40 appear robust, but the interpretation of results from treatments where N=6 may be limited due to the low sample size. This limitation is visible in Figure 5F, for example, where ABT-Aceto is different from Cont-Aceta but not PBO-Aceto because one individual shows a higher response.

      Response: We agree that probably, by increasing the sample size for inhibitor treatment experiment, may decrease these inter-individual differences and increase the overall significance value. However, our robust knock-down data showed significant results and simultaneously it complements the inhibitor study in Ae. aegypti, we do not think of any disparity in the data. Moreover, EAG response to human blend, nonanal and benzaldehyde showed similar significant results in both RNAi and inhibitor studies. Accounting, the different knock-down efficiency in dsRNA injected mosquitoes, the phenotypic assays (EAG recordings) were carried out with 40 control and 40 dsRNA-treated mosquitoes. And, we observed significant reduction in EAG response following inhibitor treatment in An. gambiae, when we tested for 6 ethanol and 6 inhibitor treated mosquitoes. Thus, we followed the similar protocol for Ae. aegypti also. However, inter-individual difference in response is affecting the significance value.

      Figure S6: how does this support that synaptic plasticity is influenced by "Time-of-day dependent modulation of serine protease genes in the brain"?

      Response: We agree with the reviewer’s concern that with only EAG data it is not possible to comment on synaptic plasticity. We apologize for it and revised the statement in the MS.


      Minor comments

      What do the authors mean by "consolidation of brain functions"? Memory consolidation? Please clarify.

      Response: The consolidation of brain function or memory consolidation means to the process of stabilizing the memory that an organism gains through the process of experience or training/learning phase. Memory consolidation initiates with rapid change in de-novo gene expression regulated by several transcription factors, effector genes and non-coding RNAs, known as molecular consolidation followed by cellular consolidation that involves cellular signal transmission within the neurons in the brain. The molecular and cellular consolidation are the basis for system level consolidation which is a slow process and involves communication among neurons located different regions of the brain. The system level consolidation is very important for the reorganization of the brain circuits to maintain long-term memory. The concept of system consolation is very much well evident in humans. Additionally, several studies in Drosophila also showed that fruit fly develop olfactory memories after classical conditioning or olfactory training through system consolidation process.

      Moreover, accumulating data from humans suggest that sleep helps in memory consolidation. Sleep is basic drive for all animals that help to build memories. There are two hypothesis and respective compelling evidences for that. First hypothesis and the supporting molecular and electrophysiological data convey that sleep facilitate the homeostatic processes of the brain involving loosening of synaptic connections between the overactive neurons, structural modification of synapse which consequently help in memory formation. The second hypothesis state the important contribution of sleep in system consolidation and long-term memory potentiation. Studying the electrical activity of the brain and the recent advancement of fMRI scan indicate reorganization of neural activity between brain regions during sleep-related memory consolidation.

      There are several experimental evidences in support of both the theory for humans as well as in fruit fry Drosophila melanogaster. In mosquitoes, the studies related to the function of brain are primarily restricted to the mechanism of odor coding and memory formation has been correlated with Dopamine neurotransmitter signalling. In view of the rapid adaptation potential, change in host-preference and evolution of temporal host-seeking behaviour, it can be hypothesized that mosquito brain also undergo the process of memory consolidation (either following any of the two hypothesized path or cumulatively apply the both) to learn new information in order to effectively shape future actions.

      Furthermore, according to the fundamental principle of modern neuroscience learning and memory are achieved either by the formation of new synaptic connections or changing in existing connections between neurons. The ability of synapses to either strengthen or weaken the communications is called plasticity which is influenced by learning and experience and facilitate organism’s adaptation and survival.

      Reference:

      1. Cervantes-Sandova, A. Martin-Peña, J. A. Berry, R. L. Davis, System-like consolidation of olfactory memories in Drosophila. J. Neurosci. 33, 9846–9854 (2013).
      2. In "Similar to previous studies (26), the expression of a limited number of rhythmic genes was visualized in Ae. aegypti" please replace "visualized" with "observed".
      3. Marshall, N. Cross, S. Binder, T. T. Dang-Vu, Brain rhythms during sleep and memory consolidation: Neurobiological insights. Physiology. 35, 4–15 (2020).
      4. Brendon O. Watson and György Buzsáki. Sleep, Memory & Brain Rhythms. Daedalus, 144(1): 67–82 (2015). doi:10.1162/DAED_a_00318

      Figure 2A, please clarify in the caption what FDR stands for.

      Response: FDR stands for “false discovery rate”. FDR is an adjusted p-value to trim false positive results.

      In "To further establish this proof-of-concept in An. gambiae, three potent CYP450 inhibitors, aminobenzotriazole(52), piperonyl butoxide(53), and schinandrin A (54), was applied topically on the head capsule of 5-6-day-old female mosquitoes" replace "was applied" with "were applied".

      Response: These changes are made in the revised manuscript.

      "Interestingly, our species-time interaction studies revealed that An. gambiae exhibits time-of-day dependent significantly high antennal sensitivity to at least four chemical odorants compared to Ae. aegypti, except phenol." is unclear. Please reword.

      Response: The statement has been revised in the MS.

      In "Similar observations were also noticed with An. stephensi." replace "noticed" with "made". Response: We have modified the statement in the revised version of the manuscript.



      Reviewer #1 (Significance (Required)):

      Such a study has the potential to be valuable for the field, but its value and significance are hindered by an accumulation of overstatements, the fact that prior work in the field has been minimized or omitted, and a lack of support for the stated conclusions.

      In this context, the advances are only slightly incremental compared to the work produced by Rund et al., and the mechanistic hypotheses emitted to link the genes selected for knockdown experiments and olfactory sensitivity are not clearly supported by the evidence presented here. The main strength of the paper is to show the role of CYP450 in olfactory sensitivity.

      The audience is fairly broad and includes insect neuro-ethologists, molecular biologists, and chronobiologists.

      Our field of expertise:

      • Mosquito chemosensation

      • Learning and memory

      • Chronobiology

      • Electrophysiology

      • Medical entomology









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

      This report combines an examination of peripheral transcriptomes and general olfactory sensitivity in an effort to underscore the importance of peri-receptor components in circadian-directed modulation of olfaction across both Aedine and Anopheline mosquitoes. While the authors do a nice job of raising the importance of the often-underappreciated spectrum of insect olfactory peri-receptor proteins, the impact of their study is undercut by technical concerns regarding methods and data presentation. That several of these concerns (detailed below) are explicitly acknowledged by the authors as limitations of this study does not mitigate their impact in eroding confidence in these data and this study.

      All in all, as a result of these concerns, I am unconvinced as to the overall merits of this somewhat interesting but generally uneven study.

      We sincerely thank the reviewer for their time and consideration, and appreciate the thorough review of our manuscript. Their insightful comments have greatly enriched our work. We also apologies for instances of overinterpreting the data. Your feedback has helped us recognize areas where clarity and caution are needed, and we are committed to addressing these concerns in our revisions. Thank you for your valuable input and guidance.

      Major concerns:

      1. That the authors use An. culicifacies for their transcriptome studies and An. gambiae (G3) for the olfactory physiology does not work. The 'technical limitations' (read studies done at two different locations) make this report an unwelcome melding of what should perhaps be two distinct studies. In order to maintain this forced marriage as a single report I would suggest the authors utilize An. culicifacies for both components. Alternatively, they can do both parts with An. gambiae but here I would strongly urge them to use any strain other than G3 which as a result of its now decades-long laboratory residence has long since lost its relevance to natural populations of Anopheline vectors. Response: We agree with the reviewer that there is significant species-specific variation in olfactory sensitivity of mosquitoes. Considering the strict nocturnal behavioral pattern of An. culicifacies and dirurnal behavior of Aedes aegypti, we performed RNA-Seq study with these respective species. However, 1) due to unavailability of EAG facility at ICMR-National Institute of Malaria Research, India (only where An. culicifacies colony is available), 2) challenges in rearing and adaptation of An. culicifacies in a new environment/laboratory (An. culicifacies take long time as it is not easily adapted, Ref: Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. Trans R Soc Trop Med Hyg. 1999;93:573–577), 3) An. culicifacies colony was not available at our collaborative laboratory, 4) to validate our hypothesis of CYP450 function in odorant detection and olfactory sensitivity of mosquitoes, we opt for the current collaborative study.

      We are also aware that species variation of Anopheles for electroantennographic study would be difficult to correlate with the molecular data on An. culicifacies. Thus, we consider An. gambiae (not other Anopheles mosquitoes like An. stephensi, An. coluzzii etc.) because of the availability of diel rhythm associated molecular data for An. gambiae (68). For better interpretation we also compare expression profiling of CYP450 and OBP genes between An. culicifacies and An. gambiae (Supplemental file 3). Importantly, we found similar expression pattern of several CYP450 and OBP/CSP genes between An. culicifacies and An. gambiae. Performing another RNA-Seq study with An. gambiae would not be possible for the current MS. Furthermore, please note that the primary focus of the current MS is to highlight the role of peri-receptor proteins in olfactory sensitivity and odor detection. And, as a proof-of-concept, we validate this hypothesis both in An. gambiae and Aed. aegypti. We believe that the basic mechanism of odor detection and peri-receptor events are similar/conserved from insects to higher vertebrates.

      The 70-80% alignment rate reported to the An. culicifacies reference genome significantly erodes this reader's confidence in the integrity of their analyses. That low level of alignment can have dramatic impacts on the estimation of transcript abundance has been repeated demonstrated (see, Srivastava, A., Malik, L., Sarkar, H. et al.. Genome Biol 21, 239, 2020, https://doi.org/10.1186/s13059-020-02151-8). This may (in part) explain why olfactory receptors have been largely absent from this data set.

      Response: We agree with the reviewer that alignment rate could have been better but this should not affect the quantitative information we are referring to in this manuscript. The alignment rates could have impacted the qualitative information which can vary due to multiple reasons including the quality of the reference genome. As it is evident from the analysis that in Ae. aegypti 90% of the reads are aligned to the reference genome, still we did not observe any difference in the abundancy of olfactory receptor genes. Previous microarray analysis in An. gambiae by Rund et.al. 2013, also did not show diel rhythmic expression of any OR genes.

      The issue of species choice is further complicated by questions regarding the An. culicifacies species complex which contains 5 cryptic species. How did the authors confirm they are indeed working with An. culicifacies species A -there is no mention regarding the molecular identification.

      Response: The An. culcifacies species A colony has been colonized at NIMR since 1999, with routine checks performed to verify its purity of species by analyzing inversion genotypes on chromosomes for the presence of sibling species (see the references). But at that time, we had three sibling species--A, B, C; subsequently, we lost B and C. Giving old references will not serve the purpose. Later we verified sibling species A by inversion genotype on chromosome and molecular tools. However, we do not have any published reference for that verified data.

      The species can be identified by performing 28S rDNA-based PCR (Singh et al, 2004) and cytochrome oxidase II-based PCR (Goswami et al 2006). Sequencing can also serve the purpose.


      Singh OP, Goswami G, Nanda N, Raghavendra K, Chandra D, Subbarao SK. An allele-specific polymerase chain reaction assay for the identification of members of Anopheles culicifacies complex. J Biosci. 2004; 29: 275—280 10.1007/bf02702609

      Goswami G, Singh OP, Nanda N, Raghavendra K, Gakhar SK, Subbarao SK. Identification of all members of the Anopheles culicifacies complex using allele-specific polymerase chain reaction assays. Am J Trop Med Hyg. 2006; 75: 454-460. doi: 10.4269/ajtmh.2006.75.454

      Adak T, Kaur S, Singh OP. Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax. Trans R Soc Trop Med Hyg. 1999;93:573–577

      The switch from dsRNAi studies in Aedes to protease inhibitor studies in Anopheles adds to the interspecies confusion.

      Response: Our main goal in this study was to evaluate the function of CYP450 in mosquito’s odor detection and olfactory sensitivity. Our data as well as previous data (Rund et.al. 2011, Rund et.al. 2013) suggesting that the basic mechanism of odor detection and peri-receptor events are similar for both An. gambiae, An. culicifacies and Ae. aegypti, and the role of detoxification genes are very much evidenced from these data. Based on our RNA-Seq data on Ae. aegypti, we shortlisted one CYP450 gene for functional knockdown assays. However, for Anopheles we used An. gambiae for functional validation. Thus, it was not possible for us to select appropriate CYP450 gene from An. gambiae. That is why, we plan for using CYP450 protein inhibitors which block the function of all the CYP450 expressing in the olfactory system of mosquitoes. Expectedly, we also observed much more pronounced reduction of olfactory sensitivity when inhibitors were applied compared to dsRNAi mediated knock-down the function of only one CYP450 protein. These data indicate that Anopheles also possess similar mechanism of perireceptor events for odor detection and CYP450 plays an important role in it.

      The olfactory shifts presented in Fig 3 are somewhat underwhelming. In An. gambiae this mostly seen at very high (to my eyes, non-biologically relevant) 10-1 dilutions. In Aedes, while statistically significant, the EAG values (especially for 4MePhenol) are very low and therefore suspect and unconvincing. It is also unclear how 'Relative EAG Responses' were derived?? Does this mean relative to solvent alone controls??

      Response: Yes, relative EAG response means relative to respective solvent control. We also make necessary changes in the text as well as in the figures for better understanding and representation.

      The same data set seems to have been presented in Figures 3 and 4, with the latter's absence of salient details e.g. haphazard odor concentrations which are seen only when legend is examined). These factors make the inclusion of Figure 4 less obvious.

      Response: Depending on the reviewer’s concern we shifted the Figure 4 into the supplemental data and we are sorry for the miscommunication.

      I am concerned that the data in Figure 5B is derived from only those samples with altered EAGs. I believe that all injected mosquitoes should be assayed in order to better understand the actual efficacy of the treatment. The cherry picking of samples is troubling.

      Response: We pooled five heads for each replicate and we performed the assay with three replicates. That mean we have taken heads from 15 mosquitoes for each experimental setup (control vs knock-down). It is true that we did not consider all the 40 mosquitoes that we used for EAG-recordings. However, we believe that 15 mosquitoes will be a good representation of the population. And the error bars among replicates of the knock-down mosquitoes, compared to the dsLacZ group, clearly indicates the disparity in knock-down efficiency among individuals.

      As is true for earlier figures, Figure 5c-f is lacking critical information about concentration (also not presented in figure legend) and should be done within the context of a multi-point dose response study. The data in its current form is not acceptable.

      Response: We apologize for the mistake for not mentioning the concentration of the inhibitors. Now, we added this information in the revised manuscript.

      The same data concerns apply to Figure 6d-g.

      Response: We apologize for the mistake for not mentioning the concentration of the inhibitors. Now, we added this information in the revised manuscript.

      The inclusion of An. stephensi data Figure S4D seems thrown in as an after-thought and without good reason.

      Response: Our RNA-Seq data on An. culicifacies and Aedes aegypti revealed similar abundance and expression pattern of rhythmic transcripts specifically for peri-receptor transcripts, as reported before by Rund et. al. 2011 & 2013 for Aedes aegypti and Anopheles gambiae. Moreover, we observed significant difference in EAG response between Aedes aegypti and Anopheles gambiae, we hypothesized that higher abundance of rhythmic peri-receptor transcripts possibly has correlation with high EAG response in Anopheles. Therefore, to get an idea about the EAG response for other Anopheles sp. we used An. stephensi, and observed similar difference in EAG response. Though, it will be interesting to compare time-dependent response between the two Anopheles species, it is not our primary interest and objectives, and is beyond the scope of the current MS and the objective can be elaborated further in future.

      I am unsure how shifts in CNS levels of P450 or serine proteases impact peripheral EAG recordings? This is especially so given that any effects on synaptic plasticity/efficacy that might occur are expected to be downstream of the peripheral antennae being recorded in EAGs. The authors do not do a great job explaining away that paradox even though that section in the discussion seems overly speculative.

      Response: We agree with the reviewer that EAG study is not enough/appropriate to comment on the effect of central-brain processing in the regulation of olfactory processes. Further validation by either neuroimaging or beavioral studies are needed to make any conclusion. And we clearly mention in the MS that our data indirectly indicating this function of serine protease and further confirmatory studies are needed to proof this hypothesis. However, it is not possible for us to perform all the experiments now, due to technical and infrastructural limitations. Thus, we hypothesized it as future research endeavour. Moreover, considering the reviewer’s concern we have modified the text and removed the overstatements and speculations.

      The authors discussion on peri-receptor protein oscillation seems premature given the data that is presented (regardless of the caveats discussed above) center on transcript abundance. There is no data on protein abundance, which while related, is an entirely different question/issue.

      Response: Yes, we agree that our hypothesis of peri-receptor protein oscillation is based on our RNA-Seq data. However, later we validated our hypothesis by knock-down studies in mosquitoes as well as we used CYP450 protein inhibitors, where also we observed significant results of decrease in olfactory sensitivity. It is true that we do not have any data on protein abundance, but several previous studies along with our data showed the similar expression profiling of peri-receptor genes, which clearly indicates that the rhythmic expression pattern of these genes are conserved among mosquitoes. None of the previous studies address the hypothesis regarding the peri-receptor events and possible function of XMEs in odorant detection, which is the uniqueness of our study. Therefore, we believe that after functional validation by dsRNAi and inhibitor study, we are able to validate our hypothesis for scientific acceptance. While, CYP450 has been reported to have crucial role in xenobiotic detoxification, its role in odor detection has not been explored yet. We agree that further biochemical validation is required to see the interaction between CYP450 and odor molecules, and how CYP450 is modifying the odorant chemicals either for its detection or for its inactivation. But, such study is out of the scope of the MS and will be our future research endeavour. However, our current data and the MS will have large impact for designing of strategies for application of insecticides, as overlapping the timing of application of insecticide and rhythmic expression/natural upregulation of XMEs could accelerate the inactivation of insecticides and rapid generation of resistant mosquitoes. Thus, we believe that the current revised MS have potential data and would be valuable for publication.

      Minor concerns:

      1. The authors routinely confuse transcript abundance derived from their RNAseq data with gene expression. The former reflects the steady-state snapshot levels of transcripts encompassing\ synthesis, use and decay while the latter is limited to the rate of transcription requiring nuclear run on or single-nucleus RNAseq approaches. Response: Thank you for your insightful comment. We appreciate your clarification regarding the distinction between transcript abundance and gene expression. In the revised manuscript, we have included a clarification stating that 'transcript abundance is referred to as gene expression, unless explicitly stated otherwise”.

      There are numerous typos, spelling errors and other grammatical mistakes-a copy editor is needed.

      Response: In the revised manuscript, we have carefully corrected the spelling errors and other grammatical mistakes.

      Many of the supplemental figures are error filled, lacking sufficient details and otherwise difficult to parse/understand. I recommend revisiting/removing many of these/

      Response: We have improvised on the supplementary figures in the revised manuscript as suggested by the reviewer.

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

      In light of the serious concerns described above there is limited significance to this study. Similarly these concerns erode almost all of any advance to the field this study might have offered. The audience of interest would be highly specialized

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

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      This report combines an examination of peripheral transcriptomes and general olfactory sensitivity in an effort to underscore the importance of peri-receptor components in circadian-directed modulation of olfaction across both Aedine and Anopheline mosquitoes. While the authors do a nice job of raising the importance of the often-underappreciated spectrum of insect olfactory peri-receptor proteins, the impact of their study is undercut by technical concerns regarding methods and data presentation. That several of these concerns (detailed below) are explicitly acknowledged by the authors as limitations of this study does not mitigate their impact in eroding confidence in these data and this study.

      All in all, as a result of these concerns, I am unconvinced as to the overall merits of this somewhat interesting but generally uneven study.

      Major concerns:

      1. That the authors use An. culicifacies for their transcriptome studies and An. gambiae (G3) for the olfactory physiology does not work. The 'technical limitations' (read studies done at two different locations) make this report an unwelcome melding of what should perhaps be two distinct studies. In order to maintain this forced marriage as a single report I would suggest the authors utilize An. culicifacies for both components. Alternatively, they can do both parts with An. gambiae but here I would strongly urge them to use any strain other than G3 which as a result of its now decades-long laboratory residence has long since lost its relevance to natural populations of Anopheline vectors.
      2. The 70-80% alignment rate reported to the An. culicifacies reference genome significantly erodes this reader's confidence in the integrity of their analyses. That low level of alignment can have dramatic impacts on the estimation of transcript abundance has been repeated demonstrated (see, Srivastava, A., Malik, L., Sarkar, H. et al.. Genome Biol 21, 239, 2020, https://doi.org/10.1186/s13059-020-02151-8). This may (in part) explain why olfactory receptors have been largely absent from this data set.
      3. The issue of species choice is further complicated by questions regarding the An. culicifacies species complex which contains 5 cryptic species. How did the authors confirm they are indeed working with An. culicifacies species A -there is no mention regarding the molecular identification.
      4. The switch from dsRNAi studies in Aedes to protease inhibitor studies in Anopheles adds to the interspecies confusion.
      5. The olfactory shifts presented in Fig 3 are somewhat underwhelming. In An. gambiae this mostly seen at very high (to my eyes, non-biologically relevant) 10-1 dilutions. In Aedes, while statistically significant, the EAG values (especially for 4MePhenol) are very low and therefore suspect and unconvincing. It is also unclear how 'Relative EAG Responses' were derived?? Does this mean relative to solvent alone controls??
      6. The same data set seems to have been presented in Figures 3 and 4, with the latter's absence of salient details e.g. haphazard odor concentrations which are seen only when legend is examined). These factors make the inclusion of Figure 4 less obvious.
      7. I am concerned that the data in Figure 5B is derived from only those samples with altered EAGs. I believe that all injected mosquitoes should be assayed in order to better understand the actual efficacy of the treatment. The cherry picking of samples is troubling.
      8. As is true for earlier figures, Figure 5c-f is lacking critical information about concentration (also not presented in figure legend) and should be done within the context of a multi-point dose response study. The data in its current form is not acceptable.
      9. The same data concerns apply to Figure 6d-g.
      10. The inclusion of An. stephensi data Figure S4D seems thrown in as an after-thought and without good reason.
      11. I am unsure how shifts in CNS levels of P450 or serine proteases impact peripheral EAG recordings? This is especially so given that any effects on synaptic plasticity/efficacy that might occur are expected to be downstream of the peripheral antennae being recorded in EAGs. The authors do not do a great job explaining away that paradox even though that section in the discussion seems overly speculative.
      12. The authors discussion on peri-receptor protein oscillation seems premature given the data that is presented (regardless of the caveats discussed above) center on transcript abundance. There is no data on protein abundance, which while related, is an entirely different question/issue.

      Minor concerns:

      1. The authors routinely confuse transcript abundance derived from their RNAseq data with gene expression. The former reflects the steady-state snapshot levels of transcripts encompassing\ synthesis, use and decay while the latter is limited to the rate of transcription requiring nuclear run on or single-nucleus RNAseq approaches.
      2. There are numerous typos, spelling errors and other grammatical mistakes-a copy editor is needed.
      3. Many of the supplemental figures are error filled, lacking sufficient details and otherwise difficult to parse/understand. I recommend revisiting/removing many of these/

      Significance

      In light of the serious concerns described above there is limited significance to this study. Similarly these concerns erode almost all of any advance to the field this study might have offered. The audience of interest would be highly specialized

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

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      In the present manuscript, the authors analyzed diel oscillations in the brain and olfactory organs' transcriptome of Aedes aegypti and Anopheles culicifacies. The analysis of their RNAseq results showed an effect of time of day on the expression of detoxification genes involved in oxidoreductase and monooxygenase activity. Next, they investigated the effect of time of day on the olfactory sensitivity of Ae. aegypti and An. gambiae and identified the role of CYP450 in odor detection in these species using RNAi. In the last part of the study, they used RNAi to knock down the expression of one of the serine protease genes and observed a reduction in olfactory sensitivity. Overall, the experiments are well-designed and mostly robust (see comment regarding the sample size and data analysis of the EAG experiments) but do not always support the claims of the authors. For example, since no experiments were conducted under constant conditions, the circadian (i.e., driven by the internal clocks) effects are not being quantified here. In addition, knocking down the expression of a gene showing daily variations in its expression and observing an effect on olfactory sensitivity is not sufficient to show its role in the daily olfactory rhythms. Knowledge gaps are not well supported by the literature, and overstatements are made throughout the manuscript. Our detailed comments are listed below.

      Major comments

      Introduction

      Several statements made in the introduction are misleading and suggest that authors are trying to exaggerate the impact of their work. For example, "Furthermore, different species of mosquitoes exhibit plasticity and distinct rhythms in their daily activity pattern, including locomotion, feeding, mating, blood-feeding, and oviposition, facilitating their adaptation into separate time-niches (7, 8), but the underlying molecular mechanism for the heterogenous temporal activity remains to be explored." is not accurate since daily rhythms in mosquitoes' transcriptomes, behavior, and olfactory sensitivity have been the object of several publications. Even though some of them are listed later in the introduction, they contradict the claim made about the knowledge gap. See:

      Rund, S. S., Gentile, J. E., & Duffield, G. E. (2013). Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC genomics, 14(1), 1-19

      Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., & Duffield, G. E. (2011). Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences, 108(32), E421-E430

      Rund, S. S., Bonar, N. A., Champion, M. M., Ghazi, J. P., Houk, C. M., Leming, M. T., ... & Duffield, G. E. (2013). Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Scientific reports, 3(1), 2494

      Rund, S. S., Lee, S. J., Bush, B. R., & Duffield, G. E. (2012). Strain-and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. Journal of insect physiology, 58(12), 1609-1619

      Leming, M. T., Rund, S. S., Behura, S. K., Duffield, G. E., & O'Tousa, J. E. (2014). A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti. BMC genomics, 15(1), 1-9

      Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

      Rivas, G. B., Teles-de-Freitas, R., Pavan, M. G., Lima, J. B., Peixoto, A. A., & Bruno, R. V. (2018). Effects of light and temperature on daily activity and clock gene expression in two mosquito disease vectors. Journal of Biological Rhythms, 33(3), 272-288

      The knowledge gap brought up in the next paragraph of the introduction doesn't reflect the questions asked by the experiments: "But, how the pacemaker differentially influences peripheral clock activity present in the olfactory system and modulates olfactory sensitivity has not been studied in detail." Specifically, the control of peripheral clocks by the central pacemaker has not been evaluated here.

      "In vertebrates and invertebrates, it is well documented that circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" should also cite work on cockroaches and kissing bugs:

      Lubinski, A. J., & Page, T. L. (2016). The optic lobes regulate circadian rhythms of olfactory learning and memory in the cockroach. Journal of Biological Rhythms, 31(2), 161-169

      Page, T. L. (2009). Circadian regulation of olfaction and olfactory learning in the cockroach Leucophaea maderae. Sleep and Biological Rhythms, 7, 152-161

      Vinauger, C., & Lazzari, C. R. (2015). Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus. Journal of Experimental Biology, 218(19), 3110-3117

      The sentence: "Previous studies showed that synaptic plasticity and memory are significantly influenced by the strength and number of synaptic connections (43, 44)." should be nuanced as the role of neuropeptides such as dopamine has also been showed to influence learning and memory in mosquitoes:

      Vinauger, C., Lahondère, C., Wolff, G. H., Locke, L. T., Liaw, J. E., Parrish, J. Z., ... & Riffell, J. A. (2018). Modulation of host learning in Aedes aegypti mosquitoes. Current Biology, 28(3), 333-344

      Wolff, G. H., Lahondère, C., Vinauger, C., Rylance, E., & Riffell, J. A. (2023). Neuromodulation and differential learning across mosquito species. Proceedings of the Royal Society B, 290(1990), 20222118

      Overall, the paragraph dealing with the idea that "circadian phase-dependent training can influence olfactory memory acquisition and consolidation of brain functions" is very confusing. This paragraph discusses mechanisms of learning-induced plasticity but seems to ignore the simplest (most parsimonious) explanations for the circadian regulation of learning (e.g., time-dependent expression of genes involved in memory consolidation). In addition, the sentence quoted above is circumvoluted to simply say that training at different times of the day affects memory acquisition and consolidation. Although the authors did look at one gene involved in neural function, learning, memory, or circadian effects were not analyzed in this study. Please reconsider the relevance of the paragraph.

      The sentence: "But, how the brain of mosquitoes entrains circadian inputs and modulates transcriptional responses that consequently contribute to remodel plastic memory, is unknown." should be rephrased. First, it should be "entrains TO circadian inputs", and second, it suggests that the study will be investigating circadian modulation of learning and memory, which is not the case. Furthermore, the term "remodel plastic memory" is unclear and doesn't seem to relate to any specific cellular or neural processes.

      Given the differences in mosquito chronobiology observed even between strains, why perform the RNAi and EAGs on a different species of Anopheles than the one used for the RNAseq (or vice versa)?

      Results

      "As reported earlier, a significant upregulation of period and timeless during ZT12-ZT18 was observed in both species (Figure 1C)." Please provide effect size and summary statistics.

      "Next, the distribution of peak transcriptional changes in both An. culicifacies and Ae. aegypti was assessed through differential gene-expression analysis. Noticeably, An. culicifacies showed a higher abundance of differentially expressed olfactory genes (Figure 1D)" Please provide effect size and summary statistics.

      "Taken together, the data suggests that the nocturnal An. culicifacies may possess a more stringent circadian molecular rhythm in peripheral olfactory and brain tissues." What do the authors mean by "stringent"? At this point, this should be stated as a working hypothesis, as the statement is not backed up by the data. It is possible that the fewer differentially expressed genes of Aedes aegypti are more central to regulatory networks and cascade into more "stringent" rhythmic control of activities and rhythms.

      The section title: "Circadian cycle differentially and predominantly expresses olfaction-associated detoxification genes in Anopheles and Aedes" doesn't make sense. The expression of genes can be modulated by circadian rhythms, but cycles don't express genes. Please rephrase. In addition, this whole section deals with "circadian rhythms" while no experiment has been conducted under constant conditions. The observed daily variations are therefore diel rhythms until their persistence under constant conditions is established.

      "The downregulated genes of Ae. aegypti did not show any functional categories probably due to the limited transcriptional change." Could the authors explain if this is actually the phenomenon or due to a lack of temporal resolution in the study design (i.e., 4 time points)?

      "a GO-enrichment analysis was unable to track any change in the response-to-stimulus or odorant binding category of genes (including OBPs, CSPs, and olfactory receptors)." This finding doesn't corroborate the statements made previously and doesn't align with previously published studies. Is it due to pitfalls in the study design?

      "In contrast, three different clusters of OBP genes in Ae. aegypti showed a time-of-day dependent distinct peak in expression starting from ZT0-ZT12 (Figure 2F)." Please provide summary statistics.

      "In the case of An. gambiae, the amplitudes of odor-evoked responses were significantly influenced by the doses of all the odorants tested (repeated measure ANOVA, p {less than or equal to} 2e-16) (Figure S4B)." Did the authors use a positive control for the EAGs? How did the authors normalize the responses across the two species? Given the way the data is presented, how were the data normalized to allow inter-species comparisons? In addition, It is highly unlikely that all the mosquito preps used in the EAG assay responded to all the odors tested. If that was the case, then the dataset includes missing data for certain odors and time points. We believe the authors have ensured there are at least a certain number of responses per odor and time point combinations. If this is true, repeated measures ANOVA is not suited for analyzing this data because this statistical technique requires all repeated measures within and across preps without missing values. Also, the authors need to correct the summary statistics for multiple comparisons within this framework to avoid inflating type-I errors. Has this been done?

      "Ae. aegypti was found to be most sensitive to all the odorants (4-methylphenol, β-ocimine, E2-nonenal, benzaldehyde, nonanal, and 3-octanol) during ZT18-20 except sulcatone (Figure 3C - 3H)." Although some of these chemicals are associated with plants and Ae. aegypti is suspected to sugar feed at night, how do the authors explain that the peak olfactory sensitivity occurs at night for compounds such as nonanal? It would be interesting to discuss how these results compare to previous studies such as:

      Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., & Vinauger, C. (2018). Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects, 9(4), 147

      "Additionally, our principal components analysis also illustrates that most loadings of relative EAG responses are higher towards the Anopheles observations (Figure S4C)." The meaning of this sentence is unclear? Please clarify.

      "Taken together these data indicate that An. gambiae may exhibit higher antennal sensitivity to at least five different odorants tested, as compared to Ae. aegypti." As mentioned above, how did the authors normalized across species to allow comparisons? If not normalized, how do you ensure that higher response magnitudes correlate with higher olfactory sensitivity, given potential differences in the morphology or size differences between the two species? Furthermore, An. gambiae has been exclusively used in the EAG assay. Besides the lack of a justification for using a species other than An. culicifacies, the authors have interpreted the EAG results under the assumption that the olfactory sensitivities of An. gambiae and An. culicifacies are comparable. This, however, is a major caveat in the experiment design, given previous studies (indicated below) have reported species-specific variations in olfactory sensitivity. In its present form, the EAG data from An. gambiae is not a piece of appropriate evidence that the authors could use to complement or substantiate the findings from other aspects of this study on An. culicifacies.

      i. Wheelwright, M., Whittle, C. R., & Riabinina, O. (2021). Olfactory systems across mosquito species. Cell and Tissue Research, 383(1), 75-90.

      ii. Wooding, M., Naudé, Y., Rohwer, E., & Bouwer, M. (2020). Controlling mosquitoes with semiochemicals: a review. Parasites & Vectors, 13, 1-20.

      iii. Gupta, A., Singh, S. S., Mittal, A. M., Singh, P., Goyal, S., Kannan, K. R., ... & Gupta, N. (2022). Mosquito Olfactory Response Ensemble enables pattern discovery by curating a behavioral and electrophysiological response database. Iscience, 25(3).

      "Similar to An. gambiae, a comparatively high amplitude response was also observed in An. stephensi (Figure S4D)." This is interesting but what would be even more relevant to the present study is to discuss how the time-dependent responses compare between the two Anopheles species.

      The paragraph titled "Daily temporal modulation of neuronal serine protease impacts mosquito's olfactory sensitivity" is confusing because the authors move on to test the effect of knocking down a serine protease gene (found to be differentially expressed throughout the day) on olfactory sensitivity. While this is interesting in and of itself, the link between the role of this gene in learning-induced plasticity, the circadian modulation of "brain functions" and olfactory sensitivity is 1) unclear and 2) not explicitly tested. We agree with the authors that what has been tested is "the effect of neuronal serine protease on circadian-dependent olfactory responses," but the two paragraphs leading to it seem to be extrapolating functional links that have yet to be determined. In this context, their conclusions that "Our finding highlights that daily temporal modulation of neuronal serine-protease may have important functions in the maintenance of brain homeostasis and olfactory odor responses." is misleading because although they used the hypothetical "may", the link between the temporal modulation of one serine protease gene and the maintenance of brain homeostasis is not explicitly tested here.

      Discussion

      The first sentence of the discussion: "In this study, we provide initial evidence that the daily rhythmic change in the olfactory sensitivity of mosquitoes is tuned with the temporal modulation of molecular factors involved in the initial biochemical process of odor detection i.e., peri-receptor events" is not true since studies from Rund and Duffield previously revealed the daily modulation of OBP gene expression. It also contradicts the next sentence: "The findings of circadian-dependent elevation of xenobiotic metabolizing enzymes in the olfactory system of both Ae. aegypti and An. culicifacies are consistent with previous literature (26, 31), and we postulate that these proteins may contribute to the regulation of odorant detection in mosquitoes."

      The use of "circadian" in the discussion of the results is also misleading as only diel rhythms were evaluated in the present study.

      "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58)." This is not really what these references show.

      "Given the potentially larger odor space in mosquitoes (like other hematophagous insects) (16, 58), it can be hypothesized that detection of any specific signal in such a noisy environment, mosquitoes may have evolved a sophisticated mechanism for rapid (i) odor mobilization and (ii) odorant clearance, to prevent anosmia (24)." One could argue that this is a requirement for all insects, regardless of the size of their olfactory repertoire.

      "Taken together, we hypothesize that circadian-dependent activation of the peri-receptor events may modulate olfactory sensitivity and are key for the onset of peak navigation time in each mosquito species." This is not entirely accurate since spontaneous locomotor activity rhythms are also observed in the absence of olfactory stimulation. While "navigation" does imply olfactory-guided behaviors, "peak navigation time" appears to be driven by other processes. See, for example, all studies testing mosquito activity rhythms in locomotor activity monitors.

      "Due to technical limitations, and considering the substantial data on the circadian-dependent molecular rhythmicity" please clarify what the technical limitations were. Is this something that prevented the authors specifically, or something tied to mosquito biology and would prevent anybody from doing it? Also, why couldn't the transcriptomic analysis be performed on An. gambiae?

      "In contrast to An. gambiae, the time-dose interactions had a higher significant impact on the antennal sensitivity of Ae. aegypti. An. gambiae showed a conserved pattern in the daily rhythm of olfactory sensitivity, peaking at ZT1-3 and ZT18-20." These two sentences are very confusing. Doesn't it simply mean that the co-variation is not linear or not the same across odors? In addition, what does it mean for a pattern to be more conserved? How can one conclude about the "conserved" nature of a pattern by looking at time-dependent variations in dose-response curves?

      "Together these data, we interpret that mosquito's olfactory sensitivity possibly does not follow a fixed temporal trait" is unclear and suggests that the authors are discussing global versus odor-specific rhythms. Please rephrase.

      "Moreover, we hypothesize that under standard insectary conditions, mosquitoes may not need to exhibit foraging flight activity either for nectar or blood, and during the time course, it may minimize their olfactory rhythm, which is obligately required for wild mosquitoes." This hypothesis is not supported by the results of the study and contradicts work by others (Rund et al., Eilerts et al., Gentile et., etc).

      The same comment applies to "Therefore, it is reasonable to think that the mosquitoes used for EAG studies may have adapted well under insectary settings and, hence carry weak olfactory rhythm." as this statement is not supported by results of the present study or comparisons of the results to previous studies based on field-caught mosquitoes. Although it is an interesting question to ask in the future, it should be stated as a future research avenue rather than a working hypothesis that results from the present study.

      "Aedes aegypti displayed a peak in antennal sensitivity at ZT18-20 to the higher concentrations of plant and vertebrate host-associated odorants tested. Given the time-of-day dependent multiple peaks (at ZT6-8 and ZT18-20 for benzaldehyde and at ZT12-14 and ZT18-20 for nonanal) in antennal sensitivity to different odorants, our data supports the previous observation of bimodal activity pattern of Ae. aegypti (50)." Rephrase by saying that results are "aligned with the previous observations of bimodal activity". Olfactory rhythms don't "support" the activity patterns because olfactory processes and spontaneous locomotor activity are independent processes.

      "our preliminary data indicate that Anopheles spp. may possess comparatively higher olfactory sensitivity to a substantial number of odorants as compared to Aedes spp." Consider removing this sentence unless the way the data has been normalized to allow for comparisons between species is clarified.

      In "A significant decrease in odorant sensitivity for all the volatile odors tested in the CYP450-silenced Ae. aegypti," please change "silenced" to "reduced" because RNAi doesn't silence (i.e. knockout) gene expression.

      The title "Neuronal serine protease consolidates brain function and olfactory detection" is extremely misleading. Do the authors refer to memory consolidation, which has not been tested here? What is brain function consolidation??

      The reference used in "Despite their tiny brain size, mosquitoes, like other insects, have an incredible power to process and memorize circadian-guided olfactory information (7)." is not appropriate. Also, "circadian-guided" is unclear. Consider replacing it with "circadian-gated".

      What is the "the homeostatic process of the brain"?

      "the temporal oscillation of the sleep-wake cycle of any organism is managed by the encoding of experience during wake, and consolidation of synaptic change during inactive (sleep) phases, respectively (70)." By experience, do the authors refer to learning? This seems out of topic as this process has not been evaluated here.

      "We speculate that after the commencement of the active phase (ZT6-ZT12), the serine peptidase family of proteins in the brain of Ae. aegypti mosquitoes may play an important function in consolidating brain actions (after ZT12) and aid circadian-dependent memory formation." The value of this statement is unclear. Circadian-dependent memory formation is not being evaluated here, and the results from the present study do not directly support this speculation, also because other processes involved in memory formation are not evaluated here. This seems at odds with the literature on learning and memory.

      "Subsequent work on electrophysiological and neuro-imaging studies are needed to demonstrate the role of neuronal-serine proteases in the reorganization of perisynaptic structure." Sure. But the link between "the role of neuronal-serine proteases in the reorganization of perisynaptic structure" and rhythms in olfactory sensitivity is unclear.

      As a general comment, EAGs seem inappropriate to evaluate the effect of the central-brain processing in the regulation of peripheral olfactory processes. This is a critical comment that needs to be considered by the authors and clarified in the manuscript. If rhythms of central brain processes are important for olfactory-guided behaviors, these should be evaluated at the level of the central brain or via behavioral metrics. The effect of the RNAi knockdowns on peripheral sensitivity is interesting, but its link with central processes is unclear and doesn't support the speculations made by the authors about learning and memory.

      Methods

      No explanations are provided for how the EAG data are normalized to allow comparisons between species.

      Figures

      Figure 1: The daily rhythm depicted in A, are not representative of the actual profiles. See: Benoit, J. B., & Vinauger, C. (2022). Chapter 32: Chronobiology of blood-feeding arthropods: influences on their role as disease vectors. In Sensory ecology of disease vectors (pp. 815-849). Wageningen Academic Publishers. Or any other paper on mosquito activity rhythms.

      Figure 3 and 4: The EAG results are plotted twice. This is redundant and misleading as it makes the reader think there is more data than actually presented.

      Figure 5: Please clarify the sample size for each panel. In C - F, what would be used as a reference? In other words, what is a Relative EAG Response of 1? And if it is "relative", are the units really mV? In E and F, it would be great to show how the Ethanol control compares to the no solvent condition. This could be placed in supplementary materials.

      Figures 5 and 6, given the dispersion in the EAG data, the treatments where N=40 appear robust, but the interpretation of results from treatments where N=6 may be limited due to the low sample size. This limitation is visible in Figure 5F, for example, where ABT-Aceto is different from Cont-Aceta but not PBO-Aceto because one individual shows a higher response.

      Figure S6: how does this support that synaptic plasticity is influenced by "Time-of-day dependent modulation of serine protease genes in the brain"?

      Minor comments

      What do the authors mean by "consolidation of brain functions"? Memory consolidation? Please clarify.

      In "Similar to previous studies (26), the expression of a limited number of rhythmic genes was visualized in Ae. aegypti" please replace "visualized" with "observed".

      Figure 2A, please clarify in the caption what FDR stands for.

      In "To further establish this proof-of-concept in An. gambiae, three potent CYP450 inhibitors, aminobenzotriazole(52), piperonyl butoxide(53), and schinandrin A (54), was applied topically on the head capsule of 5-6-day-old female mosquitoes" replace "was applied" with "were applied".

      "Interestingly, our species-time interaction studies revealed that An. gambiae exhibits time-of-day dependent significantly high antennal sensitivity to at least four chemical odorants compared to Ae. aegypti, except phenol." is unclear. Please reword.

      In "Similar observations were also noticed with An. stephensi." replace "noticed" with "made".

      Significance

      Such a study has the potential to be valuable for the field, but its value and significance are hindered by an accumulation of overstatements, the fact that prior work in the field has been minimized or omitted, and a lack of support for the stated conclusions.

      In this context, the advances are only slightly incremental compared to the work produced by Rund et al., and the mechanistic hypotheses emitted to link the genes selected for knockdown experiments and olfactory sensitivity are not clearly supported by the evidence presented here. The main strength of the paper is to show the role of CYP450 in olfactory sensitivity.

      The audience is fairly broad and includes insect neuro-ethologists, molecular biologists, and chronobiologists.

      Our field of expertise:

      • Mosquito chemosensation
      • Learning and memory
      • Chronobiology
      • Electrophysiology
      • Medical entomology
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      Reply to the reviewers

      Overall comments

      We are pleased by the reviewers' comments and appreciate their suggestions for improvements. In addition to correcting small typos throughout the manuscript, we have made the following additions or changes in response to reviewer comments and suggestions:

      1. New complementation experiments to verify the impacts of mgtA and PA4824 on bacterial fitness in fungal co-culture.
      2. New experiments to measure intracellular Mg2+ levels in corA or mgtE mutants to strengthen our conclusion that neither of these constitutive Mg2+ transporters is required for maintaining intracellular Mg2+ levels in co-culture.
      3. New experiments to confirm that the * cerevisiae mnr2D mutant does not have a fitness defect compared to WT in co-culture. This finding rules out the possibility that metabolic defects in the mnr2D mutant restore the fitness of bacterial mgtA* mutant in co-culture and strengthens our hypothesis that Mg2+ sequestration by fungal vacuole triggers Mg2+ nutritional competition with bacteria.
      4. Clarification of bacterial species we tested in our study as suggested by Reviewer #3.
      5. Revised discussion to highlight how our findings relate to any fungal-bacterial interaction both in ecological and infection contexts and any known role of mgtA in antibiotic susceptibility, as suggested by Reviewer #2. All changes in response to the reviewer's comments have been detailed in our point-by-point response (below).

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

      This manuscript investigates polymicrobial interactions between two clinically relevant species, Pseudomonas aeruginosa and Candida albicans. The findings that C. albicans mediates P. aeruginosa tolerance to antibiotics through sequestration of magnesium provides insight into a specific interaction at play between these two organisms, and the underlying mechanism. The manuscript is well composed and generally the claims throughout are supported by the provided evidence. As a result, most comments are either for clarification, or minor in nature.

      We thank the reviewer for their positive comments and their suggestions for improvement.

      Major comments:

      1) For their experiments, the authors frequently switch between 30C and 37C, but there is no rationale for why a specific temperature was used, or both were. E.g. some of the antibiotic survival assays, and fungal-bacterial co-culture assays were performed at both temperatures, while the colistin resistance, fitness competition and RNA sequencing were performed at 30C. Given the fact that the two organisms are both human pathogens and co-exist in human infections, it is not clear why 30C was used. The authors should provide clarity for why these two temperatures were used.

      We thank the reviewer for raising this point. Fungal-bacterial interactions occur in a range of temperatures in ecological contexts (e.g., in soil or on plants) or during infection in animal hosts. Both 30oC and 37oC degree temperatures are used in C. albicans studies whereas 37oC is most preferred for P. aeruginosa studies. By providing data from both temperatures for critical experiments, we demonstrate that our findings are not dependent on temperature. Our studies also allow for an easy comparison to previously published studies performed at both temperatures. We chose to screen initial co-culture conditions showing fungal antagonism at 30oC, as C. albicans cells can reach higher CFUs than at 37oC due to growth in the single-celled yeast form.

      We agree with the reviewer that 37oC is more physiologically relevant for conditions under which these two species coexist in animal hosts. Thus, we tested our findings of Mg2+ competition and antibiotic survival at 37oC.

      We now clarify our reasoning in the revised Materials and Methods section as follows: "We chose 30oC for the initial co-culture assays for two reasons. First, C. albicans cells reached higher CFU at 30oC than 37oC, which would impose a stronger competition with bacteria. Second, C. albicans cells form hyphae at 37oC, which can have multiple cells in one filament and thus confound CFU measurements. We further confirmed that our findings of Mg2+ competition are independent of temperatures by setting up co-culture assays at both 30oC and 37oC."

      2) Lines 184-191: It would be useful to measure intracellular Mg2+ (using the Mg sensor) in the corA and mgtB tn mutants in media as well as the fungal spent media, to provide stronger support for the claim that "MgtA is a key bacterial Mg2+ transporter that is highly induced under low Mg2+ conditions".

      We thank the reviewer for this suggestion. Based on our experiments, neither CorA or MgtE are induced (in RNA-seq analyses) nor required in co-culture (in Tn-seq analyses), suggesting neither is involved in Mg2+ competition with C. albicans. In contrast, MgtA is highly induced in co-culture. Loss of mgtA significantly reduces bacterial fitness in co-culture and intracellular Mg2+ levels only in C. albicans-spent BHI, but not fresh BHI. These results suggest that MgtA is the key Mg2+ transporter required for bacterial Mg2+ uptake and fitness in co-culture.

      Nevertheless, we agree with the reviewer that despite being constitutively expressed, CorA or MgtE might play an important role in importing Mg2+ in BHI and C. albicans-spent BHI. To test this possibility, we performed a new experiment suggested by the reviewer (now included in the revised manuscript) in which we measured intracellular Mg2+ levels in corA or mgtE loss-of-function mutants in BHI versus C. albicans-spent BHI, and compared them to intracellular Mg2+ levels in a mgtA loss-of-function mutant strain. We find that lack of either corA or mgtE does not significantly reduce bacterial Mg2+ levels in C. albicans-spent BHI compared to DmgtA mutant (Fig. S7C). Thus, our results strengthen our conclusion that MgtA is the key Mg2+ transporter that gram-negative bacteria use to overcome fungal-mediated Mg2+ sequestration.

      3) Line no. 276. Does the mnr2∆ S. cerevisiae mutant have a growth defect compared to the WT? This would test whether the effect of the mnr2 mutant on P. aeruginosa fitness is strictly due to Mg2 and not due to reduced growth or metabolism of the mutant.

      We agree with the possibility raised by the reviewer. In new experiments included with our revision as Figure S10, we find that the S. cerevisiae mnr2 deletion mutant exhibits similar CFU as WT in monoculture as well as co-culture. Thus, the rescuing effect of mnr2D is less likely due to reduced growth or metabolism.

      4) The authors use the term 'antibiotic resistance' throughout the manuscript. However, the assays they perform do not directly test for antibiotic resistance which is defined as the ability to grow at higher concentrations of antibiotics (e.g. as measured by MIC tests). The authors should rephrase their phenotype as antibiotic survival or antibiotic tolerance.

      We agree with the reviewer and thank them for raising this point. We replaced the phrase 'antibiotic resistance' with 'antibiotic survival' throughout the revised manuscript. We also accordingly changed our title to 'Widespread fungal-bacterial competition for magnesium lowers antibiotic susceptibility'

      5) Also, the authors have two different assays, both measuring survival in antibiotic, but one is called a colistin resistance assay (line 508) and the other a colistin survival assay (line 523). It's not obvious what is the difference between what is being assayed in the two experiments, except perhaps the growth phase of the cells when they are exposed to the antibiotic? The authors should explain the difference, and the rationale for using two different assays.

      We thank the reviewer for raising this point. In the revised manuscript, we explain the rationale of our two assays. The first assay measures the bacterial survival after colistin treatment in C. albicans-spent BHI, and the second measures the bacterial survival after colistin treatment in co-culture with C. albicans. We performed both assays because C. albicans-spent BHI mimics Mg2+-depleted conditions by C. albicans but might not represent all aspects of fungal presence in co-culture. To make sure our findings are consistent across these two experiments, we specify the difference in these two assays in the revised manuscript as the following: "Since fungal spent media cannot fully recapitulate fungal presence in co-culture conditions, we tested whether fungal co-culture also conferred increased colistin survival."

      Minor comments:

      • For almost all the figures, blue and orange dots are used for 'monoculture' and 'coculture' respectively, while orange and black dots are used for WT and the mgtA mutant. However, the black and blue dots are hard to tell apart, and for several figure sub-panels, the legends are not provided (e.g. figures 2D, 2F, S9H), making it a little confusing to figure out what is being shown. It would be best if the WT and mgtA symbols were in colors completely different from the monoculture/co-culture colors, making it easier to tell those apart.

      We have updated these figures as the reviewer suggested.

      Line no 122 and Figure 1A. The term "defense genes" in bacteria typically refers to genes conferring protection against phage infections. Perhaps the authors can use a different term (e.g. 'protective genes').

      We agree with the reviewer. We have changed "defense genes" to "fungal-defense genes" to disambiguate the terms.

      Line no 186. 'However, neither MgtA...' should be 'However, neither MgtE...'

      We thank the reviewer for pointing out this typo. We have fixed this in our revision.

      Line no 268. Does fungal-mediated Mg2+ competition extend to Gram positive bacteria?

      We thank the reviewer for raising this interesting point. MgtA is prevalent in diverse gram-negative bacteria but rare in gram-positive bacteria. Using the fitness effect of mgtA mutants in co-culture vs monoculture allowed us to infer Mg2+ competition easily for diverse gram-negative bacteria. Currently, we do not have the experimental tools to extend this finding to gram-positive bacteria. Co-culture growth kinetics for gram-positive bacteria are also likely to be different from gram-negative bacteria in a way that makes direct comparisons challenging. We have clarified our writing in the revised manuscript: "This mode of competition might be highly specific between fungi and diverse gram-negative g-proteobacteria we have tested.... Whether fungi can suppress gram-positive bacteria through the same mechanism of Mg2+ competition remains an open question."

      Line no 314. It is unclear whether the 'transient co-culture' is the same or a different assay as the colistin survival assay.

      We apologize for the confusion and have removed the word 'transient' for clarity. The assays is the same as the 'colistin survival assay in fungal co-culture,' where we co-cultured log-phase P. aeruginosa cells with C. albicans for 5 hours and treated them with colistin.

      Line no 316. For the bacterial survival assays shown in figures 3 and 4 (and other supplementary figures), please provide absolute numbers as cfu (as in figures 1 and 2), as opposed to a percentage, for cell counts. This will allow readers to appropriately interpret the data.

      We thank the reviewer for this suggestion. We now include the raw CFU counts of colistin survival assays in Fig 3 and 4 and other supplementary figures in new supplementary figures (Fig. S11, S13, S14, S15, and S17) in our revision.

      Line no 934-5: Italicize P. aeruginosa.

      This typo has been fixed in our revision.

      Reviewer #1 (Significance (Required)):

      This study identifies a novel interaction between two the co-infecting human pathogens Pseudomonas aeruginosa and Candida albicans, where C. albicans causes Mg2+ limitation for P. aeruginosa. Further, the authors show that this interaction affects levels of antibiotic resistance, as well as the adaptive mutations seen during the evolution of antibiotic resistance. This advances the field by delineating how microbial interactions can affect clinically relevant phenotypes, and potentially clinical outcomes. The study should be of interest to a broad audience of researchers studying microbial ecology, evolutionary biology, microbiology, and infectious diseases.

      We are grateful for the reviewer's positive appraisal.

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

      This paper looks at the interaction between the fungus Candida albicans (Ca) and the bacterium Pseudomonas aeruginosa (Pa), which are found together in some environments. Co-culture experiments showed that Ca can inhibit the growth of Pa. The goal of this study is to determine the reason for this phenomenon and how widespread it is. This was performed by Tnseq analysis of Pa that identified 3 genes which showed significant decreases in the presence of Ca. Interestingly these were all in an operon that was recognized by the authors as being induced by RNAseq during co-culture. One of these genes, mgtA is a known Mg2+ transporter and therefore the remainder of the paper discusses the importance of competition for Mg2+.

      The experiments seem to be well carried out and appropriately controlled.

      We thank the reviewer's appreciation of our science and the rigor of our experiments.

      The use of the Mg2+ genetic sensor reporter in Pa is an interesting approach to determine the intracellular Mg2+ concentrations, however how these levels relate to one another between different experiments is not clear. In Fig. S5, the levels are 5 AU for growth in minimal media +low (10uM) Mg2+ and 38 AU for growth in minimal media+high (10mM) Mg2+. But the levels seen in Figure 1E are all much lower. With such low levels, it is difficult to determine if the impact of ∆PA4824 and ∆mgtA (while perhaps additive) are relevant. Would differences be seen with these various strains grown under different conditions?

      We thank the reviewer for this query. The reviewer is right in that we do not use absolute quantification of intracellular Mg2+ levels. While our Mg2+ genetic sensor assay does not facilitate comparison of absolute Mg2+ levels across experiments, it provides a robust comparative measurement of relative intracellular Mg2+ levels in mutants versus WT cells, or between two different media conditions.

      Using this Mg2+ genetic sensor assay, we tested intracellular Mg2+ levels of WT P. aeruginosa under various media conditions. We found that lower intracellular Mg2+ levels in P. aeruginosa cells and the requirement of mgtA in these media are well-correlated at lower total Mg2+ levels in media (Fig. S9A-E). In contrast, there are no significant differences in intracellular Mg2+ levels between DmgtA (or DPA4824) and WT cells in BHI media, which has higher total Mg2+ levels than fungal-spent BHI media. Our experiments reveal that the lack of mgtA or PA4824 only affects intracellular Mg2+ levels when P. aeruginosa is cultured in media below a threshold level of Mg2+ concentration in media.

      The experiments suggesting that the protein PA4824 is also a Mg2+ transporter seem to be related only to alpha fold predictions.

      We clarify that our speculation that PA4824 encodes a potential novel Mg2+ transporter was first motivated by finding that it is induced in low Mg2+ conditions, its genetic importance in Tn-seq experiments independent of mgtA, and our finding that cells with loss-of-function mutations in PA4824 experience lower intracellular Mg2+ than WT cells. However, the reviewer is correct that this statement is speculative based on the Alphafold prediction. In the revised manuscript, we have clarified this point as the following: "Based on our co-culture RNA-seq and Tn-seq experiments, results from the Mg2+ genetic sensor assay, and the Alphafold prediction of PA4824 protein structure, we speculate that PA4824 potentially acts as a novel Mg2+ transporter."* * Is the statement in line 186 a typo? It is stated that "neither MgtA nor CorA was implicated in competition". Do the authors actually mean "MgtE"?

      It is a typo. We thank the reviewer for pointing this out and have changed this to "MgtE".

      Reviewer #2 (Significance (Required)):

      Ca and Pa are known to inhabit the same niches and previous studies have shown both can have antagonist effects on one another. Nutritional competition is one mechanism of antagonism that has not been that well studied between these two genera. That makes the finding of some significance and relevant to those with an interest in either of these microbes and co-infections. The authors also found that it was not just Ca that had this effect, but other fungi as well. And this effect was not just reserved to effect Pa, but also other bacteria, suggesting a more global impact.

      We thank the reviewer for an accurate summary of our findings.

      However, diminishing the impact of this finding is the question as to whether this is simply a phenomena seen under the very specific laboratory conditions tested here. Furthermore how these findings exactly relate to any infection environment is not clear.

      Fungal-bacterial interactions occur in a variety of broad biological contexts, including during infection in animal hosts or in environmental-associated microbial communities. Our study is the first to identify nutritional competition for Mg2+ as one of the most important axes of competition between fungi and bacteria. Our study also identifies MgtA as one of the key bacterial genes that mediates this interaction. MgtA is only induced upon experiencing low Mg2+ conditions; the fact that most gram-negative bacteria encode MgtA implies they must encounter low Mg2+ conditions and face fitness consequences in those conditions. To address the reviewer's concerns, we also highlight three additional points in our revised Discussion:

      1. Fungal-bacterial competition for Mg2+ is not restricted only to BHI media alone. We also found the same phenomenon in TSB media medium. Indeed, we show (Fig. S9F) also that Mg2+ competition occurs whenever the environmental Mg2+ level is lower than 0.45mM, a critical threshold for fungi and bacteria to compete for this vital ion.
      2. During infection in cystic fibrosis airways, proteomic experiments and Mg2+ measurement in CF sputum both suggest that * aeruginosa* experiences Mg2+ restriction.
      3. Many previous studies have shown that many Gram-negative bacteria, including Salmonella Typhimurium, encounter reduced magnesium concentrations upon infection of hosts (PMID: 29118452). Our discovery that fungal co-culture may generally exacerbate fitness challenges associated with low magnesium levels is of high importance to all studies of gram-negative bacteria, not just to Pa.
      4. In addition to infections in animal hosts, low Mg2+ is associated with worse outcomes of infections in plants. Our study suggests the importance of studying the role of Mg2+ competition in various infection contexts and the strategies of manipulating Mg2+ levels or fungal-bacterial interactions to constrain polymicrobial infectious diseases in diverse eukaryotic hosts and ecological conditions. The authors also seem to vastly overinterpret the significance of their findings; the impact on Pa is only to slow growth, not necessarily effect fitness, per se. The final number of bacteria appears to be the same, it just takes slightly longer to get there.

      We are puzzled by this comment from the reviewer; slow growth IS a fitness effect! Although we agree with the reviewer's point that C. albicans is more likely to inhibit bacterial growth rate than viability (bacteriostatic, not bacteriocidal), there are many bacteriostatic antibiotic mechanisms.

      In our co-culture assay, bacterial CFUs after 40 hours in co-culture are 10-100 times lower than in monoculture (this is not a subtle effect!). After 40 hours, bacterial cultures have already reached the stationary phase, which is why even slower growing bacterial cells in co-culture can 'catch up' (they are still lower by nearly 10-fold), despite fungal inhibition. Moreover, the co-culture condition provided enough of a fitness challenge to allow us to identify bacterial protective genes even in a pooled assay.

      The authors speculate that that since Mg2+ supplementation did not totally restore growth to Pa during co-culture, that other Mg2+ independent "axes of antagonism" must exist. This also tends to diminish the significance of these finding.

      Again, we are puzzled by this comment from the reviewer. Fungal-bacterial competition, like all microbial competition, is a multifactorial process, so we should not be surprised that Mg2+ isn't the only axis of competition. Indeed, our study reinforces the importance of investigating all potential axes of competition to get a complete understanding of the mechanisms of fungal-bacterial competition.

      The importance of mgtA on antibiotic susceptibility has been well studied in a number of bacteria including Pa making these findings generally confirmatory.

      We would like to clarify this comment. To the best of our knowledge, mgtA in P. aeruginosa has not been reported in antibiotic susceptibility studies. Instead, P. aeruginosa mgtE is induced upon treatment with aminoglycoside antibiotics, but its expression does not change antibiotic resistance (PMID: 24162608).

      The reviewer may be referring to studies in S. Typhimurium, where the DmgtA mutant shows increased susceptibility to nitrooxidative stress (PMID: 29118452) and to cyclohexane (PMID: 18487336), suggesting Mg2+ homeostasis might be generally important for bacterial survival to antimicrobial treatments. Although this is not the main focus of our study, we now include these references in our revised discussion to provide readers with more background on the relevance of our work: "Mg2+ has been implicated in altering the susceptibility of gram-negative bacteria to antibiotics other than colistin. For instance, in S. Typhimurium, impaired mgtA or Mg2+ homeostasis increases susceptibility to cyclohexane or nitrooxidative stress. In line with these observations, our study also highlights the importance of studying how Mg2+ homeostasis broadly impacts antimicrobial resistance in gram-negative bacteria."

      The importance of different mutations that emerge in Pa during mono vs. co-culture in the presence of colistin is not clearly explained. Why should co-culture inhibit the emergence of hypermutator Pa strains?

      We thank the reviewer for the opportunity to clarify this important point. Previous studies have shown, both in Pa as well as other bacteria, that hypermutator strains often arise when bacteria adapt to strong and continuous antibiotic stress (PMID: 28630206) to maximize exploration of mutation space necessary to acquire beneficial resistance mutations even though hypermutation itself is inherently deleterious to bacterial fitness. We show that fungal co-culture protects P. aeruginosa from high concentrations of colistin by sequestering the Mg2+ co-factor required for colistin action (Fig. 4C). Thus, under co-culture conditions, bacteria experience lower levels of colistin than the levels administered and are subject to less severe fitness challenges, allowing them to eschew the deleterious route of acquiring adaptive mutations with hypermutation.

      Our discovery that bacteria have an entirely different means of enhancing colistin resistance under fungal co-culture (or low Mg2+) conditions is one of the highlights of our study. Understanding the biological basis of this novel model of colistin resistance will be an active area of investigation to pursue in the future.

      No additional experiments are likely needed but the authors should be encouraged to place their findings more clearly in what is already known in the field as well as articulate the limitations of their study.

      We thank the reviewer for their detailed comments and suggestions. We hope our revisions have both clarified the importance and limitations of our study and provided the right context sought by the reviewer.

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

      In this study, Hsieh et al. find a critical axis of competition between Pseudomonas aeruginosa and Candida albicans is Mg2+ sequestered by Candida. The authors find that use of BHI, which is has lower Mg2+ levels compared to other media, allowed this discovery. The authors further demonstrate critical genes for this axis in multiple gammaproteobacteria and fungal species. The authors further show that fungal Mg2+ sequestration promotes polymyxin resistance in multiple gammaproteobacteria and show that it alters the course of Pseudomonas aeruginosa evolution of polymyxin resistance. Finally, they show that for populations evolved polymyxin resistance in the presence of Candida, removal of Candida by antifungal treatment re-induces sensitivity to polymyxins.

      We thank the reviewer for a concise and accurate summary of our study.

      Major comments: -The claims and conclusions are generally supported; however, a key phenotype of the ∆mgtA and ∆PA4824 mutants should be complemented in trans or in a second site of the chromosome.

      We thank the reviewer for this comment and agree with the suggestion. In our revised manuscript, we now provide results of new complementation experiments recommended by the reviewer, which find that expression of PA4824 or mgtA in trans restore the fitness cost of either deletion mutant (Fig. S4C and S4D).

      -The authors note that "This mode of competition appears to be highly specific between fungi and gram-negative bacteria." However, it does not appear that gram-positive bacteria were tested in competition with fungi. Additionally, the only gram-negative tested were gammaproteobacteria (although do represent diverse gammaproteobacteria). This could be addressed by clarifying the text or OPTIONAL additional experimentation.

      We agree with the reviewer. We had intended to highlight that we had only tested this mode of competition between fungi and gram-negative bacteria, but inadvertently phrased this to suggest that gram-positive bacteria are not subject to this competition. As we highlight in our response to Reviewer 1, we are unable to test this (so far) for gram-positive bacteria. We clarify this in our revision: ""This mode of competition might be highly specific between fungi and diverse gram-negative g-proteobacteria we have tested.... Whether fungi can suppress gram-positive bacteria through the same mechanism of Mg2+ competition remains an open question."

      -Figure 3A: is this depiction of modifications on the O-antigen correct? PhoQ- and PmrB-activated enzymes seem to modify the lipid A portion of LPS (eg PMID: 31142822)

      We thank the reviewer for noting this error, which we have now fixed in the revision.

      • For many of the figures, multiple t-tests are used and it seems like perhaps an ANOVA with multiple comparisons would be more appropriate

      We thank the reviewer for this feedback. In our revision, we now use Dunnett's one-way ANOVA test for figures with multiple comparisons; our conclusions are unchanged.

      Minor comments: - The text and figures are clear and accurate

      We thank the reviewer for this feedback.

      -the cited nutritional immunity reviews are out of date (e.g. reference 37) and there are more recent reviews on the topic (e.g. PMID: 35641670)

      We have added the suggested reference in our revision.

      -Line 293: Unclear why polymyxin resistance would be "unexpected" following the explanation of why Mg2+ depletion might confer it

      We agree and have removed 'unexpected.'

      -Line 318: "antibitoics" typo

      We thank the reviewer for pointing out this typo, which we have now corrected.

      Reviewer #3 (Significance (Required)):

      The following aspects are important:

      • General assessment: This study is very mechanistic, identifying the role of Mg2+ sequestration by fungi that limit gram-negative bacterial growth in Mg2+ deplete environments. The strengths are that relevant Mg2+ acquisition genes are identified or tested in Pseudomonas aeruginosa, the main test organism, as well as Salmonella enterica and Escherichia coli. Additionally, the authors identify a relevant Mg2+ mechanism in fungal species tested, including showing the importance with a genetic knockout. The limitations are relatively minor, and include lack of complementation, potential issues in model figure depiction of LPS modifications, and potential minor issues in statistical tests used. Future directions discussed include expanding analysis to clinical isolates, which is outside the scope of this manuscript which already showed the same mechanism in diverse gammaproteobacterial.

      We thank the reviewer for their positive appraisal.

      • Advance: This study has two major advances: The first is uncovering this critical Mg2+ sequestration axis in competition between fungal species and gammaproteobacteria. The second is the finding that the Mg+ sequestration induces polymyxin resistance and alters the evolutionary path to further polymyxin resistance. While nutrient metals as an axis of competition is not a conceptual advance, the specific role of Mg2+ and its affect on evolution of polymyxin antibiotic resistance is a conceptual advance.
      • Audience: I think this study would be of interest to a relatively broad audience. The study itself touches on multiple fields including intermicrobial competition, nutritional immunity, antimicrobial resistance, and microbial evolution. Additionally, there are clinical implications for the potential to use antifungals to resensitize polymyxin-resistant P. aeruginosa to polymyxins.
      • My field of expertise is bacterial genetics and physiology, nutritional immunity, and bacterial cell envelope. I do not have expertise in fungus.

      We appreciate the reviewer's positive and constructive feedback on our study and for highlighting the relevance of our research to a broader audience in microbiology and evolution. We do hope our mechanistic understanding of fungal-bacterial competition will spark further conversation or collaboration between evolutionary microbiologists and physician-scientists.

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      Referee #3

      Evidence, reproducibility and clarity

      In this study, Hsieh et al. find a critical axis of competition between Pseudomonas aeruginosa and Candida albicans is Mg2+ sequestered by Candida. The authors find that use of BHI, which is has lower Mg2+ levels compared to other media, allowed this discovery. The authors further demonstrate critical genes for this axis in multiple gammaproteobacteria and fungal species. The authors further show that fungal Mg2+ sequestration promotes polymyxin resistance in multiple gammaproteobacteria and show that it alters the course of Pseudomonas aeruginosa evolution of polymyxin resistance. Finally, they show that for populations evolved polymyxin resistance in the presence of Candida, removal of Candida by antifungal treatment re-induces sensitivity to polymyxins.

      Major comments:

      • The claims and conclusions are generally supported; however, a key phenotype of the ∆mgtA and ∆PA4824 mutants should be complemented in trans or in a second site of the chromosome.
      • The authors note that "This mode of competition appears to be highly specific between fungi and gram-negative bacteria." However, it does not appear that gram-positive bacteria were tested in competition with fungi. Additionally, the only gram-negative tested were gammaproteobacteria (although do represent diverse gammaproteobacteria). This could be addressed by clarifying the text or OPTIONAL additional experimentation.
      • Figure 3A: is this depiction of modifications on the O-antigen correct? PhoQ- and PmrB-activated enzymes seem to modify the lipid A portion of LPS (eg PMID: 31142822)
      • For many of the figures, multiple t-tests are used and it seems like perhaps an ANOVA with multiple comparisons would be more appropriate

      Minor comments:

      • The text and figures are clear and accurate -the cited nutritional immunity reviews are out of date (e.g. reference 37) and there are more recent reviews on the topic (e.g. PMID: 35641670)
      • Line 293: Unclear why polymyxin resistance would be "unexpected" following the explanation of why Mg2+ depletion might confer it
      • Line 318: "antibitoics" typo

      Significance

      The following aspects are important:

      • General assessment: This study is very mechanistic, identifying the role of Mg2+ sequestration by fungi that limit gram-negative bacterial growth in Mg2+ deplete environments. The strengths are that relevant Mg2+ acquisition genes are identified or tested in Pseudomonas aeruginosa, the main test organism, as well as Salmonella enterica and Escherichia coli. Additionally, the authors identify a relevant Mg2+ mechanism in fungal species tested, including showing the importance with a genetic knockout. The limitations are relatively minor, and include lack of complementation, potential issues in model figure depiction of LPS modifications, and potential minor issues in statistical tests used. Future directions discussed include expanding analysis to clinical isolates, which is outside the scope of this manuscript which already showed the same mechanism in diverse gammaproteobacterial.
      • Advance: This study has two major advances: The first is uncovering this critical Mg2+ sequestration axis in competition between fungal species and gammaproteobacteria. The second is the finding that the Mg+ sequestration induces polymyxin resistance and alters the evolutionary path to further polymyxin resistance. While nutrient metals as an axis of competition is not a conceptual advance, the specific role of Mg2+ and its affect on evolution of polymyxin antibiotic resistance is a conceptual advance.
      • Audience: I think this study would be of interest to a relatively broad audience. The study itself touches on multiple fields including intermicrobial competition, nutritional immunity, antimicrobial resistance, and microbial evolution. Additionally, there are clinical implications for the potential to use antifungals to resensitize polymyxin-resistant P. aeruginosa to polymyxins.
      • My field of expertise is bacterial genetics and physiology, nutritional immunity, and bacterial cell envelope. I do not have expertise in fungus.
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      Referee #2

      Evidence, reproducibility and clarity

      This paper looks at the interaction between the fungus Candida albicans (Ca) and the bacterium Pseudomonas aeruginosa (Pa), which are found together in some environments. Co-culture experiments showed that Ca can inhibit the growth of Pa. The goal of this study is to determine the reason for this phenomenon and how widespread it is. This was performed by Tnseq analysis of Pa that identified 3 genes which showed significant decreases in the presence of Ca. Interestingly these were all in an operon that was recognized by the authors as being induced by RNAseq during co-culture. One of these genes, mgtA is a known Mg2+ transporter and therefore the remainder of the paper discusses the importance of competition for Mg2+.

      The experiments seem to be well carried out and appropriately controlled.

      The use of the Mg2+ genetic sensor reporter in Pa is an interesting approach to determine the intracellular Mg2+ concentrations, however how these levels relate to one another between different experiments is not clear. In Fig. S5, the levels are 5 AU for growth in minimal media +low (10uM) Mg2+ and 38 AU for growth in minimal media+high (10mM) Mg2+. But the levels seen in Figure 1E are all much lower. With such low levels, it is difficult to determine if the impact of ∆PA4824 and ∆mgtA (while perhaps additive) are relevant. Would differences be seen with these various strains grown under different conditions?

      The experiments suggesting that the protein PA4824 is also a Mg2+ transporter seem to be related only to alpha fold predictions.

      Is the statement in line 186 a typo? It is stated that "neither MgtA nor CorA was implicated in competition". Do the authors actually mean "MgtE"?

      Significance

      Ca and Pa are known to inhabit the same niches and previous studies have shown both can have antagonist effects on one another. Nutritional competition is one mechanism of antagonism that has not been that well studied between these two genera. That makes the finding of some significance and relevant to those with an interest in either of these microbes and co-infections.

      The authors also found that it was not just Ca that had this effect, but other fungi as well. And this effect was not just reserved to effect Pa, but also other bacteria, suggesting a more global impact. However diminishing the impact of this finding is the question as to whether this is simply a phenomena seen under the very specific laboratory conditions tested here. Furthermore how these findings exactly relate to any infection environment is not clear.

      The authors also seem to vastly overinterpret the significance of their findings; the impact on Pa is only to slow growth, not necessarily effect fitness, per se. The final number of bacteria appears to be the same, it just takes slightly longer to get there.

      The authors speculate that that since Mg2+ supplementation did not totally restore growth to Pa during co-culture, that other Mg2+ independent "axes of antagonism" must exist. This also tends to diminish the significance of these finding.

      The importance of mgtA on antibiotic susceptibility has been well studied in a number of bacteria including Pa making these findings generally confirmatory.

      The importance of different mutations that emerge in Pa during mono vs. co-culture in the presence of colistin is not clearly explained. Why should co-culture inhibit the emergence of hypermutator Pa strains?

      No additional experiments are likely needed but the authors should be encouraged to place their findings more clearly in what is already known in the field as well as articulate the limitations of their study.

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      Referee #1

      Evidence, reproducibility and clarity

      This manuscript investigates polymicrobial interactions between two clinically relevant species, Pseudomonas aeruginosa and Candida albicans. The findings that C. albicans mediates P. aeruginosa tolerance to antibiotics through sequestration of magnesium provides insight into a specific interaction at play between these two organisms, and the underlying mechanism. The manuscript is well composed and generally the claims throughout are supported by the provided evidence. As a result, most comments are either for clarification, or minor in nature.

      Major comments:

      1. For their experiments, the authors frequently switch between 30C and 37C, but there is no rationale for why a specific temperature was used, or both were. E.g. some of the antibiotic survival assays, and fungal-bacterial co-culture assays were performed at both temperatures, while the colistin resistance, fitness competition and RNA sequencing were performed at 30C. Given the fact that the two organisms are both human pathogens and co-exist in human infections, it is not clear why 30C was used. The authors should provide clarity for why these two temperatures were used.
      2. Lines 184-191: It would be useful to measure intracellular Mg2+ (using the Mg sensor) in the corA and mgtB tn mutants in media as well as the fungal spent media, to provide stronger support for the claim that "MgtA is a key bacterial Mg2+ transporter that is highly induced under low Mg2+ conditions".
      3. Line no. 276. Does the mnr2∆ S. cerevisiae mutant have a growth defect compared to the WT? This would test whether the effect of the mnr2 mutant on P. aeruginosa fitness is strictly due to Mg2 and not due to reduced growth or metabolism of the mutant.
      4. The authors use the term 'antibiotic resistance' throughout the manuscript. However, the assays they perform do not directly test for antibiotic resistance which is defined as the ability to grow at higher concentrations of antibiotics (e.g. as measured by MIC tests). The authors should rephrase their phenotype as antibiotic survival or antibiotic tolerance.
      5. Also, the authors have two different assays, both measuring survival in antibiotic, but one is called a colistin resistance assay (line 508) and the other a colistin survival assay (line 523). It's not obvious what is the difference between what is being assayed in the two experiments, except perhaps the growth phase of the cells when they are exposed to the antibiotic? The authors should explain the difference, and the rationale for using two different assays.

      Minor comments:

      • For almost all the figures, blue and orange dots are used for 'monoculture' and 'coculture' respectively, while orange and black dots are used for WT and the mgtA mutant. However, the black and blue dots are hard to tell apart, and for several figure sub-panels, the legends are not provided (e.g. figures 2D, 2F, S9H), making it a little confusing to figure out what is being shown. It would be best if the WT and mgtA symbols were in colors completely different from the monoculture/co-culture colors, making it easier to tell those apart.

      Line no 122 and Figure 1A. The term "defense genes" in bacteria typically refers to genes conferring protection against phage infections. Perhaps the authors can use a different term (e.g. 'protective genes').

      Line no 186. 'However, neither MgtA...' should be 'However, neither MgtE...'

      Line no 268. Does fungal-mediated Mg2+ competition extend to Gram positive bacteria?

      Line no 314. It is unclear whether the 'transient co-culture' is the same or a different assay as the colistin survival assay.

      Line no 316. For the bacterial survival assays shown in figures 3 and 4 (and other supplementary figures), please provide absolute numbers as cfu (as in figures 1 and 2), as opposed to a percentage, for cell counts. This will allow readers to appropriately interpret the data.

      Line no 934-5: Italicize P. aeruginosa.

      Significance

      This study identifies a novel interaction between two the co-infecting human pathogens Pseudomonas aeruginosa and Candida albicans, where C. albicans causes Mg2+ limitation for P. aeruginosa. Further, the authors show that this interaction affects levels of antibiotic resistance, as well as the adaptive mutations seen during the evolution of antibiotic resistance. This advances the field by delineating how microbial interactions can affect clinically relevant phenotypes, and potentially clinical outcomes. The study should be of interest to a broad audience of researchers studying microbial ecology, evolutionary biology, microbiology, and infectious diseases.

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      Reply to the reviewers

      1. Point-by-point description of the revisions

      Reviewer #1:

      Evidence, reproducibility and clarity (Required):

      In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. First by studying available temperature-sensitive mutants and then by generating their own strain expressing ZYG-8 amenable to auxin-inducible degradation, they establish that defects in ZYG-8 lead to defects in spindle assembly, such as the formation of multipolar spindles, and spindle maintenance, in which spindles elongate, fall apart, and deform in meiosis. Based on these observations the authors conclude that ZYG-8 depletion leads to excessive outward force. As the lab had previously found that the motor protein KLP-18 generates outside directed forces in meiosis, Czajkowski et al initially speculate that ZYG-8 might regulate KLP-18. KLP-18 depletion generally leads to the formation of monopolar spindles in meiosis. Intriguingly, when the authors co-deplete ZYG-8 they find that in some cases bipolarity was reestablished. This led to the hypothesis that yet another kinesin, BMK-1, the homolog of the mammalian EG-5, could provide redundant outward directed forces to KLP-18. The authors then study the effect of ZYG-8 and KLP-18 co-depletion in a BMK-1 mutant background strain and observe that bipolarity is no longer reestablished under these conditions, suggesting that BMK-1 generates additional outward directed forces. The authors also conclude that ZYG-8 inhibits BMK-1. To follow up on this Czajkowski et al generate a ZYG-8 line that carries a mutation in the kinase domain, which should inhibit its kinase activity. This line shows similar effects in terms of spindle elongation but reduced impact on spindle integrity, reflected in minor effects on the number of spindle poles and spindle angle. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. Overall, the paper is well written, and the data is presented very clearly and reproducible. The experiments are adequately replicated, and statistical analysis are adequate. *The observations are very interesting. However, the authors could provide some additional insight into the function of ZYG-8. This paper is strongly focused on motor generated forces within the spindle and tries to place ZYG-8 within this context, but there is compelling evidence from other studies that ZYG-8 also affects microtubule dynamics, which would have implications for spindle assembly and structure. The paper would strongly benefit from the authors exploring this role of ZYG-8 in the context of meiosis further. If the authors feel that this would extend beyond the scope of this paper, I would suggest that the authors rephrase some of their introduction and discussion to reflect the possibility that changes in microtubule growth and nucleation rates could explain some of the phenotypes (think of katanin) and effects and that therefore it can not necessarily be concluded that BMK-1 is inhibited by ZYG-8. *

      We thank the reviewer for these positive comments on our manuscript and on the rigor of our data. We also thank them for the excellent suggestion to explore a potential role for microtubule dynamics. As detailed below in response to the specific points, we performed new experiments to explore this possibility, and found via FRAP analysis that there were substantial changes in microtubule dynamics upon ZYG-8 depletion. We have therefore added these new data and have re-written major parts of the manuscript to incorporate a discussion of microtubule dynamics throughout the paper (introduction, results, model, discussion). Our data now support two roles for ZYG-8 in regulating acentrosomal spindle assembly and stability - one in modulating microtubule dynamics and the other in tuning forces (either directly or indirectly). We are grateful to the reviewer for motivating us to do these experiments, as they have added a whole new angle to the manuscript and have greatly increased its impact, as we now have a fuller understanding of how ZYG-8 contributes to oocyte meiosis.

      Major points:

      *1.) Zyg-8, as well as the mammalian homolog DCLK-1, has been reported to play an important role for microtubule dynamics. While the introduction mentions its previously shown role in meiosis and mitosis, it is totally lacking any background on the effect on microtubule dynamics. The authors mention these findings in the discussion, but it would be helpful to incorporate this in the introduction as well. As an example, Goenczy et al 2001 demonstrated that ZYG-8 is involved in spindle positioning but also showed its ability to bind microtubules and promote microtubule assembly. Interestingly, like the authors here, Goenczy et al concluded that while the kinase domain contributes to, it is not essential ZYG-8's function. Also, Srayko et al 2005 (PMID 16054029) demonstrated that ZYG-8 depletion led to reduced microtubule growth rates and increased nucleation rates in C. elegans mitotic embryos. And in mammalian cells DCLK-1 was shown to increase microtubule nucleation rate and decrease catastrophe rate, leading to a net stabilization of microtubules (Moores et al 2006, PMID: 16957770). It would be great if the authors could add to the introduction that ZYG-8 has been suggested to affect microtubule dynamics. *

      We agree that this is a great idea. As the reviewer suggested, we decided to explore the possibility that ZYG-8 impacts microtubule dynamics within the oocyte spindle. We depleted ZYG-8 and performed FRAP experiments to determine if there were effects on microtubule turnover. We found that loss of ZYG-8 caused a dramatic decrease in the spindle's ability to recover tubulin, both at the spindle center and at the spindle poles (shown in a new Figure 7). We made substantial changes to the manuscript when adding these new data - the manuscript now discusses ZYG-8's role in modulating microtubule dynamics in the introduction, results, discussion, and model (Figure 9), and we added all of the references suggested by the reviewer. We think that the manuscript is greatly improved due to these additions and changes.

      *2a.) The authors initially study two different ts alleles, or484ts and b235ts. The experiments clearly show a significant increase in spindle length in both strains. However, the or484 strain had been previously studied (McNally et al 2016, PMID: 27335123), and only minor effects on spindle length were reported (8.5µm in wt metaphase and 10µm in zyg-8 (or484)). How do the authors explain these differences in ZYG-8 phenotype. Even though the ZYG-8 phenotype is consistent throughout this paper it would be good to explain why the authors observe spindle elongation, fragmentation and spindle bending in contrast to previous observations. *

      The reviewer is correct that McNally et.al. (2016) noted only minor effects on spindle length and did not report observing spindle bending or pole defects. However, the images presented in their paper of spindles in the zyg-8(or484) mutant (in Figure 8B) only showed spindles after they had already shrunk in preparation for anaphase; it is possible that these spindles had pole or midspindle defects prior to this shrinking, and that the authors did not note those phenotypes because their analysis focused on anaphase. In contrast, since the goal of our study focused on how ZYG-8 impacts spindle assembly and maintenance, we looked carefully at spindle morphology and quantified a larger number of metaphase spindles (in their study, only 12 metaphase spindles were measured, since metaphase was not the focus of their manuscript). Recently (after we submitted our manuscript), another study from the McNally lab was published, where they did note metaphase defects following ZYG-8 inhibition (though they did not describe the defects in detail or explore why they happened). We now mention and cite this new paper (Li et.al., 2023) in our manuscript, to show that our findings are consistent with the work of others in the field.

      *2b.) As a general note, it would be helpful if the authors could indicate if the spindles are in meiosis I or II. The only time where this is specifically mentioned is in Video 7, showing a Meiosis II spindle, which makes me assume all other data is in Meiosis I. Adding this to the figures would also help to distinguish if some of the images, i.e. Figure 1B, show multipolar spindles due to failed polar body extrusion. If this is the case then the quantification of number of poles should maybe reflect different possibilities, such as fragmented poles vs. multiple poles because two spindles form around dispersed chromatin masses. *

      We agree that it is a good idea to clarify this issue. For all of our experiments, we analyzed both MI and MII spindles. However, there were no noticeable differences in phenotype between MI and MII spindles for any of our mutant/depletion conditions - we observed bent spindles, elongated spindles, and extra poles in both MI and MII following ZYG-8 inhibition. Therefore, for the quantifications presented in the manuscript (spindle length, spindle angle, number of poles), we pooled our MI and MII data. We have now added this information to the manuscript for clarity (lines 97-99 and 139-141). In addition, we have added new images to Figure 1B that show examples of MII spindles (both at the permissive and restrictive temperature), to show that the phenotypes are indistinguishable between MI and MII.

      We agree with the reviewer that one of the spindles in the original Figure 1B looked like it could have resulted from failed polar body extrusion (the chromosomes appeared to be in two masses, something we did not originally notice, so theoretically each mass could have organized its own spindle). To determine if this was the case, we looked closely at the chromosomes in this image; we confirmed that there were only 6 chromosomes, and that all were bivalents (these can be distinguished from MII chromosomes based on size). Therefore, this spindle was not multipolar due to an issue with polar body extrusion. However, to prevent future confusion, we picked a different representative spindle (where the bivalents we not grouped into two masses), and we added a new column to the figure that shows the DNA channel in grayscale (so it is easier to see and count the chromosomes). We also now note in the materials and methods how we were able to distinguish between MI and MII in our experiments (chromosome count, size, presence of polar bodies), so that it is clear that none of our phenotypes result from failed polar body extrusion (lines 600-603).

      *3) The authors generate a line that carries a mutation leading to a kinase dead version of ZYG-8. It would be great if the authors could further test if this version is truly kinase dead. What is interesting is that the kinase dead version the authors create has less effect on the numbers of pole than the zyg-8 (b235)ts strain, which carries a mutation in a less conserved kinase region. Overall, it seems that the phenotypes are very similar, independent on mutations in the microtubule binding area, kinase area or after AID. This could of course be due to all regions being important, i.e. microtubule binding is required for localizing kinase-activity. Generating mutant versions of the target proteins, for example here BMK-1, that can not be phosphorylated or are constitutively active as well as assessment of changes in protein phosphorylation levels in the kinase dead strain would be helpful to provide deeper insight into potential regulation of proteins by ZYG-8. *

      We agree that it would be ideal to test whether the D604N mutant is truly kinase dead. However, in the interest of time, we ask to be allowed to skip that experiment. The analogous residue has been mutated in mammalian ZYG-8 (DCLK1), and has been shown to cause DCLK1 to be kinase dead in vitro; this is a highly conserved aspartic acid in the central part of the catalytic domain, so we infer that the mutation we made in ZYG-8 should be kinase dead as well. However, since we did not test this directly, we softened our language in the manuscript, explaining that we "infer" that it is kinase dead rather than stating definitively that it is. With regard to the zyg-8(b235)ts mutant having a stronger phenotype, we think that it is possible that this mutation destabilizes a larger portion of the protein (rather than just affecting the catalytic activity), since the phenotypes in this mutant are similar to depletion of the protein in the ZYG-8 AID strain. Therefore, we think that our D604N mutant reveals new information about the role of kinase activity, since it is a more specific mutation that should likely only affect catalytic activity and not the rest of the protein (based on the previous work on DCLK1).

      While we appreciate the suggestion from the reviewer to generate mutant versions of potential target proteins, we ask that this be considered beyond the scope of the study. Now that we know that ZYG-8 not only affects forces within the spindle (maybe BMK-1) but also microtubule dynamics, there are many potential targets - it would require a lot of work to figure out what the relevant targets are. Instead of exploring this experimentally in this manuscript, we added a new section to the discussion where we speculate on what some of these targets could be, to motivate future studies.

      *4a) The authors state that "BMK-1 provides redundant outward force to KLP18". Redundancy usually suggests that one protein can take over the function of another one when the other is not there. In these scenarios a phenotype is often only visible when both proteins are depleted as each can take over the function of the other one. Here however the situation seems slightly different, as depletion of BMK-1 has no phenotype while depletion of KLP-18 leads to monopolar spindles. If BMK-1 would normally provide outward directed forces, would this not be visible in KLP-18 depleted oocytes if they were truly redundant? I assume the authors hypothesize that ZYG-8 inhibits BMK-1 and thus it can not generate outward directed forces. In this case, do the authors envision that ZYG-8 inhibits BMK-1 prior to or in metaphase or only in anaphase or throughout meiosis? Do they speculate, that BMK-1 is inhibited in anaphase and only active in metaphase? *

      The reviewer makes an excellent point - we agree that we should not use the word "redundant" in this context, so we have removed this phrasing from the manuscript. We hypothesize that BMK-1 can provide outward forces during spindle assembly but is not capable of providing as much force as KLP-18 (the primary force-generating motor). We infer this based on our experiments where we co-deplete KLP-18 and ZYG-8 (using long-term depletion). Although BMK-1 is presumably activated under these conditions, it is not able to restore spindle bipolarity (there are outward forces generated, which results in minus ends being found at the periphery of the monopolar spindle, but spindles are not bipolar).

      Therefore, BMK-1 is not able to fully replace the function of KLP-18 during spindle assembly. Interestingly, our experiments imply that BMK-1 can better substitute for KLP-18 later on (when ZYG-8 is inhibited); when we remove ZYG-8 from formed monopolar spindles, bipolarity can be restored (an activity dependent on BMK-1). These findings suggest that ZYG-8 plays a more important role in suppressing BMK-1 activity after the spindle forms, to prevent spindle overelongation in metaphase. We have edited the manuscript to better explain these points.

      *4b) In addition, Figure S4 somewhat argues against a role for ZYG-8 in regulating BMK-1. ZYG-8 depletion supposedly leads to increased outward forces due to loss of BMK-1 inhibition, thus co-depletion of ZYG-8 with BMK-1 should rescue the increased spindle size at least to some extent, however neither increase in spindle length nor increase in additional spindle pole formation are prevented by co-depletion of BMK-1 suggesting that BMK-1 is not generating the forces leading to spindle length increase. Thus, arguing that after all ZYG-8 does not regulate BMK-1. This should be discussed further in the paper and the authors should consider changing the title. At this point the provided evidence that ZYG-8 is regulating motor activity is not strong enough to make this claim. *

      The reviewer is correct that Figure S4 shows that the effects of depleting ZYG-8 on bipolar spindles (spindle elongation and pole/midspindle defects) cannot solely be explained by a role for ZYG-8 in regulating BMK-1 - this was the point that we were trying to make when we included this data in the original manuscript. However, we previously did not know what this other role could be, and therefore we only speculated on other potential roles in the discussion. Now that we have done FRAP experiments and have found that ZYG-8 also affects microtubule dynamics in the oocyte spindle, we now have a better explanation for the data presented in Figure S4 - it makes sense that deleting BMK-1 would not rescue the effects of ZYG-8 depletion, since we have evidence that ZYG-8 also regulates microtubule dynamics. We now clearly explain this in the revised manuscript and we have changed the title to make it clear that ZYG-8 plays multiple roles in oocytes.

      *5) The authors are proposing that ZYG-8 regulates/ inhibits BMK-1, however convincing evidence for an inhibition is not provided in my opinion and the effect of ZYG-8 on BMK-1 could be indirect. To make a compelling argument for a regulation of BMK-1 the authors would have to investigate if ZYG-8 interacts and/ or phosphorylates BMK-1 (see 7) and if this affects its dynamics. In addition, given the reported role of ZYG-8 on microtubule dynamics it would be very important that the authors consider studying the effect of ZYG-8 degradation on microtubule dynamics. Tracking of EBP-2 would be good, however this is very difficult to do inside meiotic spindles due to their small size. In addition, the authors could maybe consider some FRAP experiments, which could provide insights into microtubule dynamics and motions, which could be indicative of outward directed forces/ sliding. *

      We thank the reviewer for these comments as they motivated us to explore a role for ZYG-8 in modulating microtubule dynamics. The reviewer is correct that tracking EBP-2 in the very small meiotic spindle is not possible due to technical limitations, so we took the suggestion to perform FRAP. These experiments revealed that microtubule turnover in the spindle is greatly slowed following ZYG-8 depletion, suggesting a global stabilization of microtubules (data presented in a new Figure 7). This change in dynamics could contribute to the observed spindle phenotypes, which we now explain in detail in the manuscript. Given these new findings, we also now note that the effects we see on BMK-1 activity could be indirect (i.e. maybe increasing the stability of microtubules allows motors to exert excess forces). We now clearly discuss these various possibilities in the discussion.

      Summary: Additional requested experiments:

      • Interaction/ phosphorylation of BMK-1 by ZYG-8, i.e. changes of BMK-1 phosphorylation in absence of ZYG-8, BMK-1 mutations that may prevent phosphorylation by ZYG-8.
      • Assessment of microtubule dynamics (EBP-2, FRAP, length in monopolar spindles...)
      • Kinase activity of the kinase dead ZYG-8 strain (OPTIONAL) We assessed the role of ZYG-8 in microtubule dynamics (bullet point #2). Because this new analysis revealed that ZYG-8 plays multiple roles in the spindle, we decided not to further investigate whether ZYG-8 phosphorylates BMK-1, since the manuscript now no longer argues that this is ZYG-8's major function. We also did not assess the kinase activity of the D604N mutant since this has been done previously for DCLK1, and instead we softened the language in manuscript when describing this mutant.

      Minor points:

      *1) In Figure 4C it seems that the ZYG-8 AID line as well as the zyg-8 (or848)ts already have a phenotype (increased ASPM-1 foci) in absence of auxin/ at the permissive temperature. Does this suggest that the ZYG-8 AID as well as the zyg-8 (or848) strains are after all slightly defective (even if Figure 1, S1 and S2 argue otherwise) and thus more responsive to the loss of KLP-18? *

      The reviewer is correct that the ZYG-8 AID strain (without auxin) and zyg-8(or848)ts strain (at the permissive temperature) are slightly defective in the klp-18(RNAi) monopolar spindle assay. To more rigorously determine whether these strains were also defective in other assays, we generated new graphs comparing the spindle lengths and angles of the two temperature sensitive strains at the permissive temperature to wild-type (N2) worms. These data are now shown in Figure S1 (new panels F and G). A comparison of our ZYG-8 AID strain to a control strain (both in the absence of auxin) are shown in Figure S2 (panels C and D). In this analysis, there wasn't a significant difference for either of these comparisons (i.e. the spindle lengths and angles were all equivalent). We do not know why these strains appear to be slightly defective in the monopolar spindle assay, though perhaps this assay is more sensitive and can detect very mild defects in protein function.

      *2) The authors observe that in preformed monopolar spindles degradation of ZYG-8 can sometimes restore bipolarity. This observation is very interesting but why do the authors not observe a similar phenotype in long-term ZYG-8 AID; klp-18 (RNAi) or zyg-8(or484)ts; klp-18(RNAi). In the latter conditions bipolarity does not seem to occur at all. Do the authors think this is due to differences in timing of events? *

      We thank the reviewer for highlighting this point. We do think that our data suggest that ZYG-8 plays a more important role maintaining the spindle that it does in spindle formation; we have now more clearly explained this in the manuscript (detailing the differences in phenotypes we observe when we deplete ZYG-8 prior to spindle assembly or after the spindle has already formed, lines 180-189 and 227-231). To emphasize this point further, we have also included a graph in Figure S3G that directly compares the number of poles per spindle in long-term auxin treated spindles to short-term auxin treated spindles (with and without metaphase arrest).

      *3) Based on the Cavin-Meza 2022 paper it looks like depletion of KLP-18 in a BMK-1 mutant background does not look different from klp-18 (RNAi) alone. However, looking at Video 8, it looks like spindles "shrink" in absence of KLP-18 and BMK-1. Or is this due to any effects from the ZYG-8 AID strain? This can also be seen in Video 9. *

      The reviewer highlights a fair point that was not clearly explained in our manuscript. In normal monopolar anaphase, chromosomes move in towards the center pole as the spindle gets smaller (C. elegans oocyte spindles shrink in both bipolar and monopolar anaphase); this was previously described in Muscat et.al. 2015, and, as the reviewer noted, in Cavin-Meza et.al. 2022 (in a strain with the bmk-1 mutation). We see this same monopolar anaphase behavior in the ZYG-8 AID strain (Figure 6). We have now better explained normal monopolar anaphase progression and we have cited the Muscat et.al. paper in the relevant sections of the manuscript (lines 221-223 and 714-717).

      *4) Line 311: " ZYG-8 loads onto the spindle along with BMK-1, and functions to inhibit BMK-1 from over elongating microtubules during metaphase." Maybe this sentence could be re-phrased as it currently sounds like BMK-1 elongates (polymerizes) microtubules. *

      In re-writing the manuscript and emphasizing that there are multiple for ZYG-8 (in addition to regulating forces within the spindle), we removed this sentence.

      *5) Line 313: "Intriguingly, in C. elegans oocytes and mitotically-dividing embryos, BMK-1 inhibition causes faster spindle elongation during anaphase, suggesting that BMK-1 normally functions as a brake to slow spindle elongation (Saunders et al., 2007; Laband et al., 2017). Further, ZYG-8 has been shown to be required for spindle elongation during anaphase B (McNally et al., 2016). Our findings may provide an explanation for this phenotype, since if ZYG-8 inhibits BMK-1 as we propose, then following ZYG-8 depletion, BMK-1 could be hyperactive, slowing anaphase B spindle elongation." This paragraph could be modified for better clarity. It is not clear how the findings of the authors, BMK-1 provides outward force but is normally inhibited by ZYG-8, align with the last sentence saying "following zyg-8 depletion, BMK-1 could be hyperactive slowing anaphase B spindle elongation", should it not increase elongation according to the authors observations? *

      In re-writing the manuscript to incorporate our new data showing that ZYG-8 plays a role in modulating microtubule dynamics, we also re-wrote this discussion so that there would be less emphasis on the potential connection between ZYG-8 and BMK-1. In making these edits to expand the focus of the manuscript, we removed this section of the discussion.

      Reviewer #1 (Significance (Required)): *In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. The authors conclude that BMK-1 generates outward directed force, redundant to forces generated by KLP-18, and that ZYG-8 inhibits BMK-1. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. This research is interesting and provides some novel insight into the role of ZYG-8. In particular the observed spindle elongation and subsequent spindle fragmentation are novel and had not yet been reported. Also, the observation that degradation of ZYG-8 in monopolar klp-18(RNAi) spindles can restore bipolarity is novel and interesting, as well as the observation that this is somewhat dependent on the presence of BMK-1. This will be of interest to a broad audience and provides some new insight into the role of importance of ZYG-8 and BMK-1. The limitation of the study is the interpretation of the results and the lack of solid evidence that the observed phenotypes are due to ZYG-8 regulation motor activity, as the title claims. To support this some more experiments would be required. In addition, ZYG-8 has been reported to affect microtubule dynamics, which can certainly affect the action of motors on microtubules. This line of research is not explored in the paper but would certainly add to its value.

      Field of expertise: Research in cell division *

      We thank the reviewer for their positive comments on the impact and novelty of our findings. We hope that the additional experiments we performed and the revisions we made to the text thoroughly address the reviewer's concerns and that they deem the revised manuscript ready for publication.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)): *In this manuscript, the authors explore the requirement for doublecortin kinase Zyg8 in C elegans oocytes. Oocytes build meiotic spindles in the absence of centrosomes, and therefore unique regulation occurs during this process. Therefore, how spindles are built and its later stability are an area of active investigation in the field. Using mutant alleles of Zyg8 and auxin-induced degron alleles, the authors demonstrate that this kinase is required to negatively regulate outward pole forces through BMK1 kinesin and that it has other functions to still explore. Overall, I find that this study takes an elegant genetic approach to tackling this important question in oocyte biology. I have some comments to consider for making the MS clear to a reader. *

      We thank the reviewer for these positive comments on our approach and the importance of our research question. We have attempted to address all of the reviewer's suggestions and we think that they increase the clarity of the manuscript.

      Major Comments:

      *1.) Although I like the graphs describing the altered angles of the spindles, it falls short in fully assessing the phenotype in a meaningful statistical way. Could the authors also graph the data to show statistical significance in the angles between conditions? Perhaps by grouping them into angle ranges and performing an Anova test? This is important in Figure 2E where it is not obvious that there is a difference. *

      The reviewer makes a good point - we have now addressed this concern by performing ANOVA tests to compare conditions on each of the angle graphs. Results of these tests have been reported in the corresponding figure legends. This analysis has confirmed all of the statements we made in the original manuscript. In Figure 1D and S1D, spindle angles were significantly different in the zyg-8 temperature sensitive mutants at the restrictive temperature, and in Figure 2, the angles were significantly different between the "minus auxin" and "plus auxin" conditions. This differs from Figure 7, where there was no significant difference in spindle angle between control spindles and kinase dead mutant spindles (p-value >0.1).

      *2.) The authors do not discuss the significance of the altered spindle angles which I think is an interesting phenotype. Would this be a problem upon Anaphase onset? What is known about spindle angle and aneuploidy or cell viability? Has this phenotype been described before in oocytes or somatic cells? Does depletion of other kinesin motors cause this? *

      The reviewer brings up a good point that warrants more discussion in the manuscript. We agree that the angled spindles are an interesting phenotype; we believe that they could be a result of the spindle elongating to a point where the spindle center becomes weakened, suggesting that the severity of the angle is representative of the severity of spindle elongation. Alternatively, the angled spindles could be a result of the loss of spindle stability factors, such as the doublecortin domain of ZYG-8. This domain is known to have microtubule binding activity; this could be required to maintain stable crosslinked microtubules in the spindle center, such that when ZYG-8 is depleted, the spindle more easily comes apart as the spindle elongates. We now discuss these possibilities in the revised manuscript.

      To the reviewer's second point, we did not examine anaphase outcomes in our manuscript. However, this was recently explored by another lab (in a study that was published after we submitted our manuscript). This study showed that spindles lacking ZYG-8 were able to initiate anaphase and segregate chromosomes (McNally et.al., 2023, https://doi.org/10.1371/journal.pgen.1011090). Perhaps when the spindle shrinks at anaphase onset, the spindle is able to reorganize and largely correct the angle defect, enabling bi-directional chromosome segregation. Interestingly, however, McNally et.al. did report conditions under which spindle bending in anaphase resulted in polar body extrusion errors. The authors reported that BMK-1, which is known to act as a brake to prevent spindle oveelongation in anaphase, is required to prevent bent spindles during anaphase by resisting the forces of cortical myosin on the spindle. Thus, there is precedence for the idea that spindle needs to remain straight throughout anaphase, to ensure proper chromosome segregation.

      *3.) How is embryo spindle positioning determined? It is not clear from the images that there is a defect so I'm not sure what to look for. Is there a way to quantify this? *

      In the original manuscript, spindle positioning within the embryo was determined qualitatively by eye, which we agree was not a precise measure. To address the reviewer's comment, we re-analyzed our images and assessed the position of the spindle within each embryo quantitatively - these data are now shown in Figure 8H and Figure S2B. Spindle position was quantified by analyzing images using Imaris software. The center of the spindle was set by creating a Surface of the DNA signal, and finding the center of that signal. The cell center was determined by measuring the length of the embryo along the long axis and the width of the embryo along the short axis, and setting the center as the halfway point of the total length and width of the embryo. Distance from spindle center to cell center was then measured and graphed. This quantification confirmed the claims we made in our original manuscript - both auxin-treated ZYG-8 AID spindles and ZYG-8 kinase-dead mitotic spindles were significantly mispositioned. The details of how we performed this quantification have been added to the materials and methods.

      *4.) In Figure 1, it appears that there are 2 spindles. Are these MI and MII spindles or ectopic spindles? How do the authors know which one to measure? *

      We thank the reviewer for pointing this out. Reviewer 1 had a similar comment, and we now understand that using that image was misleading, as it looked like as if were two separate MII spindles formed following a failed polar body extrusion event. We have gone through all of our images to stage the oocytes by looking at their chromosome morphology (i.e., to distinguish MI and MII) - the image in question had 6 bivalents and was therefore in Meiosis I; we think that this was a single spindle where the chromosomes happened to cluster into two masses. However, to prevent further confusion, we have replaced this image with a different representative image. In spindles like this with multiple poles, we measure the dominant axis of the spindle (if there are multiple poles, we pick the most prominent ones for the angle measurement). For additional details please see our response to Reviewer 1 major point #2b.

      *5.) The authors show depletion of Zyg8 by western (long) and loss of Gfp (long and short), but don't do so for the acute treatment. I'm guessing this is because the Gfp tag is taken by the spindle marker. The authors should either demonstrate or explain how they know that the acute depletion is effective in removing Zyg8 protein. *

      The reviewer makes a valid point. However, we are unable to see ZYG-8 depletion via acute auxin treatment using live imaging, as ZYG-8 localization is too dim and diffuse to see on the spindle using our typical live imaging parameters (we attempted to do this in a version of the ZYG-8 AID strain that has mCherry::tubulin and GFP::ZYG-8, so that there was no other spindle protein tagged with GFP). To see any GFP::ZYG-8 signal, we had to increase the laser power and exposure time well above what we typically use for live imaging - in doing this, we noticed that there was a limit to how high we could go before the cell began dying during the imaging time course, evident by a lack of chromosome movement, lack of tubulin turnover, and a general increase in tubulin signal throughout the cytoplasm. We do believe that ZYG-8 is being depleted using acute auxin treatment, however, as we see spindle defects very quickly upon dissection of the oocytes into auxin - we just unfortunately don't have a good way of quantifying this given these technical limitations. We have now added information to the materials and methods noting that we cannot see GFP::ZYG-8 under our live imaging conditions (lines 552-561), so that the reader better understands this caveat.

      *6.) In video 2, the chromosome signal is dimmer in the auxin treatment compared to video 1. Why is this? Is it just an experimental artifact or is there something significant about this? If it is because of video choice, consider replacing this one. *

      We thank the reviewer for their keen observations. The chromosome signal being dimmer in the auxin treatment is an experimental artifact - the brightness of the signal can vary depending on how far the spindle is from the slide (this can vary from video to video, and can also change over the time course of one video if the spindle moves during filming). Because of this, movies taken at the same intensity and exposure conditions may appear to have varying levels of brightness. So that readers of the manuscript can better see the chromosomes in this video, we have brightened the chromosome channel in this movie and noted this in the materials and methods (lines 549-551).

      7.) Please consider color palette changes for color-blind readership.

      We agree that it is important to present data in a way that can be appreciated by color-blind readers. Although we would prefer not to have to alter every image in our paper at this point, we have provided all important individual channels in grey scale. We are also planning to adopt a change in color palette for future papers.

      Reviewer #2 (Significance (Required)):

      *The strengths of this manuscript include use of multiple genetic approaches to establish temporal requirements of ZYG8 and which pathway it is acting through. Additionally, the videos and images make the phenotypes clear to evaluate. A minor limitation is that we don't know if the ZYG8 and BMK1 genetic interaction is a direct phosphorylation or not. This MS is an advancement to the field of spindle building and stability, and is particularly relevant to human oocyte quality and fertility. Previous work has shown that human oocyte spindles are highly unstable, but it is challenging to dissect genetic interactions and to conduct mechanistic studies in human oocytes. Therefore, the work here, although conducted in a nematode, can shed light on mechanism as to why human oocyte spindles are unstable and associated with high aneuploidy rates. Based on my expertise in mammalian oocyte biology, I am confident that work presented here will be of high interest to people in the field of meiotic spindle building, aneuploidy and fertility. It also will have broader interest to folks in the areas of kinesin biology, general microtubule and spindle biology. *

      We thank the reviewer for these positive comments on the strength of our data and the significance of our findings reported in our original manuscript. We think that the improvements that we have made in response to suggestions from all three reviewers has further increased this impact.

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

      *Summary: The focus of this paper is the function of a relatively understudied (at least in meiosis) kinase in acentrosomal spindle assembly (Zyg-8, or DCLK1 in mammals) in C. elegans oocytes. The authors use existing ts alleles and a newly generated GFP-Auxin fusion protein, and find that the ts alleles and auxin degron have similar phenotypes. They also examine the interaction with two related kinesins, KLP-18 and BMK-1 in order to investigate the mechanism behind the zyg-8 mutant phenotype. One can probably debate the significance and focus of their conclusions (force balance on the spindle). However, this is an important study because its the first on the meiotic function of a ZYG-8 kinase, and it may open the way to further studies of this kinase and how it regulates multiple kinesins and meiotic spindle assembly. *

      We thank the reviewer for these positive comments and for pointing out the potential future impacts of our work. In revising the manuscript, we have broadened the focus of the manuscript - we no longer solely focus on force balance within the spindle. Thus, our revisions have substantially increased the significance and impact of our work, since the manuscript is no longer narrowly focused.

      Major points:

      *1.) The main concern is the focus that the main defect in zyg-8 depleted oocytes is on outward forces (eg line 134, 277, but many other places in Results and Discussion). The arguments in favor (eg line 269-271) are reasonable. However, these data are not conclusive, and do not rule out regulation of other motor activities, such as bundling, depolymerization or chromosome movement. These are complex phenotypes, and a kinase could have multiple targets and there are often multiple interpretations. This is briefly alluded to in line 372-373 but the authors could do more. Spindle length changes could be caused by different rates of depolymerization or polymerization at the poles or chromosomes. Its not clear how poleward force regulation explains the multiple pole phenotype, although a lack of central spindle integrity could do that. In most of the Results and Discussion, it is not clearly stated on what structures these outward forces are acting. Are these forces effecting kinetochore associated microtubules, or antiparallel overlap microtubules? What do the authors mean by proper force balance? Figure 8 suggests the defect is associated with the amount of overlap and force among antiparallel microtubules - that the forces effected are from the sliding of these microtubules. *

      We agree that our original manuscript was too narrowly focused on the idea of force balance and that we did not discuss other potential roles for ZYG-8 in enough detail (except for briefly in our discussion). In response to both this comment and to a suggestion by Reviewer #1, we decided to investigate a potential role for ZYG-8 in modulating microtubule dynamics (which could be another explanation for some of the phenotypes we observed). We performed FRAP to measure the rate of tubulin turnover within the spindle near the center and at the poles. Interestingly, these experiments revealed that loss of ZYG-8 slows the rate of tubulin turnover, suggesting a general stabilization of microtubules. Thus, we have re-written our manuscript to clearly explain that ZYG-8 plays multiple roles in oocyte spindles - with these changes throughout the manuscript (in the introduction, results, and discussion), the paper is now no longer focused primarily on forces. We hypothesize in the discussion that the phenotypes we observe could be a combination of the effects on microtubule dynamics and spindle forces; if microtubules become more stable and motors produce excess outward forces, this may cause stress on the spindle structure that could cause the midspindle to bend and the poles to split (lines 379-382). We also now more clearly explain that the effect ZYG-8 has on spindle forces could be either direct or indirect (e.g., ZYG-8 could directly regulate motors or, by affecting the microtubule tracks themselves, it could affect their ability to exert forces). As for which population of microtubules are affected, we hypothesize that the excess forces act primarily on overlapping antiparallel microtubules (these microtubules run laterally alongside chromosomes in this system), as is represented in the model figure (Figure 9); we attempted to more clearly explain this in the re-written manuscript.

      *2.) Based on differences between the long term and short term knockdown phenotypes, the authors suggest ZYG-8 is more important for spindle maintenance. For example, in line 299 the authors note that there is a more severe phenotypes with zyg-8 removed from pre-formed spindles. The authors could improve the presentation of this to allow the reader to appreciate this observation. The data is spread between Figures 2 and 3 without a direct comparison of the data. One solution would be to graph the data (eg # of poles) together in one graph and indicate if there is statistical significance. In the Discussion, the authors could refer to specific figure panels. *

      The reviewer is correct that our data suggests that ZYG-8 is more important for spindle maintenance than it is for assembly. As suggested, we made a graph that includes all the pole data from Figures 2 and 3 (long-term auxin, short-term auxin, and metaphase-arrested short-term auxin) - this is now shown in Figure S3D. This makes it easier for the reader to compare these data and appreciate this point. In addition, we added text to the results section, to more clearly explain our rationale for thinking that ZYG-8 plays a more prominent role in spindle maintenance than in assembly (lines 180-189 and 227-231).

      *3.) What is the practical difference between acute and short term depletion. Does acute show weaker phenotypes because there is more residual protein? Unfortunately, the effectiveness of Auxin treatment does not appear to be measured for acute or short term. If the acute depletion adds little to Figure 3, or is not much different than long term, then its not clear what it adds to the paper. Later, in Figure 6, why is only short term and acute analyzed. In general, the authors need to provide better rationale for the different auxin conditions, particularly acute and short term (eg. line 135). If they don't add anything, they should consider not presenting them because readers may get confused by the different conditions, why they were done, and what is learned from each one. *

      The reviewer brings up a fair point that we agree requires clarification. Descriptions of the different types of auxin experiments is provided in Figure 1A. Long-term AID depletes proteins overnight, so the protein of interest is already missing from the oocyte when the spindle begins to form - this allows us to assess whether the protein is required for spindle assembly. However, to determine if a protein is required to stabilize pre-formed spindles, we need to remove the protein quickly after the spindle forms (using either acute or short-term AID). Acute AID is performed by dissecting oocytes directly into auxin-containing media; this allows us to watch what happens to the spindle live, as the protein is being depleted. However, one limitation is that we can only film for a short time before the oocytes begin to die (oocytes become unhappy with extended light exposure, so we cut off the videos after 15 minutes or so, to ensure that we are not filming past the point where they begin to arrest or die). Therefore, to assess what happens to spindles beyond this point, we perform short-term auxin treatment, where whole worms are soaked in auxin containing solution for 30-45 minutes and then the oocytes are dissected for immunofluorescence; this technique allows us to look at what happens to the spindle after more extended protein depletion (since we are not limited to the 10-15 minute window of filming). We have now clarified this in the manuscript by adding these details to the materials and methods. Unfortunately, it is not technically possible to quantify the extent of protein depletion in acute AID via western blotting since we would not be able to easily collect enough dissected oocytes to make a protein sample. (It is also technically challenging to quantify this via imaging; see our response to Reviewer #2, point #5). However, we assume that we are depleting ZYG-8 since we see dramatic spindle defects immediately upon dissection into auxin.

      Minor points:

      *3.) I am a little confused about imaging for GFP::tubulin in auxin experiments. Doesn't the ZYG-8 protein also have GFP? Should this be visible in controls? Is it measurable in the experiments? *

      The reviewer is correct that the ZYG-8 protein is also tagged with GFP in the GFP::tubulin; mCherry::histone live imaging experiments. However, we found that the GFP::ZYG-8 signal is undetectable using the live imaging conditions we are using. We determined this by analyzing a version of the ZYG-8 AID strain in which tubulin was tagged with mCherry (and thus the only GFP-tagged protein was ZYG-8). Using the same live imaging parameters we use for our movies of GFP::tubulin (same exposure time, laser power, etc), we did not detect any GFP::ZYG-8. We have now added this information to the materials and methods (lines 552-561) to clarify these points for the reader, to prevent further confusion.

      *4.) It is nice that the authors validated the results in an emb-30 background with unarrested oocytes. The authors note that the wild-type oocytes undergo anaphase (line 150). The images seem to suggest the auxin treated oocytes do not. Can the authors comment on anaphase in the depletion experiments. Even better, would be to comment on the accuracy of chromosome alignment and segregation. If zyg-8 mutant oocytes complete meiosis, is there any aneuploidy? These are important questions because otherwise the defects in zyg-8 mutants have less significance. *

      We thank the reviewer for their comment. Previous work on ZYG-8 in C. elegans examined a role for ZYG-8 in anaphase and showed that this protein is required for anaphase B spindle elongation (McNally et.al. 2016); because this was known when we launched our study, we purposely did not extensively study ZYG-8 in anaphase and instead focused on understanding how ZYG-8 contributes to spindle formation and stability. Our fixed imaging long-term AID experiments revealed that spindles were able to go through anaphase and segregate chromosomes bidirectionally despite the metaphase spindle phenotypes, consistent with this previous work (McNally et.al. 2016) and with another recent paper from the same lab (McNally et.al. 2023). However, we did not examine whether there were chromosome segregation errors. Given that anaphase is not the focus of our paper, we ask that this be deemed beyond the scope of our study.

      5.) Later, in line 184, the authors indicate that zyg-8 bipolar spindles "segregate chromosomes". Which images show anaphase I? As noted above, a limitation of these studies is not knowing the outcome of meiosis in these Zyg-8 depletions.

      We agree that in the original manuscript it was difficult to see that chromosomes were segregating bidirectionally in our movies and in the still timepoint images presented in Figure 5. Therefore, we brightened the chromosome channel in the relevant videos to make it easier to see the segregating chromosomes. Video 6 shows an oocyte in Meiosis II, as the first polar body can be seen near the spindle in this movie. At 2 minutes, the monopolar spindle becomes bipolar and begins to shrink as it goes into anaphase. Chromosomes begin to move apart and then the spindle elongates. At 11 minutes, you can see that the chromosomes have segregated bidirectionally. Thus, when monopolar spindles reorganize into bipolar spindles under these conditions, they can drive bidirectional chromosome segregation. We did not assess the fidelity of chromosome segregation under these conditions (i.e., whether chromosomes segregated accurately), as the question we were trying to answer in this experiment was whether outward forces sufficient to re-establish bipolarity could be activated upon ZYG-8 depletion (as explained above in response to point #4, we focused our study on trying to understand the effects of ZYG-8 depletion on the spindle, rather than on anaphase). We agree that analyzing anaphase outcomes would be interesting, but we ask that it be considered beyond the scope of this study.

      *6.) Line 206 suggests that ZYG-8 inhibits BMK-1. Is a simple explanation that BMK-1 is required for the bipolar spindles observed in the klp-18 zyg-8 AID oocytes? *

      Yes, the reviewer is correct that BMK-1 is required for the generation of bipolar spindles in the klp-18(RNAi) ZYG-8 AID conditions. In the original manuscript we extrapolated this result to propose that ZYG-8 regulates BMK-1. However, this comment, as well as feedback from the other reviewers and our new experiments (showing that ZYG-8 also modulates microtubule dynamics) has made us re-think the way we discuss this result, as we now agree that it does not prove this regulation (it is only suggestive). Therefore, in the revised manuscript, we no longer definitely claim that ZYG-8 regulates BMK-1 - we have switched to softer language (stating that ZYG-8 "may regulate" BMK-1, etc.). In the results section we now describe our conclusions as follows: "These data demonstrate that BMK-1 produces the outward forces that are activated upon ZYG-8 and KLP-18 co-depletion and raise the possibility that ZYG-8 regulates BMK-1 either directly or indirectly" (lines 250-252).

      *7.) Given that many mitotic and meiotic kinases are localized to specific regions or domains of the spindle, there is only limited discussion of the ZYG-8 localization pattern. Does the ZYG-8 localization pattern provide any insights into its mechanism of promoting spindle assembly? *

      The reviewer makes a good point - while we did report ZYG-8 localization, the discussion on the importance of its localization pattern was limited. To address this, we now remind readers in the discussion that ZYG-8 and BMK-1 co-localize throughout meiosis, consistent with the possibility that ZYG-8 could regulate BMK-1. Notably, this localization pattern is also consistent with the observation that ZYG-8 modulates microtubule dynamics across the spindle; this is now also noted in the discussion (lines 358-361).

      *8.) Line 96-97 - how much is the ZYG-8 depletion? *

      To address this question, we have quantified the amount of ZYG-8 protein in our ZYG-8 AID strain in control, long-term, and short-term auxin treated conditions. The western blot was quantified by comparing the raw intensity of the bands and subtracting the background signal. Short-term auxin depletion resulted in an ~63% reduction in ZYG-8 GFP signal, and long-term depletion resulted in an ~93% reduction in ZYG-8 GFP signal. This has now been reported in the manuscript on lines 785-786.

      *9.) Line 140: the authors say spindle length could not be measured, but perhaps it makes more sense to measure half spindle (chromosome to spindle pole). The images do give the impression that the chromosome to pole distance is shorter. *

      While we liked this idea and tried to perform these measurements, it turned out to be difficult in practice, since the spindle length measurements are obtained by finding the distance from pole (center of the ASPM-1 staining) to center of the chromosome signal. If you look carefully at our images you will notice that the chromosomes lose alignment following short-term AID; therefore, the chromosomes do not form one mass, which made it very difficult to determine an accurate "center" of the DNA signal. Additionally, in most cases the poles are disrupted such that ASPM-1 is found in many separate masses and/or is diffusely localized around the periphery of the spindle. Because of this, we unfortunately felt that these measurements would not be very accurate and would be hard to interpret.

      *10.) Don't see the point of lines 323-330. Could be deleted? *

      In revising our manuscript, we have rephrased these lines in an attempt to provide more context. Because DCLK1 has been shown to be upregulated in a wide variety of cancers, there are ongoing efforts to find chemical inhibitors that specifically block the kinase activity of this protein to be used as cancer therapeutics. However, no one has previously shown that the kinase activity of DCLK1 is important for its in vivo function (in any organism). Therefore, we were trying to make the point that, since we demonstrated that kinase activity is important for the functions of a DCLK1 family member in vivo, this suggests that these kinase inhibitors may in fact be beneficial in knocking down DCLK1 activity.

      11.) Figure 1: Because ts alleles could have a defective phenotype at "permissive" temperature, a wild-type control should be included. This data does appear in a later figure.

      The reviewer is correct that this data does appear in a later figure, but we agree this direct comparison would provide clarity to the reader. To address this comment, we compared the spindle lengths and angles of the two temperature-sensitive (TS) strains (at both the permissive and restrictive temperatures) to wild-type (N2) worms - these data have been added to Figure S1 (new panels F and G). The spindle lengths of both TS strains at the permissive temperature did not significantly differ from wild-type spindle lengths (p>0.1), while both TS strains at the restrictive temperature were significantly different than wild-type (p0.1), but there was a significant difference between wild-type spindles and the TS mutants at the restrictive temperature (doublecortin domain mutant (p Reviewer #3 (Significance (Required)): The strengths of this paper are the novelty of studying Zyg-8. It also addresses important questions regarding acentrosmal spindle assembly in oocytes. The weakness is mostly in the limited interpretation of results and not enough consideration of alternative interpretations. Related to this, the authors only test the force balance hypothesis with the knockout of two related kinesins. They don't experimentally investigate other mechanisms for the zyg-8 phenotype. This research should be of broad interest to anyone interested in oocyte spindle assembly, and also in a more specialized way to those who study kinases or Zyg-8 homologs in other cell types or organisms.

      We thank the reviewer for these positive comments on the strengths and novelty of our manuscript. We also appreciate the constructive suggestions of all three reviewers, which motivated us to perform new experiments that revealed additional functions for ZYG-8 - these revisions have greatly improved the manuscript and have broadened its impact.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary:

      The focus of this paper is the function of a relatively understudied (at least in meiosis) kinase in acentrosomal spindle assembly (Zyg-8, or DCLK1 in mammals) in C. elegans oocytes. The authors use existing ts alleles and a newly generated GFP-Auxin fusion protein, and find that the ts alleles and auxin degron have similar phenotypes. They also examine the interaction with two related kinesins, KLP-18 and BMK-1 in order to investigate the mechanism behind the zyg-8 mutant phenotype. One can probably debate the significance and focus of their conclusions (force balance on the spindle). However, this is an important study because its the first on the meiotic function of a ZYG-8 kinase, and it may open the way to further studies of this kinase and how it regulates multiple kinesins and meiotic spindle assembly.

      Major:

      1) The main concern is the focus that the main defect in zyg-8 depleted oocytes is on outward forces (eg line 134, 277, but many other places in Results and Discussion). The arguments in favor (eg line 269-271) are reasonable. However, these data are not conclusive, and do not rule out regulation of other motor activities, such as bundling, depolymerization or chromosome movement. These are complex phenotypes, and a kinase could have multiple targets and there are often multiple interpretations. This is briefly alluded to in line 372-373 but the authors could do more. Spindle length changes could be caused by different rates of depolymerization or polymerization at the poles or chromosomes. Its not clear how poleward force regulation explains the multiple pole phenotype, although a lack of central spindle integrity could do that. In most of the Results and Discussion, it is not clearly stated on what structures these outward forces are acting. Are these forces effecting kinetochore associated microtubules, or antiparallel overlap microtubules? What do the authors mean by proper force balance? Figure 8 suggests the defect is associated with the amount of overlap and force among antiparallel microtubules - that the forces effected are from the sliding of these microtubules.

      2) Based on differences between the long term and short term knockdown phenotypes, the authors suggest ZYG-8 is more important for spindle maintenance. For example, in line 299 the authors note that there is a more severe phenotypes with zyg-8 removed from pre-formed spindles. The authors could improve the presentation of this to allow the reader to appreciate this observation. The data is spread between Figures 2 and 3 without a direct comparison of the data. One solution would be to graph the data (eg # of poles) together in one graph and indicate if there is statistical significance. In the Discussion, the authors could refer to specific figure panels.

      3) What is the practical difference between acute and short term depletion. Does acute show weaker phenotypes because there is more residual protein? Unfortunately, the effectiveness of Auxin treatment does not appear to be measured for acute or short term. If the acute depletion adds little to Figure 3, or is not much different than long term, then its not clear what it adds to the paper. Later, in Figure 6, why is only short term and acute analyzed. In general, the authors need to provide better rationale for the different auxin conditions, particularly acute and short term (eg. line 135). If they don't add anything, they should consider not presenting them because readers may get confused by the different conditions, why they were done, and what is learned from each one.

      Minor:

      1) I am a little confused about imaging for GFP::tubulin in auxin experiments. Doesn't the ZYG-8 protein also have GFP? Should this be visible in controls? Is it measurable in the experiments?

      2) It is nice that the authors validated the results in an emb-30 background with unarrested oocytes. The authors note that the wild-type oocytes undergo anaphase (line 150). The images seem to suggest the auxin treated oocytes do not. Can the authors comment on anaphase in the depletion experiments. Even better, would be to comment on the accuracy of chromosome alignment and segregation. If zyg-8 mutant oocytes complete meiosis, is there any aneuploidy? These are important questions because otherwise the defects in zyg-8 mutants have less significance.

      3) Later, in line 184, the authors indicate that zyg-8 bipolar spindles "segregate chromosomes". Which images show anaphase I? As noted above, a limitation of these studies is not knowing the outcome of meiosis in these Zyg-8 depletions.

      4) Line 206 suggests that ZYG-8 inhibits BMK-1. Is a simple explanation that BMK-1 is required for the bipolar spindles observed in the klp-18 zyg-8 AID oocytes?

      5) Given that many mitotic and meiotic kinases are localized to specific regions or domains of the spindle, there is only limited discussion of the ZYG-8 localization pattern. Does the ZYG-8 localization pattern provide any insights into its mechanism of promoting spindle assembly?

      6) Line 96-97 - how much is the ZYG-8 depletion?

      7) Line 140: the authors say spindle length could not be measured, but perhaps it makes more sense to measure half spindle (chromosome to spindle pole). The images do give the impression that the chromosome to pole distance is shorter.

      8) Don't see the point of lines 323-330. Could be deleted?

      9) Figure 1: Because ts alleles could have a defective phenotype at "permissive" temperature, a wild-type control should be included. This data does appear in a later figure.

      Significance

      The strengths of this paper are the novelty of studying Zyg-8. It also addresses important questions regarding acentrosmal spindle assembly in oocytes. The weakness is mostly in the limited interpretation of results and not enough consideration of alternative interpretations. Related to this, the authors only test the force balance hypothesis with the knockout of two related kinesins. They don't experimentally investigate other mechanisms for the zyg-8 phenotype. This research should be of broad interest to anyone interested in oocyte spindle assembly, and also in a more specialized way to those who study kinases or Zyg-8 homologs in other cell types or organisms.

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      Referee #2

      Evidence, reproducibility and clarity

      In this manuscript, the authors explore the requirement for doublecortin kinase Zyg8 in C elegans oocytes. Oocytes build meiotic spindles in the absence of centrosomes, and therefore unique regulation occurs during this process. Therefore, how spindles are built and its later stability are an area of active investigation in the field. Using mutant alleles of Zyg8 and auxin-induced degron alleles, the authors demonstrate that this kinase is required to negatively regulate outward pole forces through BMK1 kinesin and that it has other functions to still explore. Overall, I find that this study takes an elegant genetic approach to tackling this important question in oocyte biology. I have some comments to consider for making the MS clear to a reader.

      Major Comments:

      1. Although I like the graphs describing the altered angles of the spindles, it falls short in fully assessing the phenotype in a meaningful statistical way. Could the authors also graph the data to show statistical significance in the angles between conditions? Perhaps by grouping them into angle ranges and performing an Anova test? This is important in Figure 2E where it is not obvious that there is a difference.

      2. The authors do not discuss the significance of the altered spindle angles which I think is an interesting phenotype. Would this be a problem upon Anaphase onset? What is known about spindle angle and aneuploidy or cell viability? Has this phenotype been described before in oocytes or somatic cells? Does depletion of other kinesin motors cause this?

      3. How is embryo spindle positioning determined? It is not clear from the images that there is a defect so I'm not sure what to look for. Is there a way to quantify this?

      4. In Figure 1, it appears that there are 2 spindles. Are these MI and MII spindles or ectopic spindles? How do the authors know which one to measure?

      5. The authors show depletion of Zyg8 by western (long) and loss of Gfp (long and short), but don't do so for the acute treatment. I'm guessing this is because the Gfp tag is taken by the spindle marker. The authors should either demonstrate or explain how they know that the acute depletion is effective in removing Zyg8 protein.

      6. In video 2, the chromosome signal is dimmer in the auxin treatment compared to video 1. Why is this? Is it just an experimental artifact or is there something significant about this? If it is because of video choice, consider replacing this one.

      7. Please consider color palette changes for color-blind readership.

      Significance

      The strengths of this manuscript include use of multiple genetic approaches to establish temporal requirements of ZYG8 and which pathway it is acting through. Additionally, the videos and images make the phenotypes clear to evaluate. A minor limitation is that we don't know if the ZYG8 and BMK1 genetic interaction is a direct phosphorylation or not.

      This MS is an advancement to the field of spindle building and stability, and is particularly relevant to human oocyte quality and fertility. Previous work has shown that human oocyte spindles are highly unstable, but it is challenging to dissect genetic interactions and to conduct mechanistic studies in human oocytes. Therefore, the work here, although conducted in a nematode, can shed light on mechanism as to why human oocyte spindles are unstable and associated with high aneuploidy rates.

      Based on my expertise in mammalian oocyte biology, I am confident that work presented here will be of high interest to people in the field of meiotic spindle building, aneuploidy and fertility. It also will have broader interest to folks in the areas of kinesin biology, general microtubule and spindle biology.

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      Referee #1

      Evidence, reproducibility and clarity

      In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. First by studying available temperature-sensitive mutants and then by generating their own strain expressing ZYG-8 amenable to auxin-inducible degradation, they establish that defects in ZYG-8 lead to defects in spindle assembly, such as the formation of multipolar spindles, and spindle maintenance, in which spindles elongate, fall apart, and deform in meiosis. Based on these observations the authors conclude that ZYG-8 depletion leads to excessive outward force. As the lab had previously found that the motor protein KLP-18 generates outside directed forces in meiosis, Czajkowski et al initially speculate that ZYG-8 might regulate KLP-18. KLP-18 depletion generally leads to the formation of monopolar spindles in meiosis. Intriguingly, when the authors co-deplete ZYG-8 they find that in some cases bipolarity was reestablished. This led to the hypothesis that yet another kinesin, BMK-1, the homolog of the mammalian EG-5, could provide redundant outward directed forces to KLP-18. The authors then study the effect of ZYG-8 and KLP-18 co-depletion in a BMK-1 mutant background strain and observe that bipolarity is no longer reestablished under these conditions, suggesting that BMK-1 generates additional outward directed forces. The authors also conclude that ZYG-8 inhibits BMK-1. To follow up on this Czajkowski et al generate a ZYG-8 line that carries a mutation in the kinase domain, which should inhibit its kinase activity. This line shows similar effects in terms of spindle elongation but reduced impact on spindle integrity, reflected in minor effects on the number of spindle poles and spindle angle. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis.

      • Overall, the paper is well written, and the data is presented very clearly and reproducible. The experiments are adequately replicated, and statistical analysis are adequate. The observations are very interesting. However, the authors could provide some additional insight into the function of ZYG-8. This paper is strongly focused on motor generated forces within the spindle and tries to place ZYG-8 within this context, but there is compelling evidence from other studies that ZYG-8 also affects microtubule dynamics, which would have implications for spindle assembly and structure. The paper would strongly benefit from the authors exploring this role of ZYG-8 in the context of meiosis further. If the authors feel that this would extend beyond the scope of this paper, I would suggest that the authors rephrase some of their introduction and discussion to reflect the possibility that changes in microtubule growth and nucleation rates could explain some of the phenotypes (think of katanin) and effects and that therefore it can not necessarily be concluded that BMK-1 is inhibited by ZYG-8.

      Major comments:

      1) Zyg-8, as well as the mammalian homolog DCLK-1, has been reported to play an important role for microtubule dynamics. While the introduction mentions its previously shown role in meiosis and mitosis, it is totally lacking any background on the effect on microtubule dynamics. The authors mention these findings in the discussion, but it would be helpful to incorporate this in the introduction as well. As an example, Goenczy et al 2001 demonstrated that ZYG-8 is involved in spindle positioning but also showed its ability to bind microtubules and promote microtubule assembly. Interestingly, like the authors here, Goenczy et al concluded that while the kinase domain contributes to, it is not essential ZYG-8's function. Also, Srayko et al 2005 (PMID 16054029) demonstrated that ZYG-8 depletion led to reduced microtubule growth rates and increased nucleation rates in C. elegans mitotic embryos. And in mammalian cells DCLK-1 was shown to increase microtubule nucleation rate and decrease catastrophe rate, leading to a net stabilization of microtubules (Moores et al 2006, PMID: 16957770).

      It would be great if the authors could add to the introduction that ZYG-8 has been suggested to affect microtubule dynamics.

      2) The authors initially study two different ts alleles, or484ts and b235ts. The experiments clearly show a significant increase in spindle length in both strains. However, the or484 strain had been previously studied (McNally et al 2016, PMID: 27335123), and only minor effects on spindle length were reported (8.5µm in wt metaphase and 10µm in zyg-8 (or484)). How do the authors explain these differences in ZYG-8 phenotype. Even though the ZYG-8 phenotype is consistent throughout this paper it would be good to explain why the authors observe spindle elongation, fragmentation and spindle bending in contrast to previous observations.

      As a general note, it would be helpful if the authors could indicate if the spindles are in meiosis I or II. The only time where this is specifically mentioned is in Video 7, showing a Meiosis II spindle, which makes me assume all other data is in Meiosis I. Adding this to the figures would also help to distinguish if some of the images, i.e. Figure 1B, show multipolar spindles due to failed polar body extrusion. If this is the case then the quantification of number of poles should maybe reflect different possibilities, such as fragmented poles vs. multiple poles because two spindles form around dispersed chromatin masses.

      3) The authors generate a line that carries a mutation leading to a kinase dead version of ZYG-8. It would be great if the authors could further test if this version is truly kinase dead. What is interesting is that the kinase dead version the authors create has less effect on the numbers of pole than the zyg-8 (b235)ts strain, which carries a mutation in a less conserved kinase region. Overall, it seems that the phenotypes are very similar, independent on mutations in the microtubule binding area, kinase area or after AID. This could of course be due to all regions being important, i.e. microtubule binding is required for localizing kinase-activity. Generating mutant versions of the target proteins, for example here BMK-1, that can not be phosphorylated or are constitutively active as well as assessment of changes in protein phosphorylation levels in the kinase dead strain would be helpful to provide deeper insight into potential regulation of proteins by ZYG-8.

      4) The authors state that "BMK-1 provides redundant outward force to KLP18". Redundancy usually suggests that one protein can take over the function of another one when the other is not there. In these scenarios a phenotype is often only visible when both proteins are depleted as each can take over the function of the other one. Here however the situation seems slightly different, as depletion of BMK-1 has no phenotype while depletion of KLP-18 leads to monopolar spindles. If BMK-1 would normally provide outward directed forces, would this not be visible in KLP-18 depleted oocytes if they were truly redundant? I assume the authors hypothesize that ZYG-8 inhibits BMK-1 and thus it can not generate outward directed forces. In this case, do the authors envision that ZYG-8 inhibits BMK-1 prior to or in metaphase or only in anaphase or throughout meiosis? Do they speculate, that BMK-1 is inhibited in anaphase and only active in metaphase? In addition, Figure S4 somewhat argues against a role for ZYG-8 in regulating BMK-1. ZYG-8 depletion supposedly leads to increased outward forces due to loss of BMK-1 inhibition, thus co-depletion of ZYG-8 with BMK-1 should rescue the increased spindle size at least to some extent, however neither increase in spindle length nor increase in additional spindle pole formation are prevented by co-depletion of BMK-1 suggesting that BMK-1 is not generating the forces leading to spindle length increase. Thus, arguing that after all ZYG-8 does not regulate BMK-1. This should be discussed further in the paper and the authors should consider changing the title. At this point the provided evidence that ZYG-8 is regulating motor activity is not strong enough to make this claim.

      5) The authors are proposing that ZYG-8 regulates/ inhibits BMK-1, however convincing evidence for an inhibition is not provided in my opinion and the effect of ZYG-8 on BMK-1 could be indirect. To make a compelling argument for a regulation of BMK-1 the authors would have to investigate if ZYG-8 interacts and/ or phosphorylates BMK-1 (see 7) and if this affects its dynamics. In addition, given the reported role of ZYG-8 on microtubule dynamics it would be very important that the authors consider studying the effect of ZYG-8 degradation on microtubule dynamics. Tracking of EBP-2 would be good, however this is very difficult to do inside meiotic spindles due to their small size. In addition, the authors could maybe consider some FRAP experiments, which could provide insights into microtubule dynamics and motions, which could be indicative of outward directed forces/ sliding.

      Summary:

      Additional requested experiments:

      • Interaction/ phosphorylation of BMK-1 by ZYG-8, i.e. changes of BMK-1 phosphorylation in absence of ZYG-8, BMK-1 mutations that may prevent phosphorylation by ZYG-8. -Assessment of microtubule dynamics (EBP-2, FRAP, length in monopolar spindles...) -Kinase activity of the kinase dead ZYG-8 strain (OPTIONAL)

      Minor comments:

      1) In Figure 4C it seems that the ZYG-8 AID line as well as the zyg-8 (or848)ts already have a phenotype (increased ASPM-1 foci) in absence of auxin/ at the permissive temperature. Does this suggest that the ZYG-8 AID as well as the zyg-8 (or848) strains are after all slightly defective (even if Figure 1, S1 and S2 argue otherwise) and thus more responsive to the loss of KLP-18?

      2) The authors observe that in preformed monopolar spindles degradation of ZYG-8 can sometimes restore bipolarity. This observation is very interesting but why do the authors not observe a similar phenotype in long-term ZYG-8 AID; klp-18 (RNAi) or zyg-8(or484)ts; klp-18(RNAi). In the latter conditions bipolarity does not seem to occur at all. Do the authors think this is due to differences in timing of events?

      3) Based on the Cavin-Meza 2022 paper it looks like depletion of KLP-18 in a BMK-1 mutant background does not look different from klp-18 (RNAi) alone. However, looking at Video 8, it looks like spindles "shrink" in absence of KLP-18 and BMK-1. Or is this due to any effects from the ZYG-8 AID strain? This can also be seen in Video 9.

      4) Line 311: " ZYG-8 loads onto the spindle along with BMK-1, and functions to inhibit BMK-1 from over elongating microtubules during metaphase." Maybe this sentence could be re-phrased as it currently sounds like BMK-1 elongates (polymerizes) microtubules.

      5) Line 313: "Intriguingly, in C. elegans oocytes and mitotically-dividing embryos, BMK-1 inhibition causes faster spindle elongation during anaphase, suggesting that BMK-1 normally functions as a brake to slow spindle elongation (Saunders et al., 2007; Laband et al., 2017). Further, ZYG-8 has been shown to be required for spindle elongation during anaphase B (McNally et al., 2016). Our findings may provide an explanation for this phenotype, since if ZYG-8 inhibits BMK-1 as we propose, then following ZYG-8 depletion, BMK-1 could be hyperactive, slowing anaphase B spindle elongation." This paragraph could be modified for better clarity. It is not clear how the findings of the authors, BMK-1 provides outward force but is normally inhibited by ZYG-8, align with the last sentence saying "following zyg-8 depletion, BMK-1 could be hyperactive slowing anaphase B spindle elongation", should it not increase elongation according to the authors observations?

      Significance

      In this manuscript Czajkowski et al explore the role of the doublecortin-family kinase ZYG-8 during meiosis in C. elegans Oocytes. The authors conclude that BMK-1 generates outward directed force, redundant to forces generated by KLP-18, and that ZYG-8 inhibits BMK-1. The authors conclude that ZYG-8's kinase activity is required for the function of ZYG-8 in meiosis and mitosis. This research is interesting and provides some novel insight into the role of ZYG-8. Inm particular the observed spindle elongation and subsequent spindle fragmentation are novel and had not yet been reported. Also, the observation that degradation of ZYG-8 in monopolar klp-18(RNAi) spindles can restore bipolarity is novel and interesting, as well as the observation that this is somewhat dependent on the presence of BMK-1. This will be of interest to a broad audience and provides some new insight into the role of importance of ZYG-8 and BMK-1. The limitation of the study is the interpretation of the results and the lack of solid evidence that the observed phenotypes are due to ZYG-8 regulation motor activity, as the title claims. To support this some more experiments would be required. In addition, ZYG-8 has been reported to affect microtubule dynamics, which can certainly affect the action of motors on microtubules. This line of research is not explored in the paper but would certainly add to its value.

      Field of expertise: Research in cell division

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      Reply to the reviewers

      The authors sincerely appreciate the editors’ and the reviewers’ dedication in providing constructive and insightful comments aimed at enhancing the quality of the manuscript. In response to the valuable feedback received, we have implemented significant revisions to the manuscript, including the addition of key experiments, reorganization of the figures as well as providing detailed point-to-point responses to address the reviewers’ concerns. With these changes, we are confident that we have effectively addressed the comments raised by all three reviewers and have strengthened the overall quality of the manuscript.

      Below are the major improvements we have made in the revised manuscript:

      1. Figure 4  new figure with polysome profiling assay to strengthen the link between translational regulation and mitochondrial defects.
      2. Figure 7  added confocal images showing the transfer of mitochondria into recipient cells.
      3. Figure S2  added RER data further supporting a shift of metabolism to favor fatty acid oxidation as shown by proteomics data.
      4. Figure S4  added WB data showing that protein degradation was not affected, strengthening a protein synthesis defect due to Fam210a KO.
      5. Figure S5B, S6C  added quantification to the staining and blots.

      1. Point-by-point description of the revisions

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

      In the manuscript entitled "FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse", Chen et al, found that knocking out of FAM210A specifically in muscle using Myl Cre resulted in abnormal mitochondria, hyperacetylation of cytosolic proteins, and translation defects. The manuscript uncovered the new functions of FAM210A in regulating metabolism and translation. I have the following the concerns about the manuscript.

      Comments

      One of the major phenotypes of FAM210A is the decrease of muscle mass after 6 weeks after birth. Is this phenotype caused by the accumulation of progressive loss of muscle mass from birth? Are the body weight and muscle mass reduced in FAM210A knocking out new-born mice? Is the muscle mass growth curve the same in FAM210A and WT mice from birth to 6 weeks after birth? These results will reveal more mechanism of FAM210A mediated muscle mass control. Answer: Indeed, the phenotype of the Fam210aMKO was caused by the progressive loss of muscle mass. The body weight of the mice was not different before 3-weeks of age (Figure 2B). We reasoned that myonuclei accretion occurred before Myl1Cre induced knockout of Fam210a, accounting for the relative normal muscle development and nuclei accretion prior to 21 days after birth (refer to Response Figure 2). However, due to the small muscle mass, it is hard to accurately evaluate whether the muscle mass in very young mice. Regardless, we believe that body weight and muscle weight closely mimic each other and exhibit similar slopes in WT and KO mice (Response Figure 1).

      Beyond 21 days, muscle growth is mainly attributed to hypertrophy of myofibers, a process that relies on protein synthesis. Yet the Fam210aMKO myofibers has defects in protein synthesis, explaining why the muscles cannot gain weight after 3 weeks and started to lose weight. We have shown that at 4 weeks the TA muscle weight was 13 mg in Fam210aMKO compared to 25 mg in WT control. At 6-weeks, the TA weight in the Fam210aMKO mouse was 10 mg compared to 28 mg in the WT control. Furthermore, the TA weight of the Fam210aMKO mouse was 8.7 mg compared to 36mg in the WT control. These results provide compelling evidence that the Fam210aMKO muscles are progressively wasted.

      Response Figure 1. Changes of body weights and TA muscle weights during postnatal growth. The muscle weights increased (in wildtype mice) or decreased (in KO mice) with body weights at similar trends.

      Does the muscle mass continue to decrease after 8 weeks?

      Answer: Based on the trend (see Response Figure 1), we believe the answer is “yes”. However, we were not allowed to monitor the Fam210aMKO mice after 8 weeks of age, as they were severely lethargic and can barely move, reaching the humane endpoint determined by the IACUC guidelines.

      FAM210A knockout mice displayed high lethal rate. Is there any potential mechanism for the high lethality?

      Answer: We performed extensive necropsy and could not identify a direct cause. The potential cause for the lethality could be the difficulty of breathing as the diaphragm muscle was very thin in the Fam210aMKO mouse compared to the WT control. Besides, the diminished muscle contraction force (Figure 3) might have prohibited normal activities (including eating), leading to exhaustive death.

      In Figure 2, the muscle mass decreased significantly, while the fat mass only decreased slightly in FAM210A knockout mice. However, the ratio of the lean mass and fat mass to body mass did not change in FAM210A knockout mice compared to WT mice. How do the authors reconcile this?

      Answer: Just to clarify, Figure 2D-E shows that fat mass was significantly reduced at 4-week old but not reduced at 6-week old. We interpret the significant reduction of the mass but not the ratio (to body weight) as the result of the concomitant reduction of the body weight in the Fam210aMKO mice.

      Are there changes of the number of nuclei per myotube? Is the muscle atrophy in FAM210A knockout mice caused by the defects of fusion, or the degradation of protein, or both?

      Answer: We thank the reviewer for this question. To answer this question, we isolated myofibers from WT and Fam210aMKO mouse at 4-week-old and quantify the myonuclei number. We did not observe a significant reduction of myonuclei number per myofiber in the Fam210aMKO mouse, suggesting that the myoblast fusion into myofibers was not affected in the Fam210aMKO model. (Response Figure 2)

      Response Figure 2. DAPI staining and quantification in the single myofiber isolated from WT and Fam210aMKO mice.

      The number of myonuclei in the WT and Fam210aMKO was not different, suggesting normal fusion of satellite cells in Fam210aMKO mice.

      We also did western blot to check the atrophy related protein expression in WT and Fam210aMKO mouse at different ages. Interestingly, we did not observe a significant induction of these proteins (Atrogin-1, MuRF1) in the Fam210aMKOmuscle. Therefore, we conclude that the muscle atrophy was due to protein translation defects in the Fam210aMKO, independent of myoblast fusion and protein degradation (Figure S4C).

      Are the growth curves of muscle mass growth in EDL and SOL the same in FAM210A knockout mice?

      Answer: We thank the reviewer for the question. In the Myl1Cre mediated Fam210a KO model, Fam210a was deleted in both fast (EDL) and slow (SOL) muscles (see response to Reviewer 3, second point). We think that the “growth curve” of the EDL and SOL muscle should be same (stagnant and even reduced) upon Fam210a KO as the mouse grows from 4-week to 8-week.

      The oxygen consumption and carbon dioxide production are higher in FAM210A knockout mice, suggesting a high metabolism rate. In contrast, the heat production of FAM210A knockout mice is lower, suggesting a low metabolism rate. Any explanation?

      Answer: The VCO2 and VO2 values were normalized to the body weight, and the KO value appeared high because their body weights were much lower at the time of test. While for heat production (unit: Kcal/hr), body weight was not a factor in the calculation. The seemingly contradicting/surprising result that a weak KO mouse could have higher VCO2 and VO2could be recapitulated in other mouse models (for example PMID: 22307625).

      Given the high glucose consumption in FAM210A, why is the clearance rate of blood glucose low?

      Answer: We believe there is a misunderstanding here. A smaller AUC (as seen in the KO) suggest faster blood glucose clearance. The circulating glucose level after fasting is lower in the KO mice, which suggests that the Fam210aMKO mice were consuming more glucose compared to the WT mice. In the GTT test, the Fam210aMKO mice showed a lower AUC after the injection of glucose, implying that the Fam210aMKO mice cleared the injected glucose at a faster rate, probably due to a pseudo-fasting state which would promote the uptake of circulating glucose when available.

      Are there any changes of the abilities for the FAM210A knockout mice in running endurance?

      Answer: Indeed, the Fam210aMKO mice ran less distance, shorter time, and at a lower speed when tested on a treadmill endurance running program (Figure 3)

      In page 5, the last sentence of the 2nd paragraph, the authors concluded "There results suggest that Fam210aMKO induces a metabolic switch to a more oxidative state." It is better to describe it as muscle metabolic since the whole-body metabolism has not been carefully examined.

      Answer: We thank the reviewer for pointing this out, we will change the wording to better reflect the changes observed in the Fam210aMKO mouse regarding the metabolism.

      In Fig. 6, what is the link between increased transcription level of Fgf21 and the elevated level of aberrant acetylation of proteins?

      Answer: We thank the reviewer for this interesting question! However, we did not pursue a direct causal relationship between Fgf21 level and aberrant protein acetylation. In our model, we are proposing that mitochondrial defects in the Fam210aMKO model can trigger the integrated stress response which leads to a higher Fgf21 transcript level in the muscle. This is coinciding with the acetylation increase in the muscle due to the excessive production of acetyl-CoA. A potential relationship between Fgf21 and protein acetylation warrant examination in a future study.

      After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

      Is there any link between the increased acetylation level of rebolsome proteins and the translation defects?

      Answer: Indeed, there are ample studies showing that ribosomal proteins can be acetylated, and that the acetylation of ribosomal proteins can affect the protein synthesis process, for example in PMID: 35604121 and PMID: 37742082. Here in this paper, we showed by ribosome profiling assay that the muscle has defects in the polysome formation (at 4-week and 6-week), when the protein acetylation was significantly increased in the Fam210aMKO mice (Figure 4D-4G).

      How do the abnormal mitochondria lead to increased protein acetylation? And how do these defects further cause translation problem?

      Answer: As elaborated in the discussion, we propose that upon Fam210a KO in mature myofiber, the TCA cycle in the mitochondria was disrupted, blocking utilization of acetyl-CoA and resulting in the accumulation of acetyl-CoA in the muscle. The excess acetyl-CoA lead to increased protein acetylation in the cytosol. We identified that ribosomal proteins are hyperacetylated in the muscle. We also observed that the polysome formation in the muscle was impaired, which exacerbates the translation efficiency.

      Consistently, when we treated C2C12 during in vitro culture with sodium acetate to mimic the increase of acetylation of proteins, we showed that excessive levels of acetyl-CoA can block the differentiation of C2C12 cells (Response Figure 3).

      Response Figure 3. The effect of sodium acetate on the differentiation of C2C12 myoblasts.

      The differentiation of C2C12 myoblasts into myotubes were probed by the protein abundance of Myog and MF20, which showed a decrease in the expression level when sodium acetate was added in increasing amounts.

      The defects in translation will cause general problems besides mitochondria defects. Are there any phenotypes related to the overall translation inhibition observed? If not, why?

      Answer: Just to clarify, our model suggests that mitochondrial defects in the Fam210a KO causes cytosolic translation defects, not the other way around. We showed by SUnSET experiment that the global translation was indeed reduced in the Fam210aMKO muscle at 4-week. We also observed that the p-S6 level which indicates the global protein translation was decreased. It is also true that the global translational arrest can exacerbate the mitochondrial defects and fewer mitochondrial proteins can be synthesized. This feed forward loop can explain the aggravating phenotype in the Fam210aMKO mouse as the mouse gets older.

      Are the abnormal mitochondria, increased protein acetylation, and translation inhibition observed in 2-6 weeks old mice? When were these defects first found? Are they correlated with muscle atrophy?

      Answer: At 2-week-old, the protein synthesis or degradation was not changed between WT and Fam210aMKO mice (Figure S4C). The mitochondria abnormality was first observed at 4 weeks of age, concomitant with the decrease of protein translation (decreased p-S6), polysome formation, and protein hyperacetylation. The acetylation increase was apparent at 6-week together with decreased p-S6 level, polysome assembly and mitochondrial defects. Decreased protein translation has been shown to cause muscle atrophy (PMID: 19046572).

      Reviewer #1 (Significance (Required)):

      This manuscript described many interesting phenotypes of Fam210a knockout mice. However, the links between these phenotypes are obscure. The logic of the manuscript will be greatly improved if the authors could provide explanations to logically link the phenotypes.

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

      Summary: In this manuscript, Chen et al., investigate the functions of FAM210A in skeletal muscle physiology and metabolism. FAM210A is a mitochondria-localized protein in which mutations have been associated with sarcopenia and osteoporosis. Using publicly available gene expression datasets from human skeletal muscle biopsies the authors first demonstrate that the expression of FAM210 is reduced in muscle atrophy-associated diseases and increased in muscle hypertrophy conditions. Based on this, they show that a muscle specific Fam210a deletion leads to muscle atrophy/weakness, systemic metabolic defects, and premature lethality in mouse. Further examination of the knockout myofibers reveals impaired mitochondrial respiration and translation program. Additionally, the authors demonstrate that the flow of TCA cycle is disrupted in the FAM210A-deleted myofibers, which causes abnormal accumulation of acetyl-coA and hyperacetylation of a subset of proteins. The authors claim that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins that leads to ribosomal disassembly and translational deficiency. However, this conclusion is not supported by adequate experimentation and rigorous analysis of ribosomal proteins acetylation and ribosome assembly.

      Major comments:

      -In general, figure legends are lacking information regarding number of biological replicates used and details about statistical analysis. What does three * vs. one * mean in terms of p-value? Exact p-values should be indicated.

      Answer: We thank the reviewer for pointing this out, we have added the information to the revised figure legends.

      -The mechanistic studies linking muscle phenotypes with ribosomal protein hyperacetylation and mRNA translation defects are underdeveloped and not rigorously carried.

      Answer: We agree with the reviewer and have added new data in the revised manuscript to strengthen this link. For example, we have now provided direct evidence on the defective polysome assembly in the Fam210a KO muscles (Figure 4D-4G), which should profoundly impact mRNA translation. In addition, other groups have also shown that ribosomal protein acetylation can impact mRNA translation and polysome formation (PMID: 35604121).

      We also explored the effect of acetylation on differentiation (a process accompanied by extensive protein synthesis) related to our mouse model. We used sodium acetate to elevate acetylation during C2C12 differentiation. We found that increased acetylation indeed impaired the differentiation as can be seen by the reduced expression of MF20 (myosin protein) by WB and IF. The differentiation marker Myogenin was also reduced (Response Figure 3, 4).

      Response Figure 4. Immunofluorescence staining of Myog and MF20 in the differentiated C2C12 myotubes treated with different amounts of sodium acetate.

      The number of MF20 (green) positive myotubes and Myog (red) positive nuclei was significantly reduced in the cells treated with 15mM and 30mM sodium acetate.

      -Fig S1: The validation WB of FAM210A KO is not the most convincing. Why are the FAM210A levels so low in TA compared to other tissues?

      Answer: This is due to the insufficient proteins loaded as it was obvious from the Tubulin marker. We have replaced the WB blot with more convincing blots as requested (Figure S1C).

      -Fig 2G: The authors state "Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210a KO mice up to 8 weeks". However there seems to be a progressive increase in nuclei up to 8-weeks in the KO. What is the significance of this?

      Answer: Thank you for pointing this out. We have now changed the wording and quantified the myonuclei number per myofiber. The increase of myonuclei in the H&E images is likely due to the smaller myofiber size in the Fam210aMKOmouse compared to the WT (Response Figure 5).

      Response Figure 5. Quantification of the myonuclei number in the H&E images.

      -IP-MS analysis for FAM210A interacting proteins requires validation with IP and reverse IP + WB experiment.

      Answer: We did perform the co-IP with SUCLG2 and FAM210A antibodies to try to confirm the interaction. To be more specific, we transduced C2C12 myoblasts cells with an Fam210a overexpression virus and differentiated the cells for 3 days. The myotubes were used to test the interaction by pulling down Fam210a with a myc antibody (FAM210A has a myc tag) and blot with SUCLG2 antibody. Unfortunately, the results were not promising (Response Figure 6). We reasoned that the interaction might be indirect or too transient to be reliably detected.

      Response Figure 6. co-IP of SUCLG2 and FAM210A.

      • Figure 4A requires quantification of the SDH signals from multiple samples.

      Answer: We thank the reviewer for this suggestion. We have added the quantification of the staining (Figure S5B).

      • Figure 6F: To clearly demonstrate an increase in protein acetylation in the FAM210 MKO, the authors must provide quantification data generated with more then N=1. Please add the molecular weights markings on the side of the blots.

      Answer: We thank the reviewer for this suggestion, we have provided the quantification of the Acetylated-lysine blots, and added the molecular weight markers (Figure 6F, Figure S6C).

      • Figure 6H and S5: The mitochondria transfer experiment appears to be quite efficient compared to previously published studies. It would be important to control that the signal observed in the recipient cells is not due to the leakage of the MitoTracker dye from the donor mitochondria.

      Answer: This is an interesting point though MitoTracker dye is not supposed to leak as it covalently binds to mitochondrial proteins. Even though the dye may leak to mark the endogenous mitochondrial, it does not affect our goal to demonstrate that transfer of Fam210aMKO mitochondria into healthy cells can induce protein hyperacetylation. Additional evidence argues against the leakiness of Mitotracker dye to subsequently mark other mitochondria in the recipient cells: 1) mtDNA and MitoTracker signal both increase linearly with the increasing amounts of mitochondria transferred (Figure S7A); 2) We have now also included confocal images to show the presence of both MitoTracker labeled and non-labeled mitochondria in the recipient cells. We reason that if MitoTracker leaks within a cell then it would have labeled all mitochondrial in that cell (Figure 7C).

      • Figure 6J: The increase in Fgf21 is modest. Although the difference is statistically significant, is it biologically important?

      Answer: We thank the reviewer for this question; indeed, the increase is modest. We think the reason of the modest increase compared to the drastic increase seen in vivo was because when we transplanted the WT and Fam210aMKOmitochondria to the recipient cell, the original mitochondria in the recipient were not depleted, which could explain the milder effect. However, we were able to show that the recipient cells readily increase the acetylation of proteins after receiving the Fam210aMKO mitochondria, recapitulating the phenotype we saw in the Fam210aMKO muscle.

      After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

      • Figure 6C: How significant is the difference in acetylation of RPL30 in WT vs. KO. RPS13 was not found in the WT MS? Was this normalized to Input?

      Answer: the MS was performed with same loading. The mass spectrometry results for protein identification after AcK-IP were from pooled samples from 3 independent replicates (as the KO muscles are very scarce). Therefore, there was not a significance test.

      • Figure 7D: What are the MW of the bands shown on this blot? This experiment is by no means sufficient to demonstrate and confirm that ribosomal proteins are acetylated. An increase in RPL30 and RPS13 acetylation must be directly assessed.

      Answer: We thank the reviewer for suggesting the more direct assays to look at RPL30 and RPS13 acetylation. We have shown that the ribosome fractions were indeed hyperacetylated in the Fam210aMKO mouse compared to the WT control (Figure S6D). We agree that this result cannot lead to the conclusion that the RPL30 and RPS13 are specifically hyperacetylated. Indeed, we have tried to use Acetylated lysine antibody pull down and RPS13/RPL30 blot to show the increase in the acetylated RPS13/RPL30 protein. However, we cannot show a robust increase in the acetylation, potentially due to the low number of acetylation sites on RPS13 and RPL30 protein. We therefore have reworded the conclusion in the revised manuscript to better reflect the results.

      • Fig7E: This experiment is not properly executed and in its current state does not rigorously support that "hyper-acetylation of several small ribosomal proteins leads to ribosomal disassembly". A) UV profiles of the fractionation must be provided to assess the quality of the profile. B) Provide MW markers. Which band is RPL30? The Input and free fraction bands are not at the same size. RPL30 should at least be visible on the 60S and polysomes from the WT. C) These results do not match the acetylation MS data, which seem to show that the increase in acetylation is much greater for RPS13. However, RPS13 presence on polysomes (assuming they are polysomes) is not affected in the KO. D) This type of experiment must be done for three independent biological replicates, blots from single lanes must be quantified and normalized to total signal (from all the lanes) for the same antibody.

      Answer: we appreciate the great advice on improving the experiment. As suggested, we have now added proper experimentation (UV profile, and better WB), with the help of Dr. Kotaro Fujii (included as co-author in the revised manuscript). The following results showed that in the 4-week sample, there was a decrease in the 80S monosome and polysome in the Fam210aMKO mice compared to the WT. The change was more drastic at 6-week (Figure 4D-4G). Similarly, due to the scarce amount of muscle in the KO mice, we need to pool samples from the 6-week-old mice for the experiment, and hope the reviewer can understand the situation. With the clear peaks shown in the UV profile as well as the WB results, we provide more convincing evidence that the polysome assembly was indeed impaired in the Fam210aMKO (Figure 4D-4G).

      • Fig 7F: Global translation rates are assessed by puro incorporation at week 4, a time point when differences in protein acetylation were not observed. This does not support the hypothesis that increased acetylation of ribosomal proteins causes defect in protein translation. (Referencing the authors statement p.7 lines 321-24.).

      Answer: We thank the reviewer for this question. When we quantified the protein acetylation increase in the muscle at 4-weeks, we showed that there was a significant increase. But like the reviewer said, the ribosomal fractions were not significantly acetylated by WB at 4-week. We reasoned that, at early stages (4-weeks), the ISR signaling can lead to the translational arrest, along with the polysome formation defects, leading to the decreased protein translation. These are included in the discussion.

      • Other studies have implicated Fam210A in the regulation of mitochondrial protein synthesis through an interaction with EF-Tu. The authors also identified EF-Tu as an interactor in their LC-MS analysis (FigS4). A role for this interaction accounting for mitochondrial and translation defects seems to be underestimated and unexplored here.

      Answer: We agree with this point and believe the cytoplasmic translation defects are in addition to the mitochondrial translational defects. We have shown that FAM210A KO leads to the decrease of the MTCO1 which is encoded by the mitochondrial genome. Besides, we also showed by mitochondrial proteomics that TUFM was reduced in the KO, which also contributed to translational arrest in the mitochondria (Figure 5J). To answer whether mitochondrial encoded proteins are decreased in upon Fam210a KO, we blotted the protein lysates at different stages with antibodies for a few mitochondrial encoded proteins and showed that they decreased with ages (Response Figure 7).

      Response Figure 7. WB analysis and quantification of mitochondrial encoded proteins in WT and Fam210aMKO muscle at different ages.

      The mitochondrial proteins were indeed decreased in Fam210aMKO starting from 6-weeks of age compared to the WT.

      Minor comments:

      -What is known about FAM210A, other studies assessing its role, and the rational for studying its function should be better introduced.

      Answer: We thank the reviewer for the suggestion to have more information of FAM210A functions/mechanisms in the introduction. We have added more background to the introduction.

      -In the discussion the authors states: "Moreover, when the proportion of ribosomal protein phosphorylation buildup in the Fam210aMKO, the assembly of the translational machinery is impaired therefore further dampen the cellular translation". Do they mean acetylation and not phosphorylation?

      Answer: We are sorry about the typo and have changed it. We thank the reviewer for catching this.

      • Please use the term "mRNA translation" or "protein synthesis" instead of "protein translation" in the text.

      Answer: We thank the reviewer for the suggestion to properly refer to these processes. We have changed the terms in the manuscript.

      -The methods section for RT-qPCR: It should ne M-MLV RT and not M-MLC. If the qPCR data was normalized with 18S, please provide the sequence of the primers in the table. Information on how primer efficiency was tested must be included in the method section.

      Answer: We thank the reviewer for pointing this out. We have changed the texts. We also have provided 18S sequence and provide texts about how primer efficiency was tested.

      Reviewer #2 (Significance (Required)):

      General assessment: Previous genome-wide association studies have found that mutations in FAM210A were associated with sarcopenia and osteoporosis. Because FAM210A is not expressed in the bone and highly expressed in skeletal muscle, it suggests that FAM210A likely plays an important role in muscle, which could also affect bone regulation. The authors here provide further evidence of an important role for FAM210A in diseases affecting muscle function by demonstrating that the expression of FAM210A decreases with age and in patients affected by Pompe disease, Duchenne muscular dystrophy and hereditary recessive myopathy. FAM210A is a mitochondria-localized protein and given the crucial role of mitochondria in supporting muscle metabolism, elucidating the molecular function of FAM210A may provide important insights into diseases biology that could lead to the development of therapeutic approaches. Thus, a significant protein and regulatory pathway are explored in this study that can potentially impact human health. In this manuscript, the authors provide compelling evidence of the importance of Fam210a in muscle homeostasis with their newly generate mouse model. The experiments looking at muscle physiology, function and metabolism are well-executed and for the most part rigorous, which are the strengths of this manuscript. However, the conclusion that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins, which leads to ribosomal disassembly and translational deficiency is not supported by the data presented here. As noted in the comments above, these experiments need major improvement. Additionally, there are other concerns about general scientific rigor and conclusions inconsistent with the data presented as also noted in the comments section.

      Advance: Although a previous study explored the role of FAM210A using a skeletal muscle-specific KO induced at postnatal 28 days under a HSA promoter, the model used by the authors here provide a cleaner approach and more insights into the molecular functions of FAM210A in muscle physiology. The findings that Fam210a MKO disrupts the flow of TCA cycle, which leads to an abnormal accumulation of acetyl-CoA is interesting and provide new conceptual advance on the roles of FAM210A in mitochondria function in muscle. Acetyl-CoA production is an important source of acetyl-group that can be transferred to proteins and regulate gene expression programs. Thus, this is an important finding. However, molecular mechanism by which FAM210A regulates this process through an interaction with SUCLG2 is not provided and the nature this interaction is superficially explored.

      Audience: Findings from this manuscript are likely to interest both basic research and translational/clinical audiences as it explores the physiological and molecular function of a disease-linked protein. The findings are also likely to impact the fields of metabolism, mitochondria function and regulation of gene expression by protein acetylation (if concerns raised regarding these experiments are addressed).

      The fields of expertise of this reviewer are protein and RNA modifications, ribosome biogenesis and mRNA translation.

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

      The authors state that in their manuscript "the role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers)" has been explored (Line 19-20). As experimental model, they used mice expressing Cre recombinase under the control of the myosin light chain 1 promoter. The first conclusion was that "FAM210A is positively associated with muscle mass in mice and humans". The authors say that the presented data "reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A".

      I recognize the potential of this work since the role of FAM210a has been more deeply investigated in skeletal muscle. In fact, the study by Tanaka et al, 2018 presented only a preliminary characterization of the role of FAM210a in muscle. However, I think that this work is not complete and each aspect that has been investigated is not well connected with each other. In particular, it is not clear whether the disrupted ribosomal assembly by hyperacetylation causes muscle atrophy or it is altered under catabolic states during atrophy (primary cause or consequence of?).

      Answer: We thank the reviewer for recognizing the importance of the study that characterizes the effect of FAM210A in muscle mass maintenance. In this study, we have shown that polysome formation was impaired at 4-week and therefore the translational efficiency was reduced in the muscle. This translational decrease coincides with the acetylation increase. Moreover, we showed by mitochondrial transfer experiment that the mitochondria from the Fam210aMKO mice can carry the phenotype and lead to acetylation increase in the recipient cells. Since muscle protein synthesis defects have been known to lead to muscle dystrophy, and we have shown that in the Fam210aMKO model, protein synthesis was indeed defective while there was not an induction of atrophy. Therefore, we conclude that the in the KO model, the protein synthesis defects lead to muscle atrophy.

      The other major point is represented by the fact that the Myl1-CRE expressing model provides selectivity in fast muscle fibers (see for example Barton PJR, Harris AJ, Buckingham M. Myosin light chain gene expression in developing and denervated fetal muscle in the mouse. Development. 1989;107: 819-824). Then the authors knocked out FAM210a only in fast fibers and they never take in consideration this key point! This is crucial since fast and slow muscles have different content of mitochondria with different size, shape, and metabolism! The muscle fibers can be classified based on the mitochondrial metabolism (see for example Chemello et al., 2019; PMID: 30917329).

      Regarding this point, they simply wrote at Line 75-76 "using a skeletal muscle specific Myl1 (myosin, light polypeptide 1) driven Cre recombinase specifically expressed in post-differentiation myocytes and multinucleated myofibers,...". It would be more correct to write multinucleated type 2 myofibers showing the reduction of FAM210a in different fiber types.

      I think that the authors must solve these aspect and then organize the findings accordingly. The data are in general interesting for broad type of audience.

      Answer:

      We appreciate the reviewer’s comment on the Myl1 knock-in Cre (Myl1Cre) model, which prompted us to more explicitly clarify some of the confusions around this model. We fully respect the validity of the 1989 study by Dr. Buckingham and other studies showing fast muscle specific expression of Myl1. However, we and others have shown that Myl1 not only mark the fast but also the slow myofibers (elaborated below). The discrepancy can be explained by the fact that using the Myl1Cre as a lineage marker is different from directly examining Myl1 expression at static timepoints by in situ hybridization (ISH). This is because Cre recombinase can accumulate and diffuse to all the myonuclei in a multinucleated myofiber, subsequently leading to deletion of LoxP-flanked DNA in all nuclei. Also, in the Cre/LoxP system, only a small amount of Cre recombinase is needed to induce the recombination of the target loxP sites and lead to gene KO. Another example of the discrepancy between the static mRNA pattern and the dynamic gene expression during development is the Hox gene expression. When the corresponding author (SK) of this manuscript was trained with Dr. Joshua R Sanes, he developed 3 Cre lines driven by three different Hox genes– that have been shown by ISH to be expressed in a specific rostral to caudal domain in the spinal cord during development. However, each of these Cre model ended up marking all the spinal cord without any domain specificity. In the case of Myl1Cre mouse model, we have previously published a paper on the lineage-tracing results using the Myl1Cre and showed that Myl1Cre marked all fast AND slow myofibers in mice (Wang et al, 2015, PMID: 25794679). In another lineage tracing study using nuclear GFP reporter, we report that Myl1Cre marks 96% nuclei in myofibers regardless of fiber types (Bi et al., 2016, PMID: 27644105), the remainder 4% non-marked nuclei potentially represent satellite cells. Other groups have also used the Myl1Cre model to induce KO in both fast and slow muscles (Pereira et al, 2020, PMID: 31916679). Therefore, we believe that the Myl1Cre mouse model allows us to efficiently knockout the Fam210a gene in both slow and fast muscle.

      To directly confirm that Fam210a was efficiently knocked out in both slow and fast muscles using the Myl1Cre mouse model, we isolated different muscle groups (Soleus and diaphragm that contains a large fraction of slow myofibers, TA and EDL that contain predominantly fast myofibers) and checked the expression level and the KO efficiency of Fam210a by WB. We have shown that even in slow muscles like diaphragm and SOL, the KO was very efficient, as there were no visible FAM210A bands in the WB (Figure S1C).

      In more detail:

      The data must be analyzed and discussed based on the fact that FAM210a has been deleted specifically in fast fibers. First the authors must show the protein levels of FAM210a in both fast, slow and mixed fast-slow muscles. Then for example in Figure S1C EDL, GAS and SOL muscles must be included.

      Answer: This is related to the misunderstanding of the Myl1Cre model. We understand the reviewer’s concern and therefore isolated proteins from different muscles in WT and Fam210aMKO mice at 4-weeks and checked the expression level of FAM210A. We have shown that regardless of fast or slow muscles, FAM210A was deleted.

      The blot in general must be repeated since it has poor quality (continuum of FAM210a band in the samples).

      Answer: We thank the reviewer for this suggestion and increase the data quality. We have changed the original blot with the following blots showing that FAM210A was not deleted in other non-muscle tissues (Figure S1C).

      Please provide staining of TA, GAS and SOL muscles to show how Myl1CRE-directed deletion of FAM210a affect the different myofibers.

      Answer: This point is also related to assumption that Myl1Cre only induce deletion in fast myofibers. We have done staining in both EDL and SOL muscle to show the relative changes in myofiber compositions. We found that the myofibers in EDL and SOL muscle have shifted to a more oxidative type upon Fam210a KO (Figure S3).

      In Figure 2F where decreased TA muscle weight was showed in the Fam210aMKO mice, the authors must include also the other muscles (EDL, GAS and SOL).

      Answer: We thank the reviewer for helping us be more rigorous on the phenotype examination. We understand that the reviewer initially raised this question because of the concern on Myl1Cre model. Now that we have shown the MylCremarks both the fast and slow muscles, we believe this question is no longer a concern. Besides, to indirectly answer the question, we would like for the reviewer to appreciate the size difference of the EDL as well as the SOL muscle in Figure S3 in the manuscript. As can be seen from the images, the size of the SOL muscles in the KO was significantly reduced compared to the WT, speaking in favor of the KO effect on slow muscles.

      In general, since the HSA-CRE model is generally used for gene manipulation in skeletal muscles the authors must characterize their model considering that the myosin light chain 1 promoter Myl1-Cre is mainly active in postmitotic type II myofibers. The last model can also give advantage for mosaic gene manipulation in muscles with mixed fiber types.

      Answer: We thank the reviewer for bringing this point up. We hope by the multiple lines of evidence that we provided in the previous questions, we can convince the reviewer that the KO model using the Myl1Cre does not lead to a mosaic gene manipulation in the muscle. On the contrary, the KO model is a homogeneous KO in both fast and slow muscles.

      Line 118-119 Fam210a level is positively corelated with muscle mass, as it is reduced in muscle atrophy conditions and increased in muscle hypertrophy conditions. Fig 1: I don't like since there are many different models in which the muscle mass reduction is associated with different mechanisms. Then independently of mechanisms associated with changes in muscle mass Fam210a is always linked to? Which common mechanism can explain this?

      Answer: We understand that the reviewer would like to pursue a conserved mechanism governing muscle mass maintenance, however, we by no means wanted to make a direct causal relationship between FAM210A level and different muscle disease/atrophy conditions. Indeed, the atrophic conditions presented have different mechanisms leading to muscle mass reduction, yet we wanted to present the possible connection that Fam210a level and muscle mass are co-regulated, and we later confirmed by KO mouse model that FAM210A KO indeed reduces muscle mass.

      Line 144-146 Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210aMKO mice up to 8 weeks (Figure 2G). I totally disagree! It seems that there is more inflammation upon deletion of Fam210aMKO. Please check it.

      Answer: We thank the reviewer for pointing this out to help us more rigorously describe our results. We have changed the wording to better reflect the changes observed with H&E images.

      Fig3E-L there is a huge difference between EDL and SOL. The authors can't avoid to discuss their data considering the real expression of CRE upon Myl promoter: specific deletion in fast fibers. I think that the data in FIGS3 are very important and must be linked to data in Fig3. Organize in a different way all the presented data to really describe what is happening upon deletion of Fam210a.

      Again, the authors MUST organize better their data in the manuscript: to each paragraph must correspond data in the main figures. For example: at Line 189 Fam210aMKO mice exhibit systemic metabolic defects and at Line 208 Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers. These two paragraphs discuss data showed only in supplementary figures.

      Answer: We thank the reviewer for this suggestion. As shown in the previous responses, the Myl1Cre indeed induce efficient deletion of Fam210a in slow muscles. Therefore, we did not consider this to be a myofiber-specific deletion model. We consider these two results as the effect of a mitochondrial protein (FAM210A) on the myofiber metabolism (independent of myofiber type specific deletion), and that the deletion of Fam210a results in mitochondrial stress, which can lead to myofiber switch (Figure S3).

      Physical activity mast be monitored. Show respiratory exchange ratio (RER = VCO2/VO2) and discuss the results.

      Answer: We thank the reviewer for this suggestion. By these results, we would like to demonstrate that muscle homeostasis is important for the systemic metabolism, disruption of muscle mass maintenance in the Fam210aMKO mice leads to defects in the whole-body metabolism. We have now included the RER results (Figure S2F, S2G). The results show that the Fam210aMKO mice had significantly lower RER (VCO2/VO2) value at daytime, indicating that the mice rely more on utilizing fat as the fuel source. This is consistent with the proteomics results (Figure 5K) that the Fam210aMKOmice have increased FAO pathway. Unfortunately, our metabolic chamber does not have the capacity to monitor activity. We instead include data on heat production (Figure S2E).

      "Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers". The data mast be associated with the evaluation of the expression levels of FAM210 in different fiber type to really understand what is happening upon FAM210a loss.

      Answer: We understand the reviewer’s concern on the different expression level of Fam210a as well as the KO efficiency using the Myl1Cre model. We have shown that Fam210a is knocked out in fast and slow muscles, therefore, we did not consider the effects on fast and slow myofibers separately.

      As SDH activity in type 1 fibers is higher than type 2 the and since the authors are using a model in which Fam210a is deleted only in type 2 fiber they should understand what is happening: fiber 1

      Answer: We agree with the reviewer that the SDH activity is different in different myofibers. We have shown by western blot that FAM210A was similarly KO in both fast and slow muscles. When we performed fiber type staining in EDL and SOL muscle, we saw that there was a shift towards the slower myofiber types both in the EDL and SOL muscle, due to mitochondrial defects.

      Associate a cox assay with the sdh assay

      Answer: We thank the reviewer for this suggestion. We have shown by SDH staining as well as seahorse experiments using isolated mitochondria that the complex II activity was impaired in the muscle. We understand the reviewer would like to see a COX assay to show the defects of the mitochondrial function. Though we were not able to perform the COX assay, we showed from other aspects that the mitochondrial function was impaired by running WB of the mitochondrial encoded proteins (ATP6, MTCO2, mtCYB) and showed these proteins were decreased with ages. Along with the morphological changes of the mitochondria shown by electron microscope (Figure 5 and Figure S5), we conclude that these changes must have impacted mitochondrial function.

      Figure 4b blot tubulin and FAM210a look strange. Look especially at first and second and fourth form the left side.

      Answer: We are sorry about the mistake in the images, we have changed the Tubulin blot in the Oxphos blots.

      Figure 4B OXPHOS protein levels look similar between wt and KO. Include the quantification with the significance (min 3-5 mice per genotype).

      Answer: we have quantified the change between WT and KO on different proteins (Reponse Figure 8).

      Response Figure 8. Quantification of the OxPHOS proteins in WT and Fam210aMKO muscle at different ages.

      Quantification of the blots showed that indeed the mitochondrial proteins were decreased in the Fam210aMKO. The change of mitochondrial encoded protein MTCO1 was earlier detected in the Fam210aMKO.

      Provide TEM analysis for SOL muscle. I would understand whether mitochondria are differently affected in fast and slow muscles.

      Answer: We understand the reviewer was originally concerned about the KO efficiency of Fam210a in fast and slow muscles, based on the assumption about the MylCre model. We have shown that the FAM210A protein was similarly depleted in both fast and slow muscles by western blot. In this case, we would speculate that the mitochondrial change in fast and slow muscles would be similar because the mitochondrial changes were due to the inherent defects in the mitochondria.

      In all experiments must be clear which muscle type or types was/were used:

      Line 268: "isolated from WT and Fam210aMKO muscles at 6 weeks of age".

      Line 587 "Muscle lysate acetyl-CoA contents"

      For Seahorse Mitochondrial Respiration Analysis at Line 599 "isolated mitochondria from muscle"

      For TCA cycle metabolomics at Line 615 "muscle tissue was weighed and homogenized"

      For SCS activity assay at Line 632 "mitochondria from muscles were isolated"

      For LC-MS/MS at Line 647 "Mitochondria were purified from skeletal muscles and subjected to proteomics analysis".

      For Ribosome isolation at Line 676 "Skeletal muscle from mice"

      For Polysome profiling experiment at Line 696 "muscle tissues from mice were dissected"

      It is important to know which muscles were used since confounding effects of the specific deletion of FAM210a in type 2 fibers must be identified and discussed.

      Answer: We thank the reviewer for considering the different muscle groups in our mouse model. For experiments requiring a large amount of muscle tissue, such as ribosome isolation, mitochondrial isolation and polysome profiling, we used all the muscles from the mouse. For WB experiments, we used the TA muscle. We have included this information in the method section in the manuscript. Since we have shown that FAM210A was similarly depleted in different muscles (see previous responses), we think it is justified to pool muscles from the same mouse.

      Line 296-297 The authors wrote "Consistently, the mRNA levels of Atf4, Fgf21 and the associated transcripts were highly induced in the Fam210aMKO 296 both in the 4-week and 6-week-old muscle samples". Is Fgf21 responsible for the reduction of body weight? (see for example PMID: 28552492, PMID: 28607005 and PMID: 33944779). Measure the circulating Fgf21 protein in Ko and wt mice.

      Answer: We thank the reviewer for this great suggestion. Indeed, Fgf21 can potentially lead to body weight reduction, and this can explain the smaller body weight in our mouse model as well. However, we are more concerned about the muscle changes in our mouse model, therefore we did not further validate the changes of Fgf21 in the circulation.

      After careful considerations on the mechanism proposed in the study, we decided to remove qPCR data showing the modest increase of Fgf21 mRNA level. The removal of this data will not change the conclusions we draw nor lessen the significance of the mitochondria transfer experiment.

      Moreover the authors must check Opa1 total protein level and also the ratio between long and short isoforms. Is Fam210a interacting with Opa1?

      Answer: We thank the reviewer for this interesting question. Another publication from our lab has shown that Fam210a can modulate the cleavage of OPA1 in brown adipose tissue and influence the cold-induced thermogenesis (PMID: 37816711). Indeed, OPA1 deletion in muscle can lead to muscle atrophy and postnatal death at about day 10 (PMID: 28552492) through the induction of UPR (ISR) and the induction of Fgf21. We did not check the interaction between FAM210A and OPA1 in the muscle context, and FAM210A was not found to be interacting with OPA1 in brown adipose tissue (PMID: 37816711). However, the focus of this study was the acetylation change and the FAM210A effect on muscle mass maintenance. Therefore, we did not pursue the OPA1 related mechanism in skeletal muscle.

      The final part of the paper is really interesting but need to be discussed knowing exactly the used experimental model. Then check in which fiber types FAM210a is loss.

      Answer: We thank the reviewer for the stringency on the model used. Indeed, the mitochondria can be different from different muscle groups. However, since the muscle isolated from WT and KO mice was properly controlled and therefore can balance the effects of different mitochondria. We have consistently observed the increased acetylation when mutant mitochondria were transferred.

      Regarding the mitochondrial transplantation I'm surprise to see that it happens in the direction of unhealthy mitochondria to healthy cells. Are you able to rescue the phenotype of Fam210a KO cells with healthy mitochondria?

      Answer: We thank the reviewer for bringing this interesting yet important question up! Our mitochondrial transfer results support a “gain-of-function” model where excessive Acetyl CoA produced by the Fam210a-KO mitochondrial induces hyperacetylation. Regarding the question to transfer healthy mitochondria to rescue the KO cells, we reason that even when we transfer the healthy mitochondria to the KO cells, the healthy mitochondria will not stop the mutant mitochondria from making excessive amounts of acetyl-CoA and thus protein acetylation. A clean transfer would require depletion of the mitochondria in the KO cells and concomitant restoring FAM210A level in the KO cells (as the lack of Fam210a gene in the KO cells will eventually convert the transferred mitochondrial into mutants with the normal turnover of FAM210A). This is technically highly challenging and nearly impossible to do. We hope that the reviewer can understand the difficulties.

      Reviewer #3 (Significance (Required)):

      In conclusion, the strength of the presented paper is the novelty: the authors explored the role of FAM210a in skeletal muscle. However, the major limitation is represented by the fact that they did not show in which fiber types Fam210a is knocked out. In fact, the used CRE recombinase expressing model is well-known to be specific for type 2 fibers. Then since mitochondria and metabolism are central in this manuscript and they are different in the fast and slow fiber types, the authors must dissect in details this point.

      Moreover, there are many data but they are not linked each other and discussed properly. The paper must be completely re-organized.

      This manuscript can be interesting for a broad type of audience.

      I'm an expert on mitochondria, metabolism and skeletal muscle.

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      Referee #3

      Evidence, reproducibility and clarity

      The authors state that in their manuscript "the role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers)" has been explored (Line 19-20). As experimental model, they used mice expressing Cre recombinase under the control of the myosin light chain 1 promoter. The first conclusion was that "FAM210A is positively associated with muscle mass in mice and humans". The authors say that the presented data "reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A".

      I recognize the potential of this work since the role of FAM210a has been more deeply investigated in skeletal muscle. In fact, the study by Tanaka et al, 2018 presented only a preliminary characterization of the role of FAM210a in muscle. However, I think that this work is not complete and each aspect that has been investigated is not well connected with each other. In particular, it is not clear whether the disrupted ribosomal assembly by hyperacetylation causes muscle atrophy or it is altered under catabolic states during atrophy (primary cause or consequence of?).

      The other major point is represented by the fact that the Myl1-CRE expressing model provides selectivity in fast muscle fibers (see for example Barton PJR, Harris AJ, Buckingham M. Myosin light chain gene expression in developing and denervated fetal muscle in the mouse. Development. 1989;107: 819-824). Then the authors knocked out FAM210a only in fast fibers and they never take in consideration this key point! This is crucial since fast and slow muscles have different content of mitochondria with different size, shape, and metabolism! The muscle fibers can be classified based on the mitochondrial metabolism (see for example Chemello et al., 2019; PMID: 30917329).

      Regarding this point, they simply wrote at Line 75-76 "using a skeletal muscle specific Myl1 (myosin, light polypeptide 1) driven Cre recombinase specifically expressed in post-differentiation myocytes and multinucleated myofibers,...". It would be more correct to write multinucleated type 2 myofibers showing the reduction of FAM210a in different fiber types.

      I think that the authors must solve these aspect and then organize the findings accordingly. The data are in general interesting for broad type of audience.

      In more detail:

      The data must be analyzed and discussed based on the fact that FAM210a has been deleted specifically in fast fibers. First the authors must show the protein levels of FAM210a in both fast, slow and mixed fast-slow muscles. Then for example in Figure S1C EDL, GAS and SOL muscles must be included. The blot in general must be repeated since it has poor quality (continuum of FAM210a band in the samples). Please provide staining of TA, GAS and SOL muscles to show how Myl1CRE-directed deletion of FAM210a affect the different myofibers. In Figure 2F where decreased TA muscle weight was showed in the Fam210aMKO mice, the authors must include also the other muscles (EDL, GAS and SOL). In general, since the HSA-CRE model is generally used for gene manipulation in skeletal muscles the authors must characterize their model considering that the myosin light chain 1 promoter Myl1-Cre is mainly active in postmitotic type II myofibers. The last model can also give advantage for mosaic gene manipulation in muscles with mixed fiber types. Line 118-119 Fam210a level is positively corelated with muscle mass, as it is reduced in muscle atrophy conditions and increased in muscle hypertrophy conditions. Fig 1: I don't like since there are many different models in which the muscle mass reduction is associated with different mechanisms. Then independently of mechanisms associated with changes in muscle mass Fam210a is always linked to? Which common mechanism can explain this? Line 144-146 Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210aMKO mice up to 8 weeks (Figure 2G). I totally disagree! It seems that there is more inflammation upon deletion of Fam210aMKO. Please check it. Fig3E-L there is a huge difference between EDL and SOL. The authors can't avoid to discuss their data considering the real expression of CRE upon Myl promoter: specific deletion in fast fibers. I think that the data in FIGS3 are very important and must be linked to data in Fig3. Organize in a different way all the presented data to really describe what is happening upon deletion of Fam210a. Again, the authors MUST organize better their data in the manuscript: to each paragraph must correspond data in the main figures. For example: at Line 189 Fam210aMKO mice exhibit systemic metabolic defects and at Line 208 Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers. These two paragraphs discuss data showed only in supplementary figures. Physical activity mast be monitored. Show respiratory exchange ratio (RER = VCO2/VO2) and discuss the results. "Fam210aMKO increases oxidative myofibers and decreases glycolytic myofibers". The data mast be associated with the evaluation of the expression levels of FAM210 in different fiber type to really understand what is happening upon FAM210a loss. As SDH activity in type 1 fibers is higher than type 2 the and since the authors are using a model in which Fam210a is deleted only in type 2 fiber they should understand what is happening: fiber 1 Associate a cox assay with the sdh assay Figure 4b blot tubulin and FAM210a look strange. Look especially at first and second and fourth form the left side. Figure 4B OXPHOS protein levels look similar between wt and KO. Include the quantification with the significance (min 3-5 mice per genotype). Provide TEM analysis for SOL muscle. I would understand whether mitochondria are differently affected in fast and slow muscles. In all experiments must be clear which muscle type or types was/were used: Line 268: "isolated from WT and Fam210aMKO muscles at 6 weeks of age". Line 587 "Muscle lysate acetyl-CoA contents" For Seahorse Mitochondrial Respiration Analysis at Line 599 "isolated mitochondria from muscle" For TCA cycle metabolomics at Line 615 "muscle tissue was weighed and homogenized" For SCS activity assay at Line 632 "mitochondria from muscles were isolated" For LC-MS/MS at Line 647 "Mitochondria were purified from skeletal muscles and subjected to proteomics analysis". For Ribosome isolation at Line 676 "Skeletal muscle from mice" For Polysome profiling experiment at Line 696 "muscle tissues from mice were dissected" It is important to know which muscles were used since confounding effects of the specific deletion of FAM210a in type 2 fibers must be identified and discussed. Line 296-297 The authors wrote "Consistently, the mRNA levels of Atf4, Fgf21 and the associated transcripts were highly induced in the Fam210aMKO 296 both in the 4-week and 6-week-old muscle samples". Is Fgf21 responsible for the reduction of body weight? (see for example PMID: 28552492, PMID: 28607005 and PMID: 33944779). Measure the circulating Fgf21 protein in Ko and wt mice. Moreover the authors must check Opa1 total protein level and also the ratio between long and short isoforms. Is Fam210a interacting with Opa1? The final part of the paper is really interesting but need to be discussed knowing exactly the used experimental model. Then check in which fiber types FAM210a is loss. Regarding the mitochondrial transplantation I'm surprise to see that it happens in the direction of unhealthy mitochondria to healthy cells. Are you able to rescue the phenotype of Fam210a KO cells with healthy mitochondria?

      Significance

      In conclusion, the strength of the presented paper is the novelty: the authors explored the role of FAM210a in skeletal muscle. However, the major limitation is represented by the fact that they did not show in which fiber types Fam210a is knocked out. In fact, the used CRE recombinase expressing model is well-known to be specific for type 2 fibers. Then since mitochondria and metabolism are central in this manuscript and they are different in the fast and slow fiber types, the authors must dissect in details this point. Moreover, there are many data but they are not linked each other and discussed properly.The paper must be completely re-organized.

      This manuscript can be interesting for a broad type of audience.

      I'm an expert on mitochondria, metabolism and skeletal muscle.

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      Referee #2

      Evidence, reproducibility and clarity

      Summary: In this manuscript, Chen et al., investigate the functions of FAM210A in skeletal muscle physiology and metabolism. FAM210A is a mitochondria-localized protein in which mutations have been associated with sarcopenia and osteoporosis. Using publicly available gene expression datasets from human skeletal muscle biopsies the authors first demonstrate that the expression of FAM210 is reduced in muscle atrophy-associated diseases and increased in muscle hypertrophy conditions. Based on this, they show that a muscle specific Fam210a deletion leads to muscle atrophy/weakness, systemic metabolic defects, and premature lethality in mouse. Further examination of the knockout myofibers reveals impaired mitochondrial respiration and translation program. Additionally, the authors demonstrate that the flow of TCA cycle is disrupted in the FAM210A-deleted myofibers, which causes abnormal accumulation of acetyl-coA and hyperacetylation of a subset of proteins. The authors claim that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins that leads to ribosomal disassembly and translational deficiency. However, this conclusion is not supported by adequate experimentation and rigorous analysis of ribosomal proteins acetylation and ribosome assembly.

      Major comments:

      • In general, figure legends are lacking information regarding number of biological replicates used and details about statistical analysis. What does three * vs. one * mean in terms of p-value? Exact p-values should be indicated.
      • The mechanistic studies linking muscle phenotypes with ribosomal protein hyperacetylation and mRNA translation defects are underdeveloped and not rigorously carried.
      • Fig S1: The validation WB of FAM210A KO is not the most convincing. Why are the FAM210A levels so low in TA compared to other tissues?
      • Fig 2G: The authors state "Hematoxylin and eosin (H&E) staining did not reveal any obvious myofiber pathology in the Fam210a KO mice up to 8 weeks". However there seems to be a progressive increase in nuclei up to 8-weeks in the KO. What is the significance of this?
      • IP-MS analysis for FAM210A interacting proteins requires validation with IP and reverse IP + WB experiment.
      • Figure 4A requires quantification of the SDH signals from multiple samples.
      • Figure 6F: To clearly demonstrate an increase in protein acetylation in the FAM210 MKO, the authors must provide quantification data generated with more then N=1. Please add the molecular weights markings on the side of the blots.
      • Figure 6H and S5: The mitochondria transfer experiment appears to be quite efficient compared to previously published studies. It would be important to control that the signal observed in the recipient cells is not due to the leakage of the MitoTracker dye from the donor mitochondria.
      • Figure 6J: The increase in Fgf21 is modest. Although the difference is statistically significant, is it biologically important?
      • Figure 6C: How significant is the difference in acetylation of RPL30 in WT vs. KO. RPS13 was not found in the WT MS? Was this normalized to Input?
      • Figure 7D: What are the MW of the bands shown on this blot? This experiment is by no means sufficient to demonstrate and confirm that ribosomal proteins are acetylated. An increase in RPL30 and RPS13 acetylation must be directly assessed.
      • Fig7E: This experiment is not properly executed and in its current state does not rigorously support that "hyper-acetylation of several small ribosomal proteins leads to ribosomal disassembly". A) UV profiles of the fractionation must be provided to assess the quality of the profile. B) Provide MW markers. Which band is RPL30? The Input and free fraction bands are not at the same size. RPL30 should at least be visible on the 60S and polysomes from the WT. C) These results do not match the acetylation MS data, which seem to show that the increase in acetylation is much greater for RPS13. However, RPS13 presence on polysomes (assuming they are polysomes) is not affected in the KO. D) This type of experiment must be done for three independent biological replicates, blots from single lanes must be quantified and normalized to total signal (from all the lanes) for the same antibody.
      • Fig 7F: Global translation rates are assessed by puro incorporation at week 4, a time point when differences in protein acetylation were not observed. This does not support the hypothesis that increased acetylation of ribosomal proteins causes defect in protein translation. (Referencing the authors statement p.7 lines 321-24.).
      • Other studies have implicated Fam210A in the regulation of mitochondrial protein synthesis through an interaction with EF-Tu. The authors also identified EF-Tu as an interactor in their LC-MS analysis (FigS4). A role for this interaction accounting for mitochondrial and translation defects seems to be underestimated and unexplored here.

      Minor comments:

      • What is known about FAM210A, other studies assessing its role, and the rational for studying its function should be better introduced.
      • In the discussion the authors states: "Moreover, when the proportion of ribosomal protein phosphorylation buildup in the Fam210aMKO, the assembly of the translational machinery is impaired therefore further dampen the cellular translation". Do they mean acetylation and not phosphorylation?
      • Please use the term "mRNA translation" or "protein synthesis" instead of "protein translation" in the text.
      • The methods section for RT-qPCR: It should ne M-MLV RT and not M-MLC. If the qPCR data was normalized with 18S, please provide the sequence of the primers in the table. Information on how primer efficiency was tested must be included in the method section.

      Significance

      General assessment: Previous genome-wide association studies have found that mutations in FAM210A were associated with sarcopenia and osteoporosis. Because FAM210A is not expressed in the bone and highly expressed in skeletal muscle, it suggests that FAM210A likely plays an important role in muscle, which could also affect bone regulation. The authors here provide further evidence of an important role for FAM210A in diseases affecting muscle function by demonstrating that the expression of FAM210A decreases with age and in patients affected by Pompe disease, Duchenne muscular dystrophy and hereditary recessive myopathy. FAM210A is a mitochondria-localized protein and given the crucial role of mitochondria in supporting muscle metabolism, elucidating the molecular function of FAM210A may provide important insights into diseases biology that could lead to the development of therapeutic approaches. Thus, a significant protein and regulatory pathway are explored in this study that can potentially impact human health. In this manuscript, the authors provide compelling evidence of the importance of Fam210a in muscle homeostasis with their newly generate mouse model. The experiments looking at muscle physiology, function and metabolism are well-executed and for the most part rigorous, which are the strengths of this manuscript. However, the conclusion that Fam210a deletion in skeletal muscle induces the hyper-acetylation of several small ribosomal proteins, which leads to ribosomal disassembly and translational deficiency is not supported by the data presented here. As noted in the comments above, these experiments need major improvement. Additionally, there are other concerns about general scientific rigor and conclusions inconsistent with the data presented as also noted in the comments section.

      Advance: Although a previous study explored the role of FAM210A using a skeletal muscle-specific KO induced at postnatal 28 days under a HSA promoter, the model used by the authors here provide a cleaner approach and more insights into the molecular functions of FAM210A in muscle physiology. The findings that Fam210a MKO disrupts the flow of TCA cycle, which leads to an abnormal accumulation of acetyl-CoA is interesting and provide new conceptual advance on the roles of FAM210A in mitochondria function in muscle. Acetyl-CoA production is an important source of acetyl-group that can be transferred to proteins and regulate gene expression programs. Thus, this is an important finding. However, molecular mechanism by which FAM210A regulates this process through an interaction with SUCLG2 is not provided and the nature this interaction is superficially explored.

      Audience: Findings from this manuscript are likely to interest both basic research and translational/clinical audiences as it explores the physiological and molecular function of a disease-linked protein. The findings are also likely to impact the fields of metabolism, mitochondria function and regulation of gene expression by protein acetylation (if concerns raised regarding these experiments are addressed). The fields of expertise of this reviewer are protein and RNA modifications, ribosome biogenesis and mRNA translation.

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      Referee #1

      Evidence, reproducibility and clarity

      In the manuscript entitled "FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse", Chen et al, found that knocking out of FAM210A specifically in muscle using Myl Cre resulted in abnormal mitochondria, hyperacetylation of cytosolic proteins, and translation defects. The manuscript uncovered the new functions of FAM210A in regulating metabolism and translation. I have the following the concerns about the manuscript.

      Comments

      1. One of the major phenotypes of FAM210A is the decrease of muscle mass after 6 weeks after birth. Is this phenotype caused by the accumulation of progressive loss of muscle mass from birth? Are the body weight and muscle mass reduced in FAM210A knocking out new born mice? Is the muscle mass growth curve the same in FAM210A and WT mice from birth to 6 weeks after birth? These results will reveal more mechanism of FAM210A mediated muscle mass control.
      2. Does the muscle mass continue to decrease after 8 weeks?
      3. FAM210A knockout mice displayed high lethal rate. Is there any potential mechanism for the high lethality?
      4. In Figure 2, the muscle mass decreased significantly, while the fat mass only decreased slightly. In FAM210A knockout mice. However, the ratio of the lean mass and fat mass to body mass did not change in FAM210A knockout mice compared to WT mice. How do the authors reconcile this?
      5. Are there changes of the number of nuclei per myotube? Is the muscle atrophy in FAM210A knockout mice caused by the defects of fusion, or the degradation of protein, or both?
      6. Are the growth curves of muscle mass growth in EDL and SOL the same n FAM210A knockout mice?
      7. The oxygen consumption and carbon dioxide production are higher in FAM210A knockout mice, suggesting a high metabolism rate. In contrast, the heat production of FAM210A knockout mice is lower, suggesting a low metabolism rate. Any explanation?
      8. Given the high glucose consumption in FAM210A, why is the clearance rate of blood glucose low?
      9. Are there any changes of the abilities for the FAM210A knockout mice in running endurance?
      10. In page 5, the last sentence of the 2nd paragraph, the authors concluded "There results suggest that Fam210aMKO induces a metabolic switch to a more oxidative state." It is better to describe it as muscle metabolic since the whole body metabolism has not been carefully examined.
      11. In Fig. 6, what is the link between increased transcription level of Fgf21 and the elevated level of aberrant acetylation of proteins?
      12. Is there any link between the increased acetylation level of rebolsome proteins and the translation defects?
      13. How do the abnormal mitochondria lead to increased protein acetylation? And how do these defects further cause translation problem?
      14. The defects in translation will cause general problems besides mitochondria defects. Are there any phenotypes related to the overall translation inhibition observed? If not, why?
      15. Are the abnormal mitochondria, increased protein acetylation, and translation inhibition observed in 2-6 weeks old mice? When were these defects first found? Are they correlated with muscle atrophy?

      Significance

      This manuscript described many interesting phenotypes of Fam210a knockout mice. However, the links between these phenotypes are obscure. The logic of the manuscript will be greatly improved if the authors could provide explanations to logically link the phenotypes.

  3. Mar 2024
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      Reply to the reviewers

      __Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __ Deka and colleagues report that a non-canonical NFKb signaling operates in DCs in the context of inflammation and inhibits a tolerogenic mechanism driven by b-catenin-Raldh2. The following comments are made to clarify the findings presented.

      1. The authors re-analyzed published scRNAseq data from DSS colitis to identify the expression of Relb and NFKB2 in myeloid cells. 1a- The authors are encouraged to expand this analysis to other published datasets.

      We sincerely appreciate the comment from the knowledgeable reviewer. Unfortunately, we did not find any other publicly available scRNAseq dataset from DSS-treated mice. To circumvent this problem, we instead examined a previously published microarray-based bulk transcriptomic dataset obtained using FACS-sorted DCs isolated from the mouse colon (GSE58446, Muzaki et al. 2016; doi:10.1038/mi.2015.64). We consistently found an increased expression of Relb, and to some extent Nfkb2, mRNAs in intestinal DCs upon DSS treatment (Fig R1). Because microarray analysis lacks the quantitative attributes that scRNAseq offers, we provided this newly analysed dataset for reviewer's eyes and refrained from including this data in the manuscript per se. Of note, we have also provided our own experimental data directly demonstrating p100 processing in DC isolated from colitogenic gut. (Fig 1F)

      Importantly, we could identify an additional scRNAseq dataset derived colitogenic human ulcerative colitis patients (GSE162335, Devlin et al. 2021, doi.org/10.1053/j.gastro. 2020.12.030). Interrogation of this dataset indeed confirmed that RELB and NFKB2 mRNAs were majorly expressed in intestinal DCs and not in intestinal macrophages and that IBD was associated with increased expression of multiple RelB-important genes in intestinal DCs. These analyses further supported the notion that heightened non-canonical NF-kB signalling in DCs could be fuelling aberrant gut inflammation. We have now incorporated this newly acquired data in the supplementary Figure S5A-S5C. (line# 456-460)

      1b. Additionally, the expression of Relb and NFKB2 in other cells - especially other myeloid cells -should be explored and included, even if then the authors later choose to test their function in DCs.

      Adhering to this brilliant suggestion, we have now further interrogated the mouse scRNAseq dataset (GSE148794 ; Ho et al., 2021) to compare macrophages and DCs for the expression of the non-canonical signal transducers. Indeed, we found a relatively insignificant level of Relb and Nfkb2 mRNAs in intestinal macrophages in comparison to intestinal DCs. Our data suggested that the non-canonical NF-kB pathway is likely to play a more prominent role in DCs than in macrophages in the gut. This new analysis has now been presented in the revised draft in Figure 1B. (revised text line#136-140) This comparison indeed proved useful in motivating subsequent in-depth analyses of the non-canonical NF-kB pathway in DCs in the context of experimental colitis.

      1c. Please note that there is a transition from Fig 1A to Fig 1B to focus on DCs, which is not apparent from the figure.

      Please find our response to #1b.

      Please include scale bars for all histological analyses.

      We thank the reviewer for alerting us. The scale bars were already included in the histological analyses; we have now appropriately highlighted them in this revised version for better visual clarity.

      In Fig S1I, the authors show that loss of body weight upon DSS treatment in Nfkb2DCD11c is indistinguishable from control. Why is the starting weight at 110%? Please clarify.

      We sincerely apologize for this inadvertent error. We have now rectified the axis label, representing the starting weight at day 0 as 100% (currently Figure S1J).

      In Figure 2, please indicate the database/s used for the identification of top biological pathways.

      We used "WikiPathways subset of cellular processes" available at www.gsea-msigdb.org/gsea/msigdb/mouse/collections.jsp?targetSpeciesDB=Mouse#M8 for the pathway enrichment analysis presented in Figure 2B. We also utilized a previously published RA-target gene set for the gene set enrichment analysis presented in Figure 2C (Balmer and Blomhoff, 2002; 10.1194/jlr.r100015-jlr200). While this information was included in the materials and methods section in the original draft, we have now included these descriptions in the figure legend for further clarity. (please see revised figure legends 2B and 2C)

      The authors show a more significant expansion in Tregs upon DSS treatment when non-canonical NFKb is ablated in DCs. Is this at the expense of a reduction of specific Th cells? Can the authors also report the number of cells beyond the % of cells?

      In response to the reviewer's comment, we have examined the abundance of Th17 cells in the colon of our knockout mice. As also observed earlier upon DC-specific ablation of NIK function (Jie et al., 2018), disruption of non-canonical NF-kB signaling in DCs in RelbDDC or Nfkb2DDC mice led to a reduced frequency of RoRgt+ Th17 cells in the LP (Figure S3F, new data). Our in vitro (Figure 2I) and in vivo (Figure 3F-G, 6B-6C) studies conclusively linked DC-intrinsic non-canonical NF-kB signaling to intestinal Treg via the RA pathway. Therefore, we conjectured that the observed decline in the Th17 compartment in our knockouts could be secondary to Treg expansion. We have now further discussed this point in the revised manuscript. (line#296-300)

      As the reviewer suggested, in addition to the Treg frequency, we have also presented the number of intestinal FoxP3+ CD4 T cells in the supplement (Figure S3E). Our data revealed a similar increase in the total Treg numbers in the mouse colon upon ablation of the non-canonical NF-kB pathway in DCs. (line#296-300)

      In figure 6A, it appears that not only the amount of beta-catenin expressed but also the percentage of beta-catenin positive MNL DCs is significantly expanded upon ablation of non-canonical NFkb. Please verify and if so, include.

      We thank the reviewer for this very insightful comment. We have now catalogued MLN DCs into b-cateninlow and b-cateninhigh compartments. Indeed, we found a substantial more than two-fold increase in the frequency of b-cateninhigh DCs in RelbDDC mice. Accordingly, we have revised Figure 6A and emphasised this point in the text.

      (line#428-430)

      In analogy to comment #1 above, please expand the analyses in human samples to include the expression of Relb and Nfkb2 to other myeloid cells.

      Adhering to the valuable suggestion by the reviewer, we have now analysed the scRNAseq dataset (SCP 259) comparing DCs, macrophages, and inflammatory and cycling monocytes present in the human gut for the expression of RELB and NFKB2 mRNA (Figure 7B). Consistent with our observation involving the mouse colon, we found that mRNAs encoding these non-canonical signal transducers were mostly expressed in DCs among various MNPs. This point has also been emphasized in the revised draft. (line#446-448)

      Reviewer #1 (Significance (Required)):

      Strengths of the manuscript include the conceptual novelty of the intersection between non-canonical NFkb and the tolerogenic b-catenin-Raldh2 axis. And additional strength is the methodical approach, which includes various immunological and biochemical assessments as well as genetic perturbations to dissect such relationships. While it remains unknown the relevant triggers for the non-canonical axis described, this study advances our mechanistic understanding on how activation of this axis overrides regulatory mechanisms in DCs. As such, this manuscript should be of broad interest to immunologists and in particular mucosal immunologists. We sincerely thank the reviewer for lauding our work as conceptually novel and methodical. The encouragement from the knowledgeable reviewer would certainly motivate us further to identify the relevant trigger of this pathway in the gut.


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

      The following issues are noted.

      - all animal strains and their provenance should be described and properly referenced (for example, there are at least two CD11c-Cre strains with different specificity). Along the same lines, the specificity of Cre recombination should be confirmed, at least in major cell types (DCs vs T effector or regulatory cells).

      We sincerely appreciate the reviewer's attention to these important details. We would like to point out that in our original draft, Table 1 in the Materials and Methods section provides information on the source and the identifier of all mouse strains used. In particular, we utilized CD11c-Cre mice with the identifier 008068 from the Jackson Laboratories. Alternately known as B6.Cg-Tg (Itgax-cre)1-1Reiz/J, this strain displays Cre-mediated recombination in more than 95% of conventional DCs while exhibiting only minor recombination in lymphocytes (low-activated T cells (www.jax.org/strain/008068). Importantly, our immunoblot analyses revealed efficient depletion of RelB in specifically splenic CD11c+ cells of RelbDDC mice with only a negligible reduction in CD11c- cells (Figure S1E). Our analyses involving Nfkb2DDC mice also assured of similar gene disruption specificity (Figure S1H). Notably, our results were consistent with those documented on RelbDDC and Nfkb2DDC strains earlier (Andreas et al., 2019). To further address the reviewer's concern pertaining to the T-cell compartment, we have now compared splenic CD4+ cells from Nfkb2fl/fl and Nfkb2DDC mice for the expression of p100 (Figure S1I, newly added in the revised draft). Our results confirmed that CD11c-Cre-driven ablation of the non-canonical NF-kB pathway did not perturb p100 expressions in T cells. Taken together, these allow us to emphasize that knockout phenotypes observed in our study were attributed to non-canonical NF-kB deficiency in DCs. We have accordingly modified the text to highlight gene deletion specificities in our knockouts. (line#166-167, 180-182)

      - the DSS model is prone to "batch effects" of individual cages, and proper comparison between genotypes is possible only if mice of different genotypes (eg littermates) are housed together in the same cages. The authors should clearly confirm whether this was the case, and if not, key experiments should be repeated in this setting.

      As mentioned in the materials and methods section of the original draft, littermate male mice of indicated genotypes were indeed cohoused for at least one week prior to experiments. We have now further emphasised this point in the legend of Figure 1.

      - BMDCs represent a heterogeneous mixture of DCs and macrophages (Helft et al., Immunity 2015). These populations should be clearly defined and compared between genotypes, to make sure that they do not underlie the observed gene expression differences.

      The knowledgeable reviewer has raised a very pertinent issue. We would like to emphasize that instead of generating BMDCs using GM-CSF alone following the protocol prescribed by Helft et al. (2015), we differentiated bone marrow cells to BMDCs using a cocktail of GM-CSF+IL4 adhering to the protocol published by Jin and Sprent (2018). Following the reviewer's suggestion, we have now compared BMDCs generated in these two protocols in our laboratory. As reported earlier (Jin and Sprent, 2018), unlike BMDCs generated using GM-CSF alone, BMDCs generated using the GM-CSF+IL4 cocktail did not contain CD115high macrophage-like cells (Figure S2C). (line#230-232) However, they displayed equivalent expressions of the DC marker CD135 on their surface. Moreover, when we compared BMDCs derived from Relbfl/fl and RelbDCD11c mice in flow cytometry analyses, we found comparable surface expression of CD135, assuring intact BMDC generation from bone marrow cells ex vivo in spite of the absence of RelB (Figure S2I). (line#266-268) These studies argue that macrophage-like cells did not contribute to the observed gene expression differences between WT and RelB-deficient BMDCs.

      • the analysis of DCs in mutant strains (e.g. in Fig. 3) would benefit from a better definition of populations, e.g. resident vs migratory DCs in the MLN, the Notch2-dependent CD103+ CD11b+ DCs in the LP and MLN, etc. Again, this would be important to justify differences in gene expression (e.g. Fig. 3D).

      We sincerely appreciate the comment from the knowledgeable reviewer. In a landmark paper from Prof. Fiona Powrie's group (Coombes et al., 2007), it was earlier demonstrated that CD103+ DCs present in the intestine migrate to local MLNs and play a key role in producing RA and supporting Tregs. While our BMDC data strongly supported a cell-intrinsic mechanism underlying Raldh2 upregulation upon non-canonical NF-kB deficiency (Figure 2F), our in vivo studies (Figure 3D-3E) did not entirely rule out also a possible expansion of RA-producing CD103+ DC compartment in our knockout mice. Although the proposition that non-canonical NF-kB signaling regulates the generation of specific intestinal DC subsets seems attractive, we must point out that previous studies showed a relatively unaltered frequency of CD103+ cells among steady-state migratory DCs in skin-draining lymph nodes (Döhler et al., 2017). Nevertheless, following the reviewer's suggestion, we now plan to perform advanced flow cytometry analyses to compare Relbfl/fl and RelbDCD11c mice for the frequency of CD103+CD11b-, CD103+CD11b+ and CD103-CD11b+ DCs in the intestine. To this end, we have already optimized our experimental protocol for staining intestinal DCs with anti-CD103 antibody (BD Bioscience). In the coming weeks, we are expecting to gather adequate numbers of littermate knockout mice to perform a side-by-side comparison. [NOTE: also the section - "Description of the planned revisions"]

      • the analysis of b-catenin protein expression and cellular localization at the single-cell level (e.g. by IF) would greatly strengthen the mechanistic connection between NF-kB and Wnt/b-catenin pathways.

      Adhering to the reviewer's suggestion, we have now performed immunofluorescence assay (IFA) to capture the impact of RelB deficiency on b-catenin expression and cellular localization. Because BMDCs pose challenges for IFA owing to their non-adherent nature, we instead examined mouse embryonic fibroblasts (MEFs), which provide for a genetically amenable model cell system. As presented below (Figure R2), our IFA data conclusively demonstrated an increased cellular abundance and nuclear localization of b-catenin in Relb-/- MEFs. While we are truly excited to find that our proposed mechanism is functional in another cell type, we feel that the inclusion of MEF data in the main manuscript, which describes DC-mediated immune controls, may cause significant distractions for the general audience. Accordingly, we have provided this data for the reviewer's eyes only.

      Minor: - The reanalysis of previous single-cell data is in Figs. 1 and 7 are much less convincing or exciting than the new experimental data relegated to the supplements. The distribution of the results between main and experimental figures may be reconsidered in this light.

      We concur with the knowledgeable reviewer that our scRNAseq analyses may have appeared less convincing in the original draft. In response to comments by reviewer-1 and reviewer-3, we have now added additional data panels (Figure 1B, Figure 7B and Figure 7G) and examined additional publicly available datasets (Figure S5). In the revised draft, these analyses helped us to more firmly establish a link between non-canonical NF-kB signaling in DCs to aberrant intestinal inflammation in mice and humans.

      However, we slightly diverge that many key experimental datasets were relegated to the supplement. Except for the FITC-dextran experiment, data from all other experimental analyses were presented in the main text (Figure 1). To suitably manage space in our figure panels, we opted to present quantified data averaged from experimental replicates in the main text while providing representative raw data in the supplement. Besides, immunoblot analyses confirming DC-specific ablation of target genes in our knockouts were placed in the supplement. Notably, these knockout strains were also examined earlier (Andreas et al., 2019). Those studies, along with our own analyses (Figure S1E, S1H and S1I - additional data), confirmed the most efficient gene deletion in CD11c+ cells. While maintaining these data in the supplement for want of space, we have now cited this reference in the main text to emphasize that knockout phenotypes observed in our study were attributed to non-canonical NF-kB dysfunctions in DCs.

      Reviewer #2 (Significance (Required)):

      The manuscript by Deka et al. explores the role of the non-canonical NF-kB pathway, specifically of its key mediators RelB and NF-kB2, in dendritic cells (DCs) during intestinal inflammation. The key strength of the paper is the demonstration that DC-specific deletion of RelB or NF-kB2 leads to improved acute or chronic DSS colitis. It is also shown that reducing the dose of b-catenin rescues the phenotype of RelB deletion, providing an important genetic connection between NF-kB and Wnt/b-catenin pathways. As such, the work is novel, important and of potential significance to the field.

      We express our deepest gratitude to the reviewer for his/her valuable time and insightful comments. We are indeed extremely excited that the knowledgeable reviewer finds our work novel, important and of potential significance to the field. These positive comments would inspire us to look further into potential interventions targeting the non-canonical NF-kB pathway in human ailments.


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

      Summary:

      This manuscript from Deka et al. investigates the role of dendritic cell noncanonical NFκB signaling on intestinal inflammation. Based on prior data showing altered DC function in intestinal inflammation, they interrogated existing scRNAseq data and found that DSS treatment (which yields chemical colitis) increased the expression of non-canonical NFkB family members in dendritic cells. This led to the generation of a DC-specific RelB deficient mouse and use of a DC specific NFkB2 deficient mouse, each of which showed varying degrees of protection from chemical colitis.

      Overall, they do a very nice job identifying a mechanism by which noncanonical NFκB signaling in dendritic cells contributes to intestinal inflammation via transcriptional regulation of Axin1, downregulation of β-catenin, restraint of Raldh2 synthesis, impaired retinoic acid synthesis and subsequent decrease in protective Tregs, IgA+ B cells, and microbial dysbiosis. The importance of this pathway is well supported by their focused targeting of β-catenin. After pharmacologic inhibition of β-catenin showed restoration of Raldh2 abundance, they made a DC-specific β-catenin haploinsufficiency RelBDCD11c mouse which showed impaired Raldh2 activity with restoration of colonic Tregs and fecal sIgA. When challenged with DSS, the protective phenotype seen with the RelBDCD11c was lost and the colitis phenotype returned to that of the Relbfl/fl control, further solidifying the role of β-catenin, Raldh2 and RA on intestinal inflammation. Additionally, the discussion provides a robust mechanistic explanation for the phenotypic differences between the RelB and Nfkb2 genotypes, drawing on the authors' deep knowledge of the non-canonical NFκB pathway.

      Major comments:

      1. Although they propose a novel mechanism by which dendritic cells can contribute to intestinal inflammation, it is in a model of acute epithelial injury that accentuates the contribution of the innate immune system. Would recommend including a discussion of the limitations of this model.

      We most sincerely thank the knowledgeable reviewer for raising this important issue. We argue that while erosive epithelial injury initiates colitis in the DSS model, T cells were shown to aggravate intestinal pathologies, particularly at DSS doses used in our study (Kim et al., 2006; doi: 10.3748/wjg.v12.i2.302). Furthermore, our chemically-induced colitis model offered a convenient tool for genetically dissecting the DC-intrinsic role of the non-canonical NF-kB pathway in the intestine. However, we agree entirely that no single animal model fully captures the clinical complexities of human IBD and that other models of experimental colitis should also be employed in the future to assess the generalisability of the proposed DC mechanism in regulating intestinal inflammation. In particular, future studies ought to examine composite knockout strains in the T-cell transfer model of experimental colitis to establish further the role of non-canonical NF-kB signaling in DCs in alleviating intestinal inflammation. As suggested by the reviewer, we have now articulated this point in the discussion section. (line# 553-557)

      The human work (Figure 7) shows solid evidence of heightened non-canonical NFκB signaling in DCs via abundance of RELB and NFKB2 along with a few RelB important genes, however, the RA-specific pathway identified in the mouse work is not strongly corroborated by the human data. There is demonstration of one β-activated gene (CCND1) showing decreased expression in IBD patients, however no other gene along with RA pathway was clearly identified to be differentially expressed as one would predict from the mouse work.

      We sincerely thank the knowledgeable reviewer for articulating this deficiency in our analyses of single-cell RNA-seq data derived from IBD patients (SCP259, Smillie et al., 2019). We would like to clarify that many well-known b-catenin target genes, including MYC, were not detectable in this dataset. Nevertheless, to address the reviewer's concern, we subjected this dataset to GSEA using a previously published list of RA target genes (Balmer et al., 2002). Our analyses revealed a significant enrichment of RA targets among genes that were downmodulated in DCs derived from inflamed colonic tissues of IBD patients as compared to those from non-inflamed tissues (Figure 7G, newly added in the revised version). We have now discussed this data in the result section. (line#470-475) These studies further substantiated the inverse correlation between noncanonical NF-kB signalling and the RA pathway in DCs in the inflamed human gut.

      Minor comments: 1. Their NFκB2DCD11c mouse underwent a regimen of chronic DSS treatment after acute DSS treatment only displayed subtle phenotypic changes. Was the same chronic colitis regimen also tested in the RelBDCD11c ?

      Indeed, we also examined RelbDCD11c mice in the chronic DSS treatment regime. As compared to Relbfl/fl mice, these knockout mice displayed significantly less bodyweight changes upon chronic DSS challenge. Because RelbDCD11c mice readily showed acute DSS phenotype, we did not further pursue investigations involving this strain in the chronic DSS settings and rather focused on Nfkb2DCD11c mice to illustrate chronic DSS phenotypes.

      In the introduction, it was stated that patients with UC have a marked reduction in intestinal DCs. If DCs (particularly non-canonical NFκB signaling) promote inflammation, how do you explain a decrease in this cell type in patients with active disease?

      Depending on the expression of immunogenic or tolerogenic factors, DCs may both promote or subdue inflammation in the colon. We have now revisited the relevant reference published by Magnusson et al., (2016). Indeed, the authors noted a marked reduction in the intestine of the CD103+ DC subset, which has been majorly linked to tolerogenic RA synthesis. While it is generally thought that aberrant inflammation promotes the death of mononuclear phagocytes in the intestine, it seems that either a contraction of the tolerogenic DC compartment or downmodulation of tolerogenic pathways in DCs incites gut inflammation in IBD patients. We have now revised the text in the introduction section to clarify this point. (line#81-83)

      The focus on retinoic acid is interesting, however may be oversimplifying the role of non-canonical NFκB in DCs on the mucosal immune system. It must also be mentioned that there is crosstalk between the non-canonical and canonical NFκB signaling systems, for example Nfkb2 is capable of functioning as a IkB protein and inhibiting RelA-p50 (from the last author's prior work - Basak et al, Cell, 2007). Thus would include some mention of possible effects on the canonical system that contribute to intestinal inflammation.

      We thank the reviewer for raising this important point. As mentioned in the introduction section of our original draft, the canonical NF-kB pathway in DCs aggravates experimental colitis mice (Visekruna, A. et al. 2015). Indeed, Nfkb2-dependent crosstalk was shown to modulate inflammatory RelA activity in a variety of cell types (Basak et al., 2007; Shih et al., 2009; Chawla et al., 2021). Although such cross-regulatory RelA controls by non-canonical NF-kB signaling are yet to be established in DCs, our studies involving RelB-deficient cells confirmed an essential role of p100-mediated RelB regulations in DC functions. We admit that further studies are required to determine if, independent of RelB, p100 directs immunogenic DC attributes via also RelA or another factor. We have now elaborated on p100-mediated crosstalks in the discussion section. (line#561-562)

      In the single-cell DSS data they analyzed, there was a distinct DC population seen with DSS colitis treatment. Although they are categorized as cDC2s, what genes separate them from the other DC populations?

      We curated a list of genes from Brown et al., (2019) to categorize cDC1 and cDC2 subsets in our study. We would like to clarify that the list was provided in Supplementary Table 1 in our original draft. In view of the reviewer's comment, we have now referred to this Table in the legend of Supplementary Figure 1 and also in the main text. (line#153)

      Why was the RNAseq work on BMDCs (that identified RA metabolism as a top-ranking differentially expressed pathway) done only on Nfkb2-/- BMDCs and not RelB-/-? RelB-/- had a more pronounced protected phenotype in the cell type-specific knockout and is a cleaner target (does not have the IkB capability of Nfkb2).

      We broadly agree with the knowledgeable reviewer that comparing WT and Relb-/- BMDCs for global gene expressions could have been worthwhile. We would like to clarify that we initially utilized a dataset derived using Nfkb2-/- BMDCs already available in the laboratory. These analyses were instrumental in developing a notion that non-canonical NF-kB signalling could be modulating Radh2 expression in DCs. Because previous studies involving germline Relb-/- mice suggested a role of RelB in the nonhematopoietic niche in instructing myeloid development (Briseño et al., 2017), we focused our subsequent analyses on BMDCs generated using bone marrow cells from cell type-specific knockouts. Indeed, we could confirm elevated Raldh2 expressions in BMDCs generated from both RelbDDC and Nfkb2DDC mice. Taken together, our studies suggested that Nfkb2-encoded p100 controlled Raldh2 expressions in DCs by providing RelB:p52 and less so as a regulator of the RelA activity. Although we admit that further studies are required to determine if, independent of RelB, p100 directs immunogenic DC attributes via also RelA or another factor. We have now deliberated this point in the discussion section. (line#566-567)

      Reviewer #3 (Significance (Required): As a physician-scientist who clinically cares for patients with inflammatory bowel disease, and scientifically studies signaling within innate immune cells, this manuscript does a rigorous job of identifying a mechanism by which canonical NFκB signaling in dendritic cells contributes to intestinal inflammation. This study would be very informative for both basic and translational researchers as it identifies a clear pathway by which the innate immune system contributes to intestinal inflammation, and opens up room for inquiry into triggers of non-canonical NFκB in IBD and modulation of the RA pathway as a potential novel therapeutic target.

      We are humbled that the knowledgeable reviewer finds our work to be informative for basic and translational research. These encouragements would undoubtedly motivate us further to identify the relevant trigger of this pathway in the gut and explore potential interventions.

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

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      Referee #3

      Evidence, reproducibility and clarity

      Summary:

      This manuscript from Deka et al. investigates the role of dendritic cell noncanonical NFκB signaling on intestinal inflammation. Based on prior data showing altered DC function in intestinal inflammation, they interrogated existing scRNAseq data and found that DSS treatment (which yields a chemical colitis) increased expression of non-canonical NFkB family members in dendritic cells. This led to the generation of a DC specific RelB deficient mouse and use of a DC specific NFkB2 deficient mouse, each of which showed varying degrees of protection from chemical colitis. Overall, they do a very nice job identifying a mechanism by which noncanonical NFκB signaling in dendritic cells contributes to intestinal inflammation via transcriptional regulation of Axin1, downregulation of β-catenin, restraint of Raldh2 synthesis, impaired retinoic acid synthesis and subsequent decrease in protective Tregs, IgA+ B cells, and microbial dysbiosis. The importance of this pathway is well supported by their focused targeting of β-catenin. After pharmacologic inhibition of β-catenin showed restoration of Raldh2 abundance, they made a DC specific β-catenin haploinsufficiency RelBCD11c mouse which showed impaired Raldh2 activity with restoration of colonic Tregs and fecal sIgA. When challenged with DSS, the protective phenotype seen with the RelBCD11c was lost and the colitis phenotype returned to that of the Relbfl/fl control, further solidifying the role of β-catenin, Raldh2 and RA on intestinal inflammation. Additionally, the discussion provides a robust mechanistic explanation for the phenotypic differences between the RelB and Nfkb2 genotypes, drawing on the authors' deep knowledge of the non-canonical NFκB pathway.

      Major comments:

      1. Although they propose a novel mechanism by which dendritic cells can contribute to intestinal inflammation, it is in a model of acute epithelial injury that accentuates the contribution of the innate immune system. Would recommend including a discussion of the limitations of this model.
      2. The human work (Figure 7) shows solid evidence of heightened non-canonical NFκB signaling in DCs via abundance of RELB and NFKB2 along with a few RelB important genes, however the RA specific pathway identified in the mouse work is not strongly corroborated by the human data. There is demonstration of one β-activated gene (CCDN1) showing decreased expression in IBD patients, however no other gene along with RA pathway was clearly identified to be differentially expressed as one would predict from the mouse work.

      Minor comments:

      1. Their NFκB2CD11c mouse underwent a regimen of chronic DSS treatment after acute DSS treatment only displayed subtle phenotypic changes. Was the same chronic colitis regimen also tested in the RelBCD11c ?
      2. In the introduction, it was stated that patients with UC have a marked reduction in intestinal DCs. If DCs (particularly non-canonical NFκB signaling) promote inflammation, how do you explain a decrease in this cell type in patients with active disease?
      3. The focus on retinoic acid is interesting, however may be oversimplifying the role of non-canonical NFκB in DCs on the mucosal immune system. It must also be mentioned that there is crosstalk between the non-canonical and canonical NFκB signaling systems, for example Nfkb2 is capable of functioning as a IkB protein and inhibiting RelA-p50 (from the last author's prior work - Basak et al, Cell, 2007). Thus would include some mention of possible effects on the canonical system that contribute to intestinal inflammation.
      4. In the single cell DSS data they analyzed, there was a distinct DC population was seen with DSS colitis treatment. Although they are categorized as cDC2s, what genes separate them from the other DC populations?
      5. Why was the RNAseq work on BMDCs (that identified RA metabolism as a top ranking differentially expressed pathway) done only on Nfkb-/- BMDCs and not RelB-/-? The RelB-/- had a more pronounced protected phenotype in the cell type specific knockout, and is a cleaner target (does not have the IkB capability of Nfkb2).

      Significance

      As a physician scientist who clinically cares for patients with inflammatory bowel disease, and scientifically studies signaling within innate immune cells, this manuscript does a rigorous job of identifying a mechanism by which canonical NFκB signaling in dendritic cells contributes to intestinal inflammation. This study would be very informative for both basic and translational researchers as it identifies a clear pathway by which the innate immune system contributes to intestinal inflammation, and opens up room for inquiry into triggers of non-canonical NFκB in IBD and modulation of the RA pathway as a potential novel therapeutic target.

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

      Learn more at Review Commons


      Referee #2

      Evidence, reproducibility and clarity

      The following issues are noted.

      • all animal strains and their provenance should be described and properly referenced (for example, there are at least two CD11c-Cre strains with different specificity). Along the same lines, the specificity of Cre recombination should be confirmed, at least in major cell types (DCs vs T effector or regulatory cells).
      • the DSS model is prone to "batch effects" of individual cages, and proper comparison between genotypes is possible only if mice of different genotypes (eg littermates) are housed together in the same cages. The authors should clearly confirm whether this was the case, and if not, key experiments should be repeated in this setting.
      • BMDCs represent a heterogeneous mixture of DCs and macrophages (Helft et al., Immunity 2015). These populations should be clearly defined and compared between genotypes, to make sure that they do not underlie the observed gene expression differences.
      • the analysis of DCs in mutant strains (e.g. in Fig. 3) would benefit from better definition of populations, e.g. resident vs migratory DCs in the MLN, the Notch2-dependent CD103+ CD11b+ DCs in the LP and MLN, etc. Again, this would be important to justify differences in gene expression (e.g. Fig. 3D).
      • the analysis of b-catenin protein expression and cellular localization at single-cell level (e.g. by IF) would greatly strengthen the mechanistic connection between NF-kB and Wnt/b-catening pathways.

      Minor:

      • the reanalyses of previous single-cell data in Figs. 1 and 7 are much less convincing or exciting than the new experimental data relegated to the supplements. The distribution of the results between main and experimental figures may be reconsidered in this light.

      Significance

      The manuscript by Deka et al. explores the role of the non-canonical NF-kB pathway, specifically of its key mediators RelB and NF-kB2, in dendritic cells (DCs) during intestinal inflammation. The key strength of the paper is the demonstration that DC-specific deletion of RelB or NF-kB2 lead to improved acute or chronic DSS colitis. It is also shown that reducing the dose of b-catenin rescues the phenotype of RelB deletion, providing an important genetic connection between NF-kB and Wnt/b-catenin pathways. As such, the work is novel, important and of potential significance to the field.

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

      Learn more at Review Commons


      Referee #1

      Evidence, reproducibility and clarity

      Deka and colleagues report that a non-canonical NFKb signaling operates in DCs in the context of inflammation and inhibits a tolerogenic mechanism driven by b-catenin-Raldh2. The following comments are made to clarify the findings presented.

      1. The authors re-analyzed published scRNAseq data from DSS colitis to identify the expression of Relb and NFKB2 in myeloid cells.
        • a. The authors are encouraged to expand this analysis to other published datasets.
        • b. Additionally, the expression of Relb and NFKB2 in other cells - especially other myeloid cells -should be explored and included, even if then the authors later choose to test their function in DCs.
        • c. Please note the there is a transition from Fig 1A to Fig1B to focus on DCs, which is not apparent from the figure.
      2. Please include scale bars for all histological analyses.
      3. In Fig S1I, the authors show that loss of body weight upon DSS treatment in Nfkb2deltaCD11c is indistinguishable from control. Why is the starting weight at 110%? Please clarify.
      4. In Figure 2, please indicate the database/s used for identification of top biological pathways.
      5. The authors show a more significant expansion in Tregs upon DSS treatment when non-canonical NFKb is ablated in DCs. Is this at the expense of a reduction of specific Th cells? Can the authors also report the number of cells, beyond the % of cells?
      6. In figure 6A, it appears that not only the amount of beta-catenin expressed, but also the percentage of beta-catenin positive MNL DCs is significantly expanded upon ablation of non-canonical NFkb. Please verify and if so, include.
      7. In analogy to the comment #1 above, please expand the analyses in human samples to include the expression of Relb and Nfkb2 to other myeloid cells.

      Significance

      Strengths of the manuscript include the conceptual novelty of the intersection between non-canonical NFkb and the tolerogenic b-catenin-Raldh2 axis. And additional strength is the methodic approach, which includes various immunological and biochemical assessments as well as genetic perturbations to dissect such relationships. While it remains unknow the relevant triggers for the non-canonical axis described, this study advances our mechanistic understanding on how activation of this axis overrides regulatory mechanisms in DCs. As such, this manuscript should be of broad interest to immunologists and in particular mucosal immunologists.

  4. www.b