把单个矩阵切碎给多卡用

(1) attention一次性把所有向量矩阵（token）输入模型，实现并行计算。self-attention的核心是矩阵运算，把所有词存入矩阵一次性计算。

(2)每个token之间是直接连接的，可以直接（一步）计算每两个token的注意力分数，而RNN不同距离的token存在远近距离，因此会遗忘。

I don't even know, do other competitors have a CRM system or is this a unique point?

In a way this is really good, I want that we discuss weaknesses also of the core here in the last section. But at the same time, I don't want to say or to argue that price is the biggest weakness. Let's start with weaknesses that are functional and then we add the pricing as an additional weakness or potential weak point. I'm not even convinced that the pricing here is a weakness.

This is all part of the ERP functionality, the first sentence seems a bit off. What does it mean, let's rewrite.

This is a specific strength about bookkeeping, it is good that we mentioned it but I still want to start every major section of a competitor with the basics. So what do they cover in terms of ERP functionalities and multi channel functionalities. only then can we go into specific strengths and weaknesses

All right, let's get rid of superlatives again, one only sing it that we need to consider is which ratings do we want to use, we cannot have OMR, Capterra, and trust pilot mixed in differently. So either we use all three for all competitors or we focus on one and use it for all competitors. Otherwise it doesn't make any sense.

The problem for me is that this is to negative, first of all is it really downside if the channels come the partners, also plenty one has a lot of partner channels. Secondly I would much prefer if these core topics are just descriptive, so basically ERP functionalitiesand multi channel options should be descriptive first and then only in the last paragraph do we list downsides potentially, including those limitations we see in the core abilities.

This is a good start, the question is, are they really better at this kind of ERP questions and functionalities, compared to plenty one? At any rate I want to get rid of superlatives.

About general section structure, we have laid out some basic criteria above, and I want to mention the good parts of each competitors against those criteria. So basically mention the parts they are good at, with a specific focus on multi channel and ERP functionalities. So in essence we start with the good parts and where the company is stronger anyway, and then we discussed the downsides or let's just say the limitations.

This is to negative, I am not sure if I want to keep these kind of scores anyway. Let's discuss.

I am not sure if we should mention this, it is not irrelevant point for most companies.

OK, I see we are having the point I made before already in a text. Let's still put this part towards the top so it becomes immediately clear that you can bring your own shop or you can use this shop system.

Also add here that why we offer a shop, we also integrate with auto shops, for example Shopify, so if you have a different shop it is not a problem, that is also very important, since a lot of brands will have everything ready and don't want to switch. They might only need some ERP functionalities, or some multi channel functionalities.

Please doublecheck this. I am not sure if this is accurate.

We need to decide what we mean by channel options, if we want to speak about ERP site integrations, so for example of payment portals or bookkeeping software as well, then the number is fine, but in that case, I doubt that we are the only ones.

I suppose this is to show the ERP basis let's make it a bit more clear, please.

I want to mention in this section that it's about multi channel integration, so having a lot of sales channels available easily, and on the other hand ERP abilities to manage the whole process and what is going on on the backend with the brand. I don't think you're covering it at the moment, but instead of focusing on some differences to supposedly other brands comparied direct consumer brands.

So let's think about what we can write to really cover the basis and point out what differences there are. Though I must say, there are not so many differences in my opinion to other brands, except maybe that auto brand sell more to wholesalers and direct to consumer cells to consumers of course. But this does not mean that they don't sell with other channels, for example Amazon.

what do you mean, this does not make sense

About the sales channels here, let's use different numbers, I don't mind if they are connected via partners and not native, as long as they are mentioned on the website.

Here as well the question is whether this is true. Do direct consumer brands sell on fewer sales channels and marketplace then normal brands? In my experience it does not make a difference, the difference comes from them not selling the wholesalers. But they will sell on Amazon on their own shop on other shops. I don't see a difference in that sense. Pushback if you see that differently.

Let's discuss what is that? What is really different between what are we comparing here? It's the question I get we're talking direct consumers. Some of the arguments make sense but other users of Shopify are not wholesalers. They might be reselling some things, but in a sense, I'm not sure if that's very different from what director consumers doing, since they alsooutsourced production mostly and just at their own brand on top of existing product. So in essence, I want to understand if this is truly the key difference that we should address.

What is this? the word chargenfuehrung??

Not happy with this sentence. I doubt that this is the main problem that companies are facing. We can argue that it's about complexity with growth, complexity with adding multiple channels, complexity with adding a 10 more complex processes, that might require ERP functionalities.

It's not a key point here that the brands are producing themselves direct. The consumer only means that they sell directly to consumers. It doesn't say anything about their production process.

Maybe we can integrate multi channel here instead so instead of saying shop marketplace et cetera, we say multi channel and full ERP. I don't know make some proposal what's the best solution?

I need a different name here. It must include the word multi channel and maybe something more like full system or full EP multi channel yeah I don't know.

the html is somewhat off, no padding on the left side it seems. lets check against other html docuemnts pleas

In human history we have continually come up with myths to explain natural cycles and to explain the meaning behind rituals we perform. In the various stories posed "The Golden Bough" each culture uses their myths to pose warnings to people, such as the Babylonian-Syrian myth of Tammuz and Ishtar in which every year Ishtar's death led to Tammuz having to venture into the underworld to revive him. In Tammuz's absence, the world fell into disarray and carnage, becoming a barren land where, "the passion of love ceased to <br /> operate: men and beasts alike forgot to reproduce <br /> their kinds: all life was threatened with extinction" (Frazer, 9). This barren loveless land is an early depiction of an unproductive wasteland, and is an explanation for times in human history when humans struggled to survive. Wastelands don't have to be literal, but rather can be metaphors and explanations for struggles throughout the history, when other explanations are outside of human understanding and comprehension. Eliot's wasteland can be perceived as the physical destruction left behind by World War I, but to me it is more than a physical description. Eliot uses the title to discuss a time in human history when the world as a whole was in a confusing freefall, every country having to regroup after the terror of the war. After such destruction, it takes time for literary and artistic practices to return to their full force, and in this time there is a barrenness of art and culture, as mourning and recovery takes precedent.

Putting myself in T. S. Eliot’s shoes, The Waste Land seems like a fitting title for his pessimistic view of the modern world. The title comes from the medieval story La Mort d’Arthur, in which the land becomes barren after King Hurlame strikes King Labor, a faithful Christian king. Because King Labor represents Christian authority, the blow violates the religious order holding society together. This directly parallels Eliot’s concern about the rise of atheism: just as Hurlame’s rejection of a Christian king causes the medieval kingdom to become physically barren, Europe’s movement away from Christianity leaves Eliot’s modern world spiritually barren. This connection makes me wonder whether Eliot wished society had remained Christian, or at least believed that Christianity had once provided a shared moral structure that modern society could not replace/ shared purpose.

World War I deepened this crisis, as supposedly Christian nations destroyed one another. Eliot’s waste land is therefore both a ruined Europe and a spiritually empty society. People continue working and forming relationships, but without the shared faith that once gave those actions meaning.

The title also suggests a drought-stricken landscape, almost like Mad Max: Fury Road. :) In From Ritual to Romance, Jessie Weston explains that rain restores the barren land and symbolizes rebirth. Eliot uses the same symbolism more pessimistically. His world desperately needs rain, but renewal would force it to confront what it has become. In Weston, rain promises rebirth; in Eliot, even rebirth feels uncertain and painful.

The Waste Land, (as a title) seems in explicit condemnation of human behavior, morality, and faculty. The narrative itself—in it's three disjunct poems—is an account, or portrait, of humanity's cycles. The first poem, "The Burial of the Dead," and final, "What the Thunder Said," comment on a human's predispositions. In the first, it's asked "What are the roots that clutch, what branches grow/ Out of this stony rubbish? Son of man,/ You cannot say.."; can the human justify its innate "roots," and its blood of Adam and Eve? This question is evident in many following and preceding stanzas. In the final poem, man is placed upon a desolate image:"What is that...Murmur of maternal lamentation/ Who are those hooded hordes swarming/ Over endless plains, stumbling in cracked earth...Jerusalem Athens Alexandria/ Vienna London/ Unreal." The stanza is a brutalist depiction of man's vice, and in the final lines Eliot seems to extend this barbarity to the hubs of human 'intellect' and capacity. These themes, also found within The Golden Bough, are just one understanding of "The Waste Land"'s syncretic title.

In the case of “The Waste Land”, the title refers to a place that is empty, barren and unable to support life. This is used by Eliot as an analogy in order to symbolize the desolation of modern times in relation to the spiritual and emotional aspects of life. In addition to this, the title also makes references to the legend of Fisher King in which an ailing king causes his kingdom to become barren and infertile. The only way of restoring fertility of the land lies in curing the king by using the Holy Grail as a means of spiritual redemption. It shows that the modern world is analogous to the kingdom of Fisher King in the sense that it is isolated, destroyed, and lacks spirituality but still holds possibilities of renewal.

What antagonism is he talking about?

conclusio minima concreta alineada con todo el contenido

explica mejor que por mas que cresca la congestion hay zonas que no se ven afectadas lo que le pone un tope a la saturacion real de la red vial

en comparacio con que? cual es el monto promedio de la gam segun la referencia nacional ?

eliminar

referencia cruzada al cuadro al que hace referencia

eliminar

mucho para una sola pregunta mejor partirla en varias

hace falta 2 fuente aca o con una basta?

In the painting, there is a plough-man, quite unperturbed, unconcerned with Icarus falling from the sky.

The last line grabs my attention because it talks about a ship sailing by and seeing a boy falling out of the sky but the boat has to be somewhere so it ignores the boy. I think this refers to the bad problems happening in the world, but everyone just keeps moving on.

One of the most requested features we get from customers is an IPNS-like solution that is flexible and performant. While IPFS is immutable, there are plenty of valid use cases for being able to swap out the content of an IPFS CID in an auditable way without having to change the entire URL. This works well for websites and NFTs as well as dozens of other use cases we’ve heard customers ask about in the past.

IPFS is a public, permissionless protocol. This means that while it can host content that should be there, it can also host content that you or your users might find inappropriate. Running a gateway to distribute content has often meant manual content moderation. The Moderator Plugin solves this.

Pinata hot swap gateway plugin

plugin

https://hyp.is/nqni-iPSEfG22bNsWZlHTg/www.youtube.com/watch?v=Ucgyd5ML0j4

https://youtu.be/Ucgyd5ML0j4?si=vEkJfS4ud_9xFTLY

cost of answers tends to zero value is in the questions ai youtube

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

Learn more at Review Commons

Reply to the reviewers

We thank the reviewers for the constructive criticism. All issues are addressed in the following point-by-point response.

We addressed a major issue of the reviewers by adding a domain-deletion study. This study shows that Ig-like domain 3 of CADM1 is responsible for the interaction. Thus, we now propose a clear mechanism for CD82 regulation of CADM1 adhesion function.

Point-by-point description of the revisions

Reviewer #1:

Major Comments:

• Using a variety of elegant and sophisticated studies, the authors clearly present data that strongly support their conclusions of the interaction between CADM1 and CD82. Additionally, they speculate on the specific interaction sites/mechanism through which CD82 may affect the spacing of CADM1, which is supported by cryo EM. However, they do not go on to demonstrate a physiological role for this inhibitory interaction.

In this MS we identify an interaction between CADM1 and CD82. Though we find an inhibitory activity, indeed we do not present in vivo data supporting a physiological role.

Our observation of CADM1-CD82 interaction in HEK cells and Jurkat Tcells, co-expression of the two proteins in several tissues (based on human protein atlas) and shared relevance in metastasis support a physiological role.

Minor Comments:

• In Figure 4, there seems to be a significant size difference between WT and CD82KO Jurkat cells. If this is the case, this needs to be described a bit more. Additionally, how does this potential size difference impact the quantification of the percentage space covered by CADM1 clusters? Also, for these experiments, it is more appropriate to use the mean of each independent replicate rather than simply counting each cell as an n. Including these data as super plots so the reader can better understand the spread and reproducibility of the data would be ideal. Best practices suggest that statistical analysis should be performed on data from independent experiments (Lord et al 2020 PMID: 32346721).

With regard to the size difference, this may indeed represent a phenotype associated with CD82 knockout. A modest difference in cell size was observed, although this did not reach statistical significance (Reviewer Fig. A). As all quantitative analyses in Figure 4 were performed at the cell surface, cell volume was not a parameter used in the primary analysis. To provide a more complete visualisation of the cellular context, we additionally assessed 3D cell dimensions and used these measurements to examine whether the observed clustering phenotype was influenced by cell size. Restricting the analysis to cells with a Z-projected area of 100-150 µm² yielded the same trends in percentage space covered (Reviewer Fig. B), cluster density (Reviewer Fig. C), and cluster size (Reviewer Fig. D). These findings support the conclusion that the differences reported in Figure 4 are maintained independently of the modest differences in cell size observed. We have not included these additional analyses in the revised manuscript, as they do not alter the overall conclusions and may distract from the main message, particularly as the size difference could represent an indirect consequence of CD82 loss. We would, however, be happy to include these data should the editor feel they would strengthen the manuscript. In addition, we have replaced the representative image of the CD82 KO cell with one that more accurately reflects the modest size difference observed.

We have now updated the graphs (visualizing the independent experiments) to be in-line with Lord et al. 2020.

Reviewer #2:

__ __ 2.1 Line 101-102- In MS analysis, Table 1, several common proteins are present in all three bands analyzed by MS including CD82. Authors should explain why CD82 is observed in 250 KDa band in relatively high abundance?

The intensity of CD82 in the 250-kDa band is 30-50x lower than observed in the other two bands (90-100 and 100-110 kDa).

The detection of some CD82 in the 250-kDa band likely originates from partial aggregation due to freeze thawing or temporary storage at 4 degrees. Reducing sample buffer was added to all samples but samples were not boiled and partial aggregation is visible in SDS-PAGE gel.

2.2 Line 118-119, authors should make a better explanation why the lower MW CADM1 band is not observed in co-expression, especially that lower MW band is also absent in CADM1-Strep purification FigS2b? Although the migration pattern of CD82 and CADM1 is similar to glycosylated proteins authors should confirm this band doubling is due to glycosylation. It is crucial because a great portion of the manuscript relies on protein separation on SEC that can be greatly impacted by glycosylation state of the proteins.

A lower band was observed in the SDS-PAGE gel in fig. S2b (Now Fig. S3b). We now clearly indicate the double band with a bracket instead of the single arrow.

Regarding the N-glycosylation of CD82, Wang et al. (https://doi.org/10.1016/j.jprot.2011.11.013) demonstrate band shifts of the upper band of CD82 by side-directed mutagenesis.

In case of CADM1, we tried to validate the bands with additional experiments not included in the manuscript. If the reviewer finds it necessary this figure can be included. We demonstrated that at least the upper band at 90 kDa purified with CD82-gfp-strep shifts to 45 kDa, the expected MW of CADM1, when mutating all N-glycosylation sites to Glu. However, this mutant showed low expression and low stability. To further confirm that the double-band pattern is caused by glycosylation, we treated CADM1(ECD) and CADM1(F42S, ECD) with O-glycosidase, neuroaminidase and PNGaseF. The SDS-PAGE gels show a double band before and a single band after digestion.

With respect to N-glycan variants in SEC:

We often observed narrow double peaks for CD82-strep purifications (Fig. S6A, S8B,G), likely caused by the two N-glycosylation variants. However, when fused to eGFP, we can no longer distinguish the two states on SEC (although we still see a double band on SDS-PAGE gel). Consistently, we observed single peaks for different constructs of CADM1 in SEC, while observing double bands on SDS-PAGE gel. Likely, the two variants migrate very close to each other and can only be separated when the added MW of glycans is relatively high. We therefore don’t think that the large shifts of peaks for CADM1 or CADM1-CD82 complex can be caused by N-glycosylation.

2.3 Line 120-considering the lower yield of CADM1 in co-expression studies and presence of CD82 protein in MS analysis of band 3, it raises the question whether the peak eluted at 10 mL in F-SEC chromatogram of co-expressed proteins is from a legit CD82-CADM1 complex or it is simply CD82 oligomerization/interaction with other proteins. I would suggest authors purify GFP-CD82 and CADM individually and run the mixture over F-SEC to confirm complex formation rather than running co-purified protein. The same experiment should be done with CD82-CADM1mixture without fusing GFP to rule out the possible influence of GFP on dimerization.

The detection of CD82 in the 250 kDa is likely due to aggregation (see 2.1).

Indeed, based on SEC results resulting from our small-scale expressions (Fig. S2E) we cannot conclude the composition of the 10 mL peak. In our large-scale experiment, we put single fractions of the SEC on gel and only see bands of CADM1 and CD82 for earlier peaks (Fig.1A-B). From this, we conclude the 12.5 mL peak in fig. 1A is composed of CADM1 and CD82.

Mixing two detergent-solubilized membrane proteins to analyze complex formation is not straight forward due to the possible interference of micelles.

2.4 Figure 3. The color scheme used makes it really difficult to follow curves for different concentrations. I would suggest using distinct colors for each concentration.

We changed the colors of graphs showing different concentrations in figure 2, figure 3, S7 and S8 to a better distinguishable color palette.

2.5 274-275. Authors argue that the CADM clusters occupy more surface area in CD82KO cells compared to normal cells. From figure 4C. it seems that CD82KO cells are smaller in size than normal cells? Is that true? If that's the case, authors should provide evidence that the expression/surface area is the same for both normal and CD82KO cells before making the conclusion. If the expression level remains the same between two cell types but the size of KO cell is smaller, it is expected that CADM will occupy more surface area on KO cells. Consequently, the clusters are expected to be bigger. Therefore, the smaller cluster size in normal cells is not directly caused by CD82 but by smaller cell size due to CD82KO.

With regard to the size difference, this may indeed represent a phenotype associated with CD82 knockout. A modest difference in cell size was observed, although this did not reach statistical significance (Reviewer Fig. A). As all quantitative analyses in Figure 4 were performed at the cell surface, cell volume was not a parameter used in the primary analysis. To provide a more complete visualisation of the cellular context, we additionally assessed 3D cell dimensions and used these measurements to examine whether the observed clustering phenotype was influenced by cell size. Restricting the analysis to cells with a Z-projected area of 100-150 µm² yielded the same trends in percentage space covered (Reviewer Fig. B), cluster density (Reviewer Fig. C), and cluster size (Reviewer Fig. D). These findings support the conclusion that the differences reported in Figure 4 are maintained independently of the modest differences in cell size observed. We have not included these additional analyses in the revised manuscript, as they do not alter the overall conclusions and may distract from the main message, particularly as the size difference could represent an indirect consequence of CD82 loss. We would, however, be happy to include these data should the editor feel they would strengthen the manuscript. In addition, we have replaced the representative image of the CD82 KO cell with one that more accurately reflects the modest size difference observed.

2.6 Line 205-207. Authors show that CADM1(ECD) is not interacting with CD82 and conclude that interaction between transmembrane helices is required. It raises the question that how in cryoEM, authors could capture interaction between Ig domain of CADM1 and CD82 between two different micelles? Providing such details about experimental setup helps readers to adopt the method for structural determination of elusive complexes.

For the cryo EM sample a complex of full-length CADM1 and CD82 was purified. The 2D classes we observe showing two connected micelles are likely caused by interaction of the CADM1 ectodomain, forming a complex of two dimers of CADM1 and CD82 in one micelle (CD82-CADM1)—(CADM1-CD82).

Reviewer #3:

Major comments:

3.1 In Figure 1, the second co-purification is much less efficient than the first (1C vs 1A). Perhaps removing Figure 1C and the associated SEC trace (or moving to the supplement) would make for an easier-to-understand figure panel. I was initially confused about the low efficiency, and it raised some doubts while I was first interpreting this figure. The large-scale purification in 1F and 1G looks much more convincing than what is shown in 1C.

We moved all small-scale experiments into a supplemental figure (S2) and truncated the text where possible to leave the main focus on the large-scale purification.

3.2 I found the experiments in Figure 2 very hard to follow, and the accompanying text needs improvement before publication. Why was CADM1 ECD rather than full-length used for SEC-MALS? It seems like the authors are trying to claim that CD82 reduces CADM1 oligomerization based on a smaller shoulder peak at ~10-12 mL with the fusion construct compared to CADM1 alone. However, it looks like 10 mL is the void volume of this column- wouldn't another explanation be the CADM1 sample simply has a higher percentage of misfolded or aggregated protein, and it not necessarily an "oligomer"?

We modified the text describing the results in Fig. 2 to improve readability.

Performing SEC-MALLS with membrane proteins is not straight forward, due to the presence of empty micelles causing an unstable baseline and the possibility that micelles fuse or separate. Therefore, we only performed SEC-MALLS with soluble CADM1(ECD) to have a clear readout.

The void volume of the column is at ca. 8.5 mL EV. The void peak is indeed barely visible as we use the protein directly after the SEC for purification without concentrating. In the SECs for protein purification the void peak at 8.5 mL is more visible (Fig. S3B,D). The same column has been used for both experiments connected to different systems (AKTA and HPLC).

Further, we think the 10 mL peaks are complexes instead of aggregation because we see a shift to higher EV after dilution during the SEC after reinjection as expected for concentration dependent complex formation.

3.3 The liposome assay is a clever way to assess if CD82 alters CADM1 oligomerization. However, details of how this assay was performed need clarification, and I am not convinced of the authors' conclusions given the current presentation. In the S4A, the authors show that CADM1 ECD His does not co-purify with CD82, but then CADM1 ECD His was used in liposome experiments to assess how CD82 changes clustering. Why was full-length CADM1 not used? For quantification, the average liposome radius is reported, but to me, a more robust quantification would be the percentage of liposome area contacting another liposome (i.e., ~35 nm distance between two liposomes), if the authors are trying to assess how CD82 affects CADM1 clustering. How does the average liposome radius reflect CADM1 clustering?

Reconstituting both membrane proteins in full-length would indeed be the more native approach. However, we chose to anchor CADM1(ECD)-his to the surface of the liposome for several practical reasons. We used CADM1(ECD)-his because it gave us the possibility to “titrate” different CADM1 concentration against one sample of reconstituted liposomes. We cannot estimate how much CD82 is successfully reconstituted into the liposome. When we reconstitute full length CADM1 mixed with CD82 it was difficult to validate if both proteins and in which ratio they are reconstituted.

We were trying to use cryo-EM pictures to quantify cluster sizes using the grey gradient but this proved difficult even using a single grid with consistent imaging settings because of the ice gradients over the grid and ice contamination. Observed clusters are 3-dimensional and we can only observe single liposomes at the very edge of it.

The clusters we observed when looking and reconstituted liposomes looked very different from non-reconstituted liposomes. We added examples to figure S5. The clusters are overall less dense and liposomes cluster but not with a consistent distance as we observe it for WT CADM1.

The DLS reads out the particle sizes and shows two peaks at 100 and 300 nm after adding CADM1(ECD)-His. We interpret this as CADM1 connecting micelles as observed on cryoEM images and therefore causing an increase in average particle size.

3.4 The AlphaFold model shown in main text Figure 5 is not high confidence, and I suggest moving this to the supplement. To validate that the LEL of CD82 interacts with Ig1 of CADM1 as suggested by the EM density, the authors should perform mutagenesis or a domain swap with a different tetraspanin, and also binding assays with Ig1, Ig2, and Ig3 of CADM1 to validate this claim in the absence of high-resolution structural data.

We removed the AlphaFold predictions from figure 5 and added it to the other predictions in Fig. S13. We did not see interactions of soluble CADM1(ECD) (Fig. S12) or single Ig domains (not included in MS) with purified CD82. Instead, we added a domain-truncation study in Fig. 5. The study shows the same level of co-purifications of CD82-StrepII3 and CADM1-His, CADM1ΔIg1 and CADM1Δ1-2 but strongly reduced co-purification when deleting all 3 Ig like domains. No CADM1-His constructs were detected in absence of CD82-StrepII3 and expression controls are shown in Fig. S12. The additional data made the overall interaction mechanism more clear and we rephrased it in the discussion.

3.5 The EM processing workflow figure (S10) is missing many important details. FSC curve needs to be shown for the final map, and details like what was used for motion correction, CTF estimation, and how 31k particles became 45k particles (were these classes used as templates for picking?), and how many classes were used in heterogeneous refinement (and what those volumes looked like) should be included.

We created a new processing figure. The figure includes the FSC curve and particle distribution for the finale volume. We added the first ab initio and heterogeneous refinement steps for each round of particle picking and marked volumes of which particles were selected for further processing. We indicated the number of selected and total particles for the final 2D classes and then indicated which group of particles was used and the percentage of particles in the resulting 3D volumes instead of indicating the particle number for each volume for better overview. We added what jobs were used for motion correction and CTF estimation.

3.6 A short model figure at the end of the manuscript would be helpful to summarize the authors' findings.

We added a figure displaying how CD82 hinders the tight formation of CADM1 multimers between membranes as Fig. 6.

Minor comments:

__ __3.7 Line 67: "In cancer, CADM1 can function as an oncogene as well as a tumor suppressor by regulating different signaling cascades, such as the Hippo pathway that promotes cell proliferation [27]." I found this sentence confusing- I think it is worth including another sentence or two explaining how CADM1 can have both tumor suppressor or promoter effects for readers that aren't familiar with CADM1 biology.

We expended this part of the introduction to better explain the different functions of CADM1 and how it acts as a tumor suppressor and promotor simultaneously using examples:

“Cell adhesion molecule 1 (CADM1) is a widely expressed adhesion molecule that has a dual function in cell signaling and adhesion and is involved in several biological functions. CADM1 overexpression promotes recruitment of T cells or mast cells in autoinflammatory diseases, like type 1 diabetes, asthma or neuroinflammatory diseases [24, 25, 26]. In most cancers, low CADM1 expression is associated with more aggressive tumors as it promotes apoptosis and inhibits proliferation [27].However, in T-cell lymphoma high CADM1 expression is associated with poor outcomes as its overexpression can lead to increased organ infiltration of tumors. The role of CADM1 in signaling and adhesion might explain its dual association with poor or good cancer prognosis and in both cases the change in expression level makes it a suitable therapeutic target and a biomarker [28, 29]. First in vivo studies using antibody-drug conjugates or chimeric-antigen receptors show a specific response against cancer cells and are considered promising candidates for clinical trials [30, 31, 32]. Considering these recent successes, it is crucial to understand the different functions and regulation of CADM1.”

3.8 The introduction spends time discussing glycosylation sites on CD82 and CADM1. When I first read the intro, I thought glycosylation would be a focus of some of the experiments, but it wasn't. Instead, I think the paper would be stronger if you spent a bit more time in the intro talking about what is known about CADM1- as some examples, are there other interaction partners known to regulate its oligomerization? Therapeutic targeting is briefly mentioned, but has there been any success or progress in that area?

The glycosylation explains why we consistently observe double bands in our SDS-PAGE gels and therefore needs to be mentioned in the introduction.

We added more information about CADM1 as a cancer therapy target and recent developments in the introduction (see 3.6).

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Tetraspanin CD82 reduces the formation of CADM1 oligomers Lamottke et al. present data identifying CADM1 as a partner of the tetraspanin CD82. They then characterize this interaction using binding experiments, liposome assays, microscopy, and single-particle cryo-EM. Overall, I found this work to be an interesting read, and in general, the experiments were well explained and well controlled. Overall, nice work and an interesting finding! Below are my comments for improvements:

Major comments:

• In Figure 1, the second co-purification is much less efficient than the first (1C vs 1A). Perhaps removing Figure 1C and the associated SEC trace (or moving to the supplement) would make for an easier-to-understand figure panel. I was initially confused about the low efficiency, and it raised some doubts while I was first interpreting this figure. The large-scale purification in 1F and 1G looks much more convincing than what is shown in 1C
• I found the experiments in Figure 2 very hard to follow, and the accompanying text needs improvement before publication. Why was CADM1 ECD rather than full-length used for SEC-MALS? It seems like the authors are trying to claim that CD82 reduces CADM1 oligomerization based on a smaller shoulder peak at ~10-12 mL with the fusion construct compared to CADM1 alone. However, it looks like 10 mL is the void volume of this column- wouldn't another explanation be the CADM1 sample simply has a higher percentage of misfolded or aggregated protein, and it not necessarily an "oligomer"?
• The liposome assay is a clever way to assess if CD82 alters CADM1 oligomerization. However, details of how this assay was performed need clarification, and I am not convinced of the authors' conclusions given the current presentation. In the S4A, the authors show that CADM1 ECD His does not co-purify with CD82, but then CADM1 ECD His was used in liposome experiments to assess how CD82 changes clustering. Why was full-length CADM1 not used? For quantification, the average liposome radius is reported, but to me, a more robust quantification would be the percentage of liposome area contacting another liposome (i.e., ~35 nm distance between two liposomes), if the authors are trying to assess how CD82 affects CADM1 clustering. How does the average liposome radius reflect CADM1 clustering?
• The AlphaFold model shown in main text Figure 5 is not high confidence, and I suggest moving this to the supplement. To validate that the LEL of CD82 interacts with Ig1 of CADM1 as suggested by the EM density, the authors should perform mutagenesis or a domain swap with a different tetraspanin, and also binding assays with Ig1, Ig2, and Ig3 of CADM1 to validate this claim in the absence of high-resolution structural data.
• The EM processing workflow figure (S10) is missing many important details. FSC curve needs to be shown for the final map, and details like what was used for motion correction, CTF estimation, and how 31k particles became 45k particles (were these classes used as templates for picking?), and how many classes were used in heterogeneous refinement (and what those volumes looked like) should be included.
• A short model figure at the end of the manuscript would be helpful to summarize the authors' findings.

Minor comments:

• Line 67: "In cancer, CADM1 can function as an oncogene as well as a tumor suppressor by regulating different signaling cascades, such as the Hippo pathway that promotes cell proliferation [27]." I found this sentence confusing- I think it is worth including another sentence or two explaining how CADM1 can have both tumor suppressor or promoter effects for readers that aren't familiar with CADM1 biology
• The introduction spends time discussing glycosylation sites on CD82 and CADM1. When I first read the intro, I thought glycosylation would be a focus of some of the experiments, but it wasn't. Instead, I think the paper would be stronger if you spent a bit more time in the intro talking about what is known about CADM1- as some examples, are there other interaction partners known to regulate its oligomerization? Therapeutic targeting is briefly mentioned, but has there been any success or progress in that area?

Significance

This work reports a new direction interaction between CADM1 and CD82, which is interesting and signficant because relatively few direct interactions between tetraspanins and their partners are well characterized. The manuscript can be improved by relating this new interaction discovery back to how this may help CADM1 biology or developing new agents targeting CADM1, which I mention above in my comments.

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 manuscript by Lamottke et al. characterizes the interaction of CD82 with CADM1 protein. Although, the data provides important insight into how CD82 regulates CADM1 clustering, I have few questions that needs to be addressed to improve the manuscript for readers before acceptance of the paper.

Line 101-102- In MS analysis, Table 1, several common proteins are present in all three bands analyzed by MS including CD82. Authors should explain why CD82 is observed in 250 KDa band in relatively high abundance?

Line 118-119, authors should make a better explanation why the lower MW CADM1 band is not observed in co-expression, especially that lower MW band is also absent in CADM1-Strep purification FigS2b? Although the migration pattern of CD82 and CADM1 is similar to glycosylated proteins authors should confirm this band doubling is due to glycosylation. It is crucial because a great portion of the manuscript relies on protein separation on SEC that can be greatly impacted by glycosylation state of the proteins.

Line 120-considering the lower yield of CADM1 in co-expression studies and presence of CD82 protein in MS analysis of band 3, it raises the question whether the peak eluted at 10 mL in F-SEC chromatogram of co-expressed proteins is from a legit CD82-CADM1 complex or it is simply CD82 oligomerization/interaction with other proteins. I would suggest authors purify GFP-CD82 and CADM individually and run the mixture over F-SEC to confirm complex formation rather than running co-purified protein. The same experiment should be done with CD820-CADM1mixture without fusing GFP to rule out the possible influence of GFP on dimerization.

Figure 3. The color scheme used makes it really difficult to follow curves for different concentrations. I would suggest using distinct colors for each concentration.

Line 274-275. Authors argue that the CADM clusters occupy more surface area in CD82KO cells compared to normal cells. From figure 4C. it seems that CD82KO cells are smaller in size than normal cells? Is that true? If that's the case, authors should provide evidence that the expression/surface area is the same for both normal and CD82KO cells before making the conclusion. If the expression level remains the same between two cell types but the size of KO cell is smaller, it is expected that CADM will occupy more surface area on KO cells. Consequently, the clusters are expected to be bigger. Therefore, the smaller cluster size in normal cells is not directly caused by CD82 but by smaller cell size due to CD82KO.

Line 205-207. Authors show that CADM1(ECD) is not interacting with CD82 and conclude that interaction between transmembrane helices is required. It raises the question that how in cryoEM, authors could capture interaction between Ig domain of CADM1 and CD82 between two different micelles? Providing such details about experimental setup helps readers to adopt the method for structural determination of elusive complexes.

Significance

This solid study provides new insight into the interaction between CD82 and CADM1, how self-assembly and clustering is regulated using a complemenatry biochemical approaches. While the structural details remain wanting, the sound biochemical work lends confidence in the study's findings.

The greater community interested in tetraspanin biochemistry will appreciate this work.

We have experience understanding related plasma membrane remodeling proteins (from the Prom family) and their interactions with other binding partners.

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: In this study, the authors identify cell adhesion molecule 1 (CADM1) as an interaction partner of tetraspanin CD82. They used a combination of biochemical, cellular and structural studies to support this conclusion and identify an inhibitory effect of CD82 on CADM1 oligomerization.

Major Comments: Using a variety of elegant and sophisticated studies, the authors clearly present data that strongly support their conclusions of the interaction between CADM1 and CD82. Additionally, they speculate on the specific interaction sites/mechanism through which CD82 may affect the spacing of CADM1, which is supported by cryo EM. However, they do not go on to demonstrate a physiological role for this inhibitory interaction.

Minor Comments: In Figure 4, there seems to be a significant size difference between WT and CD82KO Jurkat cells. If this is the case, this needs to be described a bit more. Additionally, how does this potential size difference impact the quantification of the percentage space covered by CADM1 clusters? Also, for these experiments, it is more appropriate to use the mean of each independent replicate rather than simply counting each cell as an n. Including these data as super plots so the reader can better understand the spread and reproducibility of the data would be ideal. Best practices suggest that statistical analysis should be performed on data from independent experiments (Lord et al 2020 PMID: 32346721).

Significance

General comments useful to editors and readers: This is a well written manuscript with compelling data that supports the overall conclusion of a protein-protein interaction between CADM1 and CD82. The abstract could be strengthened by the addition of a bit more biological rationale for the presented work and some context of impact.

Advance: Through compelling structural work, the authors establish an interaction between CADM1 and CD82 and suggest based on the cryo-EM that the interaction occurs between the CD82(LEL) and an Ig-like domain of CADM1. The manuscript does not provide a physiological impact of the identified interaction, as this was suggested to be outside the scope of the current work. As such this limits the overall advance in knowledge some to a thorough description of a protein-protein interaction that impacts protein clustering.

Audience: This is largely a structural study evaluating protein-protein interactions and the impact of a membrane scaffold protein on the clustering of a cell adhesion molecule. Thus, it is well suited for structural biologists and cell biologists studying cell adhesion molecules, membrane scaffold proteins and their regulation.

Describe your expertise: My expertise is in cell biology, so admittedly, some of the structural data was difficult from me to critically evaluate.

They write a prologue saying Pyramus isn't really dead and Bottom is just Bottom. They're deleting the play so the ladies don't get scared by the play.

Bottom says make it eight and eight. He has no idea what that changes. He just wants more.

Helena hears a real declaration as mockery. She's the only one confused without a flower.

clothes. Robin picks the wrong man off an outfit. Oberon said find an Athenian and never said which one, so this is partly on him.

lie down / tell a lie. He's punning his way closer to her. She says he "riddles very prettily," which means she caught it.

"Ounce" = lynx, "pard" = leopard. Oberon is picturing a predator. He gets a weaver with a donkey head.

"Reremice" = bats. Titania's first order is send someone to skin bats for fairy coats. Not as gentle a scene as it looks.

Question 16: D (The tiny landlocked area next to the Jordan River)Question 17: B (The easternmost country on this map, bordering Iran)Question 18: D (The large northern country above the Mediterranean Sea)💧 Water & Geography (Questions 19–24)Question 19: a. When harmful things are dumped into rivers, lakes, or oceansQuestion 20: b. It can cause diseases and health problemsQuestion 21: c. It can harm plants and make food unsafeQuestion 22: b. Some people have plenty of water, others have little or noneQuestion 23: b. They don’t have easy access to clean water nearbyQuestion 24: c. They struggle to grow crops due to lack of water🏜️ Climate & Rivers (Questions 25–30)Question 25: a. Because it’s hot and there is very little waterQuestion 26: c. They use camels to carry goodsQuestion 27: Trucks and airplanes make it easier to move goods (First choice)Question 28: They provide fresh water for drinking and farming (Third choice)Question 29: Near rivers (Third choice)Question 30: Boats can carry goods up and down the river (First choice)📊 Economic Systems (Questions 31–35)Question 31: The government (Second choice)Question 32: People who have money buy what they need or want (Fourth choice)Question 33: A system that combines market and command features (Second choice)Question 34: Israel (Second choice)Question 35: The government controls most of the economy, especially oil (First choice)🤝 Trade, OPEC, & Human Capital (Questions 36–42)Question 36: A country focuses on making one or a few products really well (First choice)Question 37: It helps countries trade for what they need and use resources wisely (Third choice)Question 38: To control how much oil is produced and keep prices stable (Third choice)Question 39: Better jobs and higher standard of living (First choice)Question 40: How comfortable and healthy people’s lives are (Second choice)Question 41: The total value of goods and services a country produces (Second choice)Question 42: It improves skills, raises income, and grows the economy (Third choice)

Question 16: D (The tiny landlocked area next to the Jordan River)Question 17: B (The easternmost country on this map, bordering Iran)Question 18: D (The large northern country above the Mediterranean Sea)💧 Water & Geography (Questions 19–24)Question 19: a. When harmful things are dumped into rivers, lakes, or oceansQuestion 20: b. It can cause diseases and health problemsQuestion 21: c. It can harm plants and make food unsafeQuestion 22: b. Some people have plenty of water, others have little or noneQuestion 23: b. They don’t have easy access to clean water nearbyQuestion 24: c. They struggle to grow crops due to lack of water🏜️ Climate & Rivers (Questions 25–30)Question 25: a. Because it’s hot and there is very little waterQuestion 26: c. They use camels to carry goodsQuestion 27: Trucks and airplanes make it easier to move goods (First choice)Question 28: They provide fresh water for drinking and farming (Third choice)Question 29: Near rivers (Third choice)Question 30: Boats can carry goods up and down the river (First choice)📊 Economic Systems (Questions 31–35)Question 31: The government (Second choice)Question 32: People who have money buy what they need or want (Fourth choice)Question 33: A system that combines market and command features (Second choice)Question 34: Israel (Second choice)Question 35: The government controls most of the economy, especially oil (First choice)🤝 Trade, OPEC, & Human Capital (Questions 36–42)Question 36: A country focuses on making one or a few products really well (First choice)Question 37: It helps countries trade for what they need and use resources wisely (Third choice)Question 38: To control how much oil is produced and keep prices stable (Third choice)Question 39: Better jobs and higher standard of living (First choice)Question 40: How comfortable and healthy people’s lives are (Second choice)Question 41: The total value of goods and services a country produces (Second choice)Question 42: It improves skills, raises income, and grows the economy (Third choice)

1.b

💻/asus/🧊/me/📓/2026/9/🔍/-/fission.codes-did

💻/asus/🧊/me/📓/2026/9/🔍/-/fission.codes-did

any/of any kind

careful

smoothly

collaborative, assistant, subsidiary

to give one’s hand

m, -s apprentice, trainee

to suffer, incur, sustain

eLife Assessment

This important study presents a convincing methodological approach to probe the structural features of the full-length human Hv1 channel as a purified protein. The method is supported by rigorous biochemical assays and spectral FRET analysis, which will interest biophysicists and physiologists studying Hv1 and other ion channels and membrane proteins. Overall, the work introduces an interesting labeling strategy and provides a methodology that is of value in investigating hHV1 in particular and can be extended to other ion channels. The authors also provide preliminary observations regarding conformational changes induced by zinc.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review, shown below.]

In this study, the noncanonical amino acid acridon-2-ylalanine (Acd) was inserted at various positions within the human Hv1 protein using a genetic code expansion approach. The purified mutants with incorporated fluorophore were shown to be functional using a proton flux assay in proteoliposomes. FRET between native tryptophan and tyrosine residues and Acd were quantified using spectral FRET analysis. Predicted FRET efficiencies calculated from an AlphaFold model of the Hv1 dimer were compared to the corresponding experimental values. Spectral FRET analysis was also used to test whether structural rearrangements caused by Zn2+, a well-known Hv1 inhibitor, could be detected. The experimental data provide a good validation of the approach, but further expansion of the analysis will be necessary to differentiate between intra- and intersubunit structural features.

Interestingly, the observed rearrangements induced by Zn2+ were not limited to the protein region proximal to the extracellular binding site but extended to the intracellular side of the channel. This finding agrees with previous studies showing that some extracellular Hv1 inhibitors, such as Zn2+ or AGAP/W38F, can cause long-range structural changes propagating to the intracellular vestibule of the channel (De La Rosa et al. J. Gen. Physiol. 2018, and Tang et al. Brit J. Pharm 2020). The authors should consider adding these references.

Since one of the main goals of this work was to validate Acd incorporation and the spectral FRET analysis approach to detect conformational changes in hHv1 in preparation for future studies, the authors should consider removing one subunit from their dimer model, recalculating FRET efficiencies for the monomer, and comparing the predicted values to the experimental FRET data. This comparison could support the idea that the reported FRET measurements can inform not only on intrasubunit structural features but also on subunit organization.

Reviewer #2 (Public review):

This manuscript by Carmona, Zagotta, and Gordon is generally well-written. It presents a crude and incomplete structural analysis of the voltage-gated proton channel based on measured FRET distances. The primary experimental approach is Förster Resonance Energy Transfer (FRET), using a fluorescent probe attached to a noncanonical amino acid. This strategy is advantageous because the noncanonical amino acid likely occupies less space than conventional labels, allowing more effective incorporation into the channel structure.

Fourteen individual positions within the channel were mutated for site-specific labeling, twelve of which yielded functional protein expression. These twelve labeling sites span discrete regions of the channel, including P1, P2, S0, S1, S2, S3, S4, and the dimer-connecting coiled-coil domain. FRET measurements are achieved using acridon-2-ylalanine (Acd) as the acceptor, with four tryptophan or four tyrosine residues per monomer serving as donors. In addition to estimating distances from FRET efficiency, the authors analyze full FRET spectra and investigate fluorescence lifetimes on the nanosecond timescale.

Despite these strengths, the manuscript does not provide a clear explanation of how channel structure changes during gating. While a discrepancy between AlphaFold structural predictions and the experimental measurements is noted, it remains unclear whether this mismatch arises from limitations of the model or from the experimental approach. No further structural analysis is presented to resolve this issue or to clarify the conformational states of the protein.

The manuscript successfully demonstrates that Acd can be incorporated at specific positions without abolishing channel function, and it is noteworthy that the reconstituted proteins function as voltage-activated proton channels in liposomes. The authors also report reversible zinc inhibition of the channel, suggesting that zinc induces structural changes in certain channel regions that can be reversed by EDTA chelation. However, this observation is not explored in sufficient depth to yield meaningful mechanistic insight.

Overall, while the study introduces an interesting labeling strategy and provides valuable methodological observations, the analysis appears incomplete. Additional structural interpretation and mechanistic insight are needed.

Author response:

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

We greatly appreciate both reviewers for their insightful comments. As noted by the reviewers and editors, this work validates a new approach to study conformational dynamics of full-length hH_v1 using acridon-2-ylalanine (Acd), including its advantages and limitations for future structural studies. Accordingly, we have changed the manuscript to “Tools and Resources”. The incorporation of the reviewers’ suggestions has greatly improved the quality of the updated manuscript.

Public Reviews:

Reviewer #1 (Public review):

We thank Reviewer #1 for the feedback and comments. We addressed the points the reviewer raised below.

Interestingly, the observed rearrangements induced by Zn2+ were not limited to the protein region proximal to the extracellular binding site but extended to the intracellular side of the channel. This finding agrees with previous studies showing that some extracellular Hv1 inhibitors, such as Zn2+ or AGAP/W38F, can cause long-range structural changes propagating to the intracellular vestibule of the channel (De La Rosa et al. J. Gen. Physiol. 2018, and Tang et al. Brit J. Pharm 2020). The authors should consider adding these references.

We added the suggested references to the Results section.

Since one of the main goals of this work was to validate Acd incorporation and the spectral FRET analysis approach to detect conformational changes in hHv1 in preparation for future studies, the authors should consider removing one subunit from their dimer model, recalculating FRET efficiencies for the monomer, and comparing the predicted values to the experimental FRET data. This comparison could support the idea that the reported FRET measurements can inform not only on intrasubunit structural features but also on subunit organization.

We calculated the predicted intrasubunit FRET efficiency and presented the results in the new Figure S10. Pearson’s coefficient decreased from 0.48 for the dimer to 0.18 for the monomer, suggesting the experimental FRET contains information about subunit organization. This was added to the text.

Reviewer #2 (Public review):

We appreciate the detailed revision, comments, and feedback from Reviewer #2. We addressed the reviewer’s major and minor points below, which we believe improve the manuscript.

(1) Tryptophan and tyrosine exhibit similar quantum yields, but their extinction coefficients differ substantially. Is this difference accounted for in your FRET analysis? Please clarify whether this would result in a stronger weighting of tryptophan compared to tyrosine.

We accounted for differences in the extinction coefficients of Trp and Tyr in our calculations, which are detailed in the Supplementary Text. The assumptions result in a stronger contribution from Trp than from Tyr.

(2) Is the fluorescence of acridon-2-ylalanine (Acd) pH-dependent? If so, could local pH variations within the channel environment influence the probe's photophysical properties and affect the measurements?

The acridone fluorescence, which is the fluorophore in Acd, is not pH-dependent between pH 2 and 9 (Stephen G.S. and Sturgeon R.J. Analytica Chimica Acta. 1977). This was added to the text.

(3) Several constructs (e.g., K125Tag, Y134Tag, I217Tag, and Q233Tag) display two bands on SDS-PAGE rather than a single band. Could this indicate incomplete translation or premature termination at the introduced tag site? Please clarify.

Yes, the additional bands in the WB are due to the termination of translation for the mentioned protein constructs. We added a note in the legend of Figure 2 regarding this point.

(4) In Figure 5F, the comparison between predicted FRET values and experimentally determined ratio values appears largely uninformative. The discussion on page 9 suggests either an inaccurate structural model or insufficient quantification of protein dynamics. If the underlying cause cannot be distinguished, how do the authors propose to improve the structural model of hHv1 or better describe its conformational dynamics?

We understand the confusion about this point. We are not planning to improve the structural model with FRET between Trp/Tyr and Acd. We modified the text to avoid confusion regarding this point. We plan to use Acd as a transition metal ion FRET (tmFRET) donor to study the conformational dynamics of hHv1 in the future (Discussion).

(5) Cu²⁺, Ru²⁺ and Ni²⁺, are presented as suitable FRET acceptors for Acd. Would Zn²⁺ also be expected to function as an acceptor in this context? If so, could structural information be derived from zinc binding independently of Trp/Tyr?

Transition metal ion FRET (tmFRET) uses a fluorophore as the donor and a transition metal ion chelator as the acceptor. For FRET to occur between these donor-acceptor pairs, the fluorescence spectrum of the donor must overlap the absorption spectrum of the metal ion (Zagotta et al., eLife. 2021; Zagotta et al., Biophys J. 2024; Gordon et al., Biophys J. 2024). Zn²⁺ does not absorb visible light, so tmFRET cannot occur for this divalent metal.

(6) The investigated structure is most likely dimeric. Previous studies report that zinc stabilizes interactions between hHv1 monomers more strongly than in the native dimeric state. Could this provide an explanation for the observed zinc-dependent effects? Additionally, do the detergent micelles used in this study predominantly contain monomers or dimers?

Our full-length hH_v1 in Anz3-12 detergent micelles is predominantly a dimer, as demonstrated in the new panel of Figure S5. From our data, we cannot compare the effects of zinc between monomers and dimers.

(7) hHv1 normally inserts into a phospholipid bilayer, as used in the reconstitution experiments. In contrast, detergent micelles may form monolayers rather than bilayers. Could the authors clarify the nature of the micelles used and discuss whether the protein is expected to adopt the same fold in a monolayer environment as in a bilayer?

We used Anzergent 3-12 detergent micelles, which stabilize hH_v1 in solution. We indicated this in the Results and Materials and Methods sections. We are also intrigued by whether protein folding and conformational dynamics differ between detergent micelles and proteoliposomes, but our data do not provide an answer to this question. We found that the proteoliposomes used for measuring the hHv1 function don’t have enough Acd signals to record their spectra, preventing us from performing the same FRET measurements between Trp/Tyr and Acd in liposomes. Still, detergent-solubilized hH_v1 is functional upon reconstitution, demonstrating that its functional folding is not irreversibly altered in micelles.

Recommendations for the Authors:

Reviewer #2 (Recommendations for the authors):

(1) On page 9, the reference to Figure S11 should be corrected to Figure S10.

We thank the reviewer for catching this mistake. It was corrected in the updated version.

(2) On page 9, multiple prior studies describing zinc binding to hHv1 should be acknowledged, for example:

Musset et al. (2010), J. Physiol., 588, 1435-1449; Jardin et al. (2020), Biophys. J., 118, 1221-1233.

References were added to the text.

(3) On page 11, the statement "with Acd incorporated ... we can interrogate its gating mechanism in unprecedented detail" appears overly strong relative to the data presented. Another phrasing might be appropriate.

The sentence was changed. It now reads: “With Acd incorporated at multiple sites in full-length hH_v1, it will be possible to interrogate conformational changes across the protein’s different structural domains using Acd as a tmFRET donor to understand its molecular mechanisms.”

A good example to study, fairly simple calculus.

eLife Assessment

This important study compares how different classes of drugs act on the SARS-CoV-2 main protease, a key antiviral target, and shows that many of them work by controlling whether the enzyme assembles into its active dimeric form. The evidence, based on a range of complementary biophysical methods, is convincing and points to the interface between the two protein protomers, including a newly found binding site, as a promising target for broad-spectrum antiviral drugs. This work will be of interest to biochemists and virologists working on treatments for coronaviruses.

Reviewer #1 (Public review):

Summary:

Since dimerization is essential for SARS-CoV-2 Mpro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the Mpro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how Mpro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.

Strengths:

This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.

Weaknesses:

None. The requested mutagenesis data have been provided in the revised manuscript, and all of my previous concerns have been satisfactorily addressed.

Reviewer #2 (Public review):

Summary:

This manuscript presents a sophisticated investigation into the mechanisms by which different inhibitor classes affect the SARS-CoV-2 main protease (Mpro), a pivotal antiviral drug target. This study reveals that effective inhibition can be achieved by modulating the stabilization of the essential dimeric state. It also indicates the dimer interface could be a druggable allosteric site, which may offer a strategy for developing broad-spectrum anticoronaviral agents.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

Comments on revised version:

The authors have very nicely addressed most of the previous comments raised. But one comment remains to be clarified relating to original point 5 and the authors' response:

"We agree with the reviewer about the need for quantitative rigor in reporting HDX changes. We have calculated the fractional deuterium uptake difference for each peptide fragment discussed in the text between the inhibitor-bound and unbound states. These values, along with their statistical significance (p-values from a two-tailed t-test), have been provided in the revised manuscript (Legends for Figures 3 and 4). Although the HDX change of residues 296-306 is relatively small (<5%), this region showed a reproducible difference with low experimental variability and statistical significance (p < 0.05). Given its location within the C-terminal dimerization interface and its consistency with native MS, we interpret this change as a subtle local conformational perturbation."

Two questions remain for the statements in line 376-380. First, while it is stated "residues 296-304 in the C-terminal region of Mpro were more flexible upon ebselen binding", the segment of 296-306 is shown Figure 4c. Second, the HDX change for this segment upon ebselen binding is very subtle in the figure (in contrast to the significant HDX change of the same segment in the protein upon PF-07321332 binding), thus making the strong conclusion that "This suggests that ebselen targeting C300 may induce structural changes in the C-terminal helical segment, weakening key hydrogen bonds at the dimer interface and ultimately inhibiting activity" not convincing. The reviewer would suggest the authors either delete this conclusion or largely tone it down.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Since dimerization is essential for SARS-CoV-2 M^pro enzymatic activity, the authors investigated how different classes of inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, boceprevir), non-peptidomimetic inhibitors (carmofur, ebselen, and its analog MR6-31-2), and allosteric inhibitors (AT7519 and pelitinib), influence the M^pro monomer-dimer equilibrium using native mass spectrometry. Further analyses with isotope labeling, HDX-MS, and MD simulations examined subunit exchange and conformational dynamics. Distinct inhibitory mechanisms were identified: peptidomimetic inhibitors stabilized dimerization and suppressed subunit exchange and structural flexibility, whereas ebselen covalently bound to a newly identified site at C300, disrupting dimerization and increasing conformational dynamics. This study provides detailed mechanistic evidence of how M^pro inhibitors modulate dimerization and structural dynamics. The newly identified covalently binding site C300 represents novelty as a druggable allosteric hotspot.

Strengths:

This manuscript investigates how different classes of inhibitors modulate SARS-CoV-2 main protease dimerization and structural dynamics, and identifies a newly observed covalent binding site for ebselen.

Weaknesses:

The major concern is the absence of mutagenesis data to support the proposed inhibitory mechanisms, particularly regarding the role of the inhibitor binding site.

We thank the reviewer for the recognition and comments. We agree that mutagenesis is critical for validating the proposed role of C300. We therefore generated the C300S and C300F mutants and characterized their oligomeric states and proteolytic activities. C300S was designed to remove the reactive thiol group while minimally affecting M^pro structure and dimerization. C300F was introduced to mimic the steric perturbation associated with C300 modification and assess its impact on M^pro dimerization. Native PAGE showed that WT and C300S M^pro predominantly formed dimers, whereas C300F was mainly monomeric. Consistently, C300S retained approximately 70% of WT activity, whereas C300F retained only approximately 10%. Because C300F itself strongly disrupted dimerization, C300S was used as the principal mutant to evaluate the specific contribution of the C300 thiol to ebselen action. Native MS showed that ebselen could still bind to both monomeric and dimeric C300S M^pro but did not markedly shift its monomer-dimer equilibrium toward the monomeric state. In parallel, ebselen reduced WT activity to approximately 53% of the untreated control, whereas C300S retained approximately 78% activity at the same 1:3 M^pro-to-ebselen molar ratio. These results provide experimental support for the contribution of C300 to ebselen-induced dimer destabilization and functional inhibition, while the residual binding and inhibition observed for C300S suggest the involvement of additional C300-independent interactions. The corresponding revisions have been made to Methods (Lines 627–648), and Results (Lines 397–453) of the manuscript, together with the newly added figures (Figures S14–S16).

Reviewer #2 (Public review):

Summary:

This is a mechanistic study that provides new insights into the inhibition of SARS-CoV-2 M^pro.

Strengths

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

We thank the reviewer for the positive comments and recognition of our study.

Weaknesses:

The primary weaknesses relate to linking the biophysical observations more directly to functional enzymatic outcomes and providing more quantitative rigor in some analyses. While the study is overall strong, addressing its weaknesses and limitations would elevate the impact and translational relevance of the current manuscript.

We thank the reviewer for these comments, which have helped to iM^prove the quality and impact of our manuscript.

(1) Correlation with Functional Activity:

The most significant gap is the lack of direct enzymatic activity assays under the exact conditions used for MS and HDX. While EC50 values are listed from literature, demonstrating how the observed dimer stabilization (by peptidomimetics) or dimer disruption (by ebselen) directly correlates with inhibition of proteolytic activity in the same experimental setup would solidify the functional relevance of the biophysical observations. For instance, does the fraction of monomer measured by native MS quantitatively predict the loss of activity? Also, the single inhibitor concentration used in each MS experiment needs to be specified in the main text and legends. A discussion on whether the inhibitor concentrations required to observe these dimerization effects (in native MS) or structural dynamics (in HDX-MS) align with EC50 values would be helpful for contextualizing the findings.

We thank the reviewer for these important points. To link the biophysical observations more directly to function, we compared the oligomeric states and proteolytic activities of WT, C300S, and C300F M^pro. C300F was predominantly monomeric and retained only approximately 10% of WT activity, whereas C300S remained predominantly dimeric and retained approximately 70% activity. We further evaluated ebselen inhibition using a matched 1:3 M^pro-to-ebselen molar ratio. Ebselen reduced WT activity to approximately 53% of its untreated control but reduced C300S activity only to approximately 78%, demonstrating that removal of the C300 thiol significantly attenuated the functional effect of ebselen. These data support a relationship between C300-dependent dimer destabilization and reduced proteolytic activity. The Methods (Lines 627–648), and Results (Lines 397–453) have been revised accordingly, with Figures S14–S16 newly added, in the revised manuscript. We did not expect a linear relationship between the monomer fraction measured by native MS and enzymatic activity loss, because ebselen can modify multiple cysteine residues, and individual modification events may have distinct effects on M^pro dimerization and catalytic function. The concentrations and molar ratios used in the native MS, HDX-MS, and activity assays have now been stated in the figure legends. The ebselen concentrations used for native MS and HDX-MS were optimized for biophysical characterization and comparison, and therefore, these concentrations might not be directly related to their IC₅₀ or EC₅₀ values. In these experiments, ebselen was applied at a 3-fold molar excess relative to M^pro, consistent with the enzymatic assay. The observed dimer disruption and conformational changes were consistent with functional inhibition, supporting their mechanistic relevance.

(2) For the two Cys residues found to be targeted by ebselen, what are their respective modification stoichiometry related to the ebselen concentration? Especially for the covalent binding site C300, which is proposed in this study to represent a novel allosteric inhibition mechanism of ebselen, more direct experimental evidence is needed to support this major hypothesis. Does mutation or modification of C300 affect the M^pro dimerization/monomer equilibrium and alter the enzymatic activity? If ebselen acts as a covalent inhibitor linked to multiple Cys, why is its activity only in the μM range?

We thank the reviewer for the insightful comments. Our LC-MS/MS data identified C44 and C300 as ebselen-modified residues, but they do not permit reliable site-resolved occupancy measurements because modified and unmodified peptides can differ in digestion efficiency and MS response. We have therefore clarified that these data provide qualitative site identification rather than absolute modification stoichiometry. To obtain direct functional evidence for C300, we generated C300S and C300F mutants. C300S preserved dimer formation and substantial activity, whereas C300F was mainly monomeric and showed severe activity loss. Importantly, although ebselen-bound C300S species were still detected by native MS, ebselen did not markedly redistribute C300S toward the monomeric state, and its inhibition was reduced from approximately 47% for WT to approximately 22% for C300S. These results indicate that C300 is an important contributor to ebselen-induced dimer disruption, while residual binding and inhibition indicate additional reactive sites. Corresponding revisions have been made to the (Lines 627–648), and Results (Lines 397–453) of the manuscript, together with the newly added figures (Figures S14–S16). The moderate micromolar potency of ebselen is consistent with its heterogeneous, multi-site covalent reactivity: modification occupancy and functional consequence are site-dependent, and not every adduct produces complete inhibition.

(3) For the allosteric inhibitor pelitinib with low-μM activity, no significant differences in deuterium uptake of M^pro were observed. In terms of the binding affinity, what is the difference between pelitinib and ebselen? Some explanations could be provided about the different HDX-MS results between the two non-peptidomimetic inhibitors with similar activities.

We agree with the reviewer that the absence of significant HDX changes for pelitinib requires clarification. Different from ebselen that forms covalent bond with multiple cysteine residues of M^pro, which could lead to sustained conformational changes that are more readily detected by HDX-MS, pelitinib non-covalently binds M^pro and might not induce significant perturbations in backbone dynamics that are detectable at the peptide level by HDX-MS. These points have been integrated into the revised manuscript (Lines 333-337).

(4) Native MS Quantification: 

The analysis of monomer-dimer ratios from native MS spectra appears qualitative or semi-quantitative. A more rigorous and quantified analysis of the percentage of dimer/monomer species under each condition, with statistical replicates, would strengthen the equilibrium shift claims. For native MS analysis of each inhibitor, the representative spectrum can be shown in the main figure together with quantified dimer/monomer fractions from replicates to show significance by statistical tests.

We thank the reviewer for the suggestion. We have performed a quantitative analysis of the monomer-dimer equilibrium based on triplicate native MS measurements for each condition. Representative spectra, quantified monomer/dimer ratios, and statistical analyses have been added to Figures 1 and S3. The quantitative results have also been described in the Results section (Lines 158–161, 165-168, 172-174, 177-179, 199-200).

(5) Changes of HDX rates in certain regions seem very subtle. For example, as it states 'residues 296-304 in the C-terminal region of M^pro were more flexible upon ebselen binding (Figure 4c)', the difference is barely observable. The percentage of HDX rate changes between two conditions (with p values) can be specified in the text for each fragment discussed, and any change below 5% or 10% is negligible.

We agree with the reviewer about the need for quantitative rigor in reporting HDX changes. We have calculated the fractional deuterium uptake difference for each peptide fragment discussed in the text between the inhibitor-bound and unbound states. These values, along with their statistical significance (p-values from a two-tailed t-test), have been provided in the revised manuscript (Legends for Figures 3 and 4). Although the HDX change of residues 296–306 is relatively small (<5%), this region showed a reproducible difference with low experimental variability and statistical significance (p < 0.05). Given its location within the C-terminal dimerization interface and its consistency with native MS, we interpret this change as a subtle local conformational perturbation.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major points:

(1) The study lacks validation through inhibitor binding site mutagenesis assays, especially peptidomimetic inhibitor PF-07321332 and ebselen, which would strengthen the mechanistic conclusions.

We appreciate this suggestion. For PF-07321332, the inhibitor forms a covalent interaction with the catalytic residue C145 and inhibits M^pro activity through a distinct mechanism. Previous studies have shown that mutation of C145, such as C145A, completely abolishes M^pro catalytic activity (Bhandari, D, et al. Communications Biology 2025, 8, 1061), making it difficult to directly evaluate the contribution of this residue to inhibitor-induced inhibition using enzymatic assays alone. This limitation and the relevant literature have now been discussed in the revised manuscript (Lines 272–277). Therefore, we focused on C300-dependent regulation of ebselen, which represents a distinct inhibitory mechanism involving modulation of M^pro structural dynamics and dimer stability.

To validate the role of C300 in ebselen-mediated regulation of M^pro, we generated C300S and C300F mutants and performed additional biochemical and structural characterization. The enzymatic assay showed that the C300F mutation significantly affected M^pro activity, and the inhibitory effect of ebselen on C300S M^pro was markedly reduced compared with WT M^pro. Furthermore, native MS analysis demonstrated that ebselen could still bind to C300S M^pro but failed to induce a significant shift in the monomer-dimer equilibrium observed for WT M^pro. These results indicate that C300 is not the only site involved in ebselen binding but is critical for mediating ebselen-induced structural perturbation and dimer destabilization. The manuscript has been revised accordingly for the (Lines 627–648), and Results (Lines 397–453), with new figures (Figures S14–S16) included, further supporting the functional contribution of C300 in ebselen-mediated M^pro regulation.

(2) MR6-31-2 is an ebselen derivative and exhibits a lower EC50 (1.78 μM) compared to ebselen (4.67 μM). It would be helpful to discuss why their activities differ, probably based on the assay conditions or binding behavior.

We agree with the reviewer that the difference in antiviral activity between MR6-31-2 and ebselen requires further clarification. The lower EC₅₀ of MR6-31-2 may result from iM^proved cellular properties, including compound stability, permeability, intracellular exposure, and potentially altered interactions with M^pro and/or iM^proved cellular properties. Although MR6-31-2 shares the ebselen scaffold, the modified chemical structure may affect its binding behavior and biological activity. However, EC₅₀ values obtained from cellular assays cannot directly reflect the biochemical inhibition potency against purified M^pro. These points have been integrated into the revised Introduction (Lines 98–101).

(3) In Figures 2, S1, S2, S4, S6, and S11, adding the drug name under each panel would make the data much clearer for readers.

The corresponding drug names have been added to panels to iM^prove figure clarity.

Minor points:

(1) Line 62-63 refers to the "long linker loop," while Figure 1a labels it as the "long loop linker." Please keep this consistent.

The terminology has been unified as “long loop linker” throughout the manuscript.

(2) Table 1 should be cited at line 80, and PDB code 7BAK should be included in Table 1.

PDB code 7BAK has been included in Table 1, and Table 1 has been cited in the context, as suggested.

(3) Figure 1a should include the corresponding PDB code in the figure legend.

The corresponding PDB code has been added to the Figure 1a legend, as suggested.

(4) It would be helpful to indicate in Figure 1a that the upper structure represents the dimer and the lower structure represents the monomer.

The upper and lower structures in Figure 1a have been indicated as dimeric and monomeric M^pro, respectively, as suggested.

(5) In the Figure S1 legend, it should mention that some inhibitor structures (like ebselen and MR6-31-2) are not fully resolved. Also, the Se atom in ebselen should be shown in Figure S1f (PDB: 7BAK).

The Figure S1 legend has been revised to indicate that some inhibitor structures, including ebselen and MR6-31-2, are partially unresolved, and the selenium atom of ebselen has also been shown in Figure S1f, as suggested.

(6) Pelitinib is an allosteric, non-covalently binding inhibitor. However, in Figure S3, the native MS profile shows dimer species (13+ to 15+) compared with unbound M^pro (14+ to 17+). Please clarify this difference.

We thank the reviewer for raising this good point. Protein charge-state distributions can be influenced by solution-phase conformation, conformational flexibility, solvent properties, and electrospray droplet charging (Susa AC, et al. J Am Soc Mass Spectrom 2017, 28, 332-340). The observed shift in charge state distribution in native MS might suggest that the addition of pelitinib caused changes in the protein conformation, solvent property and electrospray droplet charging. The relevant literature and discussion have been added in the revised manuscript (Lines 200–204).

(7) Line 172: "S1are" should be corrected to "S1 are."

Corrected.

Does including the "logic equation" in here add or subtract from it? To understand it you need to read all of App Matt A but without it seems unjustified to make the statement about the two conditions.

eLife Assessment

This is a fundamental study on the sensory roles of cerebrospinal-fluid-contacting neurons (CSF-cNs) in mammals, revealing how the apical extension is used as an amplifier of chemical changes in content of the CSF. Specifically, the authors show compelling evidence that PKD2L1 is predominantly a pH-sensing channel in CSF-cNs and link its apical localization to dual phasic and sustained responses underlying CSF chemosensation.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

This study by Vitar et al. probes the molecular identity and functional specialization of pH-sensing channels in cerebrospinal fluid-contacting neurons (CSFcNs). Combining patch-clamp electrophysiology, laser-based local acidification, immunohistochemistry, and confocal imaging, the authors propose that PKD2L1 channels localized to the apical protrusion (ApPr) function as the predominant dual-mode pH sensor in these cells.

The work establishes a compelling spatial-physiological link between channel localization and chemosensory behavior. The integration of optical and electrical approaches is technically strong, and the separation of phasic and sustained response modes offers a useful conceptual advance for understanding how CSF composition is monitored.

Reviewer #2 (Public review):

Summary:

Cerebrospinal fluid contacting neurons (CSF-cNs) are GABAergic cells surrounding the spinal cord central canal (CC). In mammals, their soma lies sub-ependymally, with a dendritic-like apical extension (AP) terminating as a bulb inside the CC.

How this anatomy-soma and AP in distinct extracellular environments-relates to their multimodal CSF-sensing function remains unclear.

The authors confirm in the GATA3:GFP mice where these cells are labeled that CSFcNs exhibit prominent spontaneous electrical activity mediated by PKD2L1 (TRPP2) channels, non-selective cation channels with ~200 pS conductance modulated by protons and mechanical forces.

They investigated PKD2L1 pH sensitivity and its effects on CSFcN excitability. They uncovered that PKD2L1 generates both phasic and tonic currents, bidirectionally modulated by pH with high sensitivity near physiological values.

Combining electrophysiology (intact and isolated AP recordings) with elegant laser-photolysis, they show functional PKD2L1 channels localize specifically to the apical extension (AP).

This spatial segregation, coupled with PKD2L1's biophysical properties (high conductance, pH sensitivity) and the AP's unique features (very high input resistance), renders CSFcN excitability highly sensitive to PKD2L1 modulation. Their findings reveal how the AP's properties are optimised for its sensory role.

Strengths:

This is a very convincing demonstration using elegant and challenging approaches (uncaging, outside out patch of the AP) together to form a complete understanding on how these sensory cells can detect so finely the changes of pH in the CSF.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

This study by Vitar et al. probes the molecular identity and functional specialization of pH-sensing channels in cerebrospinal fluid-contacting neurons (CSFcNs). Combining patch-clamp electrophysiology, laser-based local acidification, immunohistochemistry, and confocal imaging, the authors propose that PKD2L1 channels localized to the apical protrusion (ApPr) function as the predominant dual-mode pH sensor in these cells.

The work establishes a compelling spatial-physiological link between channel localization and chemosensory behavior. The integration of optical and electrical approaches is technically strong, and the separation of phasic and sustained response modes offers a useful conceptual advance for understanding how CSF composition is monitored.

Comments on revised version:

I thank the authors for their extensive revisions and detailed responses to the reviewers' comments. The manuscript has been substantially improved, and most of the major concerns raised in the initial review have been adequately addressed. In particular, the additional analyses of PKD2L1 channel activity, the incorporation of physiologically relevant pH conditions, the clarification of ASIC involvement, and the expanded Discussion have significantly strengthened the study.

Major scientific concerns largely addressed:

Quantification of PKD2L1 channel activity

The authors appropriately addressed my previous concerns regarding the use of Po as the sole measure of channel activity. The inclusion of additional parameters such as apparent Po, open time, nmax, holding current, and membrane charge provides a more robust assessment of PKD2L1 activity and substantially strengthens the conclusions.

Physiological relevance of pH modulation

The inclusion of experiments at pH 6.5 and the additional analyses of holding current and resting membrane potential are valuable additions. These experiments considerably improve the physiological relevance of the study.

ASIC contribution

The additional pharmacological experiments using ASIC blockers are helpful and support the conclusion that the photolysis-evoked response in the apical process is predominantly mediated by PKD2L1 channels.

Functional implications

The expanded Discussion regarding Ca2+-dependent signaling, neurosecretion, and the potential physiological roles of CSFcNs considerably improves the manuscript.

Remaining concerns:

Continued overstatement regarding "exclusive" localization and function:

Although the authors softened some statements in the revised manuscript, the term "exclusive" remains in several key locations, including the title.

For example:

"PKD2L1 channels segregated to the apical compartment are the exclusive dual-mode pH sensor..."

The data clearly demonstrate strong enrichment of functional PKD2L1 channels in the apical process. However, the available evidence does not fully justify the term "exclusive," particularly because:

- PKD2L1 immunoreactivity is still detectable outside the apical process.

- ASIC-mediated responses are present in CSFcNs.

- The authors themselves use more appropriate terminology such as "predominantly located" in the Discussion.

Therefore, I recommend replacing "exclusive" with more conservative terminology such as:

- predominant

- predominantly localized

- enriche

- functionally segregated

throughout the manuscript, including the title, Abstract, Introduction, Results, and Discussion.

We agree with the reviewer that the world “exclusive” is misleading and should be replaced. Following the reviewer’s suggestions, we have deleted the word “exclusive from the title, which now reads: “PKD2L1 channels segregated to the apical compartment are the functional dual-mode pH sensors in cerebrospinal fluid-contacting neurons.”

In addition, the word “exclusive” has been changed with more conservative terminology in other parts of the text: lines 80, 420, 466 and 551.

Use of the term "tonic current"

The manuscript continues to use the term "PKD2L1 tonic current."

While the dibucaine-sensitive holding current is clearly present, the precise mechanism generating this current remains uncertain. Indeed, the authors themselves acknowledge in the Discussion that:

- an alternative conducting state may exist, or

- unresolved brief channel openings may account for the current.

Therefore, the data support the existence of a sustained PKD2L1-associated current, but do not yet definitively establish a distinct tonic gating mode of the channel.

I therefore recommend replacing:

"tonic current" with a more neutral expression such as:

- sustained current

- PKD2L1-associated holding current

- sustained PKD2L1-mediated current throughout the manuscript.

Continued use of "off-current" and "off-response":

The revised manuscript has improved considerably in this regard. However, the terms "off-current" and "off-response" still remain in portions of the text and figure legends.

Because the manuscript itself demonstrates that the response reflects recovery from transient acidification rather than a separate OFF signaling mechanism, these terms remain potentially misleading.

I recommend replacing them with terminology such as:

- photolysis-evoked PKD2L1 current

- recovery current

- proton-removal-induced current

throughout the manuscript, including figure legends.

We apologize, as the word “tonic” and the terminology “off-current” should have completely disappeared after the first round of revisions. We have now replaced those all along the text. “Tonic” has been replaced by “sustained”.

“Off-current” or “off-response” have been replaced by appropriate terms in lines: 339, 342, 544, 545, 546, 549, 552, 555, 556, 560, 576, 580, 585, 588, 807 and 929. We have nevertheless conserved the term “off-current” in line 552 as we are referring to terminology used by other authors.

Minor editorial corrections

Figure 1Bd Please change: "po" to "Po" for consistency with standard channel physiology nomenclature.

Figure 1Ca Please add units (mV) to the voltage labels shown on the left side of the traces.

Figure 3E Please change: "Norm po" to "Norm Po".

Figure 4Fb Please replace: "sec" with "s" to conform with SI unit conventions.

Done.

The authors have addressed the majority of my previous concerns and the manuscript has been substantially improved. The remaining issues are primarily related to terminology and overinterpretation rather than experimental deficiencies.

Reviewer #2 (Public review):

Summary:

Cerebrospinal fluid contacting neurons (CSF-cNs) are GABAergic cells surrounding the spinal cord central canal (CC). In mammals, their soma lies sub-ependymally, with a dendritic-like apical extension (AP) terminating as a bulb inside the CC.

How this anatomy-soma and AP in distinct extracellular environments-relates to their multimodal CSF-sensing function remains unclear.

The authors confirm in the GATA3:GFP mice where these cells are labeled that CSFcNs exhibit prominent spontaneous electrical activity mediated by PKD2L1 (TRPP2) channels, non-selective cation channels with ~200 pS conductance modulated by protons and mechanical forces.

They investigated PKD2L1 pH sensitivity and its effects on CSFcN excitability. They uncovered that PKD2L1 generates both phasic and tonic currents, bidirectionally modulated by pH with high sensitivity near physiological values.

Combining electrophysiology (intact and isolated AP recordings) with elegant laser-photolysis, they show functional PKD2L1 channels localize specifically to the apical extension (AP).

This spatial segregation, coupled with PKD2L1's biophysical properties (high conductance, pH sensitivity) and the AP's unique features (very high input resistance), renders CSFcN excitability highly sensitive to PKD2L1 modulation. Their findings reveal how the AP's properties are optimised for its sensory role.

Strengths:

This is a very convincing demonstration using elegant and challenging approaches (uncaging, outside out patch of the AP) together to form a complete understanding on how these sensory cells can detect so finely the changes of pH in the CSF.

Weaknesses:

Not weaknesses, there are only minor requests to complete the beautiful study.

(1) The apical extension's response to removal of acidification is nicely illustrated in Figure 4C,G. There's something puzzling there: while the response to Glutamate is immediate, the channel responses to H+ is extremely delayed by 100ms - 2s, and even sometimes came in bursts separated by few hundreds of ms. H+ diffuse even faster than glutamate. Why is that?

I don't quite understand how the response is so delayed & how to explain the recurring bursts of channel opening in the figure panel ?

The kinetic of the response to proton uncaging is analyzed in Figure 4E, where the charge of the current traces is plotted against time. What this analysis shows is that the response lasts a few hundred ms (τ 250 ms) and then the PKD2L1 activity increase subsides to baseline. The peak of the response is at 100 ms (Figure 4G), but the increase in activity happens as soon as the uncaging pulse ends (Figure 4D, G and H). This behavior has already been shown in expression systems, where the channel activity is blocked by protons and the blockage is released when the acid is withdrawn. In an intact cell as the CSFcNs studied here, the exact kinetics of the recovery response are probably more complex (and variable) than in expression systems. Indeed, it is known that the recovery of this current depends, for example, on pH and extracellular calcium. Also, PKD2L1 are inhibited by intracellular calcium (de Caen et al, eLife 2016) but are themselves permeable to Ca⁺⁺ ions. The interaction of these effects could give rise to the “bursts” that are observed in some cases. However, this is merely speculative at this point.

- The authors should show in Fig 4C,G the traces for 1-2 s before uncaging occurs so we can appreciate whether such events occur as well in baseline and discuss this further in revisions.

Following the reviewer’s suggestion, we have added a trace in Figure 4C (upper blue trace) showing the spontaneous activity of the cell, prior to uncaging, as it is already shown for another example in Figure 4D.

- Could the authors use a fluorescent pH sensor to monitor pH in the extracellular space and in the cell ?

This is an important point that was already addressed by the reviewing editors in the previous round of revisions. Indeed, we have attempted to perform pH calibrations in the setup using the pHsensitive dye pyranine (or HPTS: 8-Hydroxypyrene-1,3,6-trisulfonic acid). HPTS is a very useful tool for pH calibrations in the physiological range: its pKa value is close to 7.2 and it can be used as a ratiometric dye (its fluorescence is pH-independent at 405–410 nm and pH-dependent at 450 nm). Unfortunately, the calibration under the conditions of a real experiment is not possible because the photolysis in the slice occurs in a tiny volume (approximately 1 µm³ in a total bath volume of more than 1 ml). In these conditions, the 405 nm uncaging pulse bleaches the dye in the photolysis spot and any useful information is lost. In addition, our imaging system is not fast enough to follow the pH change. As discussed in the Materials and Methods section, subsection “Estimation of the pH drop induced by photolysis” (line 791), the fast protonation of bicarbonate indicates that the pH change induced by the photolysis recovers in the submillisecond range.

- Could the authors investigate whether in the apical extension, PKD2L1 channels are mainly at the outer membrane in the apical extension OR whether many channels are located in inner membranes ?

PKD2L1 channels are probably subject to a high rate of turnover, and they are certainly localized in the plasma membrane of the apical process as well as in the inner membranes. Although this is a very interesting point, we believe it is out of the scope of this work.

(2) Suppl Fig 4 is very cool and should be moved to main figure. The coupling of Soma and AP is very tight, yet there is a clear difference in targeting of channels that respond to cues in the CSF. In the context of an intact spinal cord, we can wonder how and when the contribution from ASIC in the some would be relevant to physiology. Can the authors think of experiments with an intact central canal to test the sensitivity and condition of recruitment of pH sensing in the soma (ASIC) versus the apical extension (PKD2L1)?

We have followed the suggestion of the reviewer and have made Supplementary Figure 4 a main figure.

The fact that the normal interphase between the spinal cord parenchyma and the cc is lost is already acknowledged in the discussion, lines 486 to 489. As the reviewer suggests, PKD2L1 and ASIC channels seem both to be important in the response of CSFcN to pH changes. However, both channels are activated in very different physiological contexts, as is discussed in the section “The involvement of ASIC channels”. Keeping the central canal intact in order to be as close as possible to physiological conditions, as suggested by the reviewer, would be ideal. However, as CSFcNs are in the middle of the cord, it would require the use of optical techniques that allow to penetrate deep into the tissue (e.g., 2-photon microscopy) that unfortunately are not available in our labs.

(3) The Reissner fiber is missing after slicing the spinal cord. From our observations in fish, the fiber being under tension triggers lots of activity in CSF-cNs (Bellegarda et al Elife 2023) that also relies on PKD2L1 (Bohm et al NC 2016; Sternberg et al NC 2019). Could the authors discuss the contribution of the Reissner fiber to the PKD2L1 mediated modulation of CSFcN excitability ? Could the authors conceive a way to slice along the anteroposterior axis (sagitally) the spinal cord to keep the Reissner fiber in the central canal when recording CSF-cN apical extension ?

- The authors should show in Fig 4C,G the traces for 1-2 s before uncaging occurs so we can appreciate whether such events occur as well in baseline and discuss this further in revisions.

As discussed in the previous point, the in vitro slice preparation has technical limitations that are mainly related to the alterations of the normal structure of the tissue. Although keeping the Reissner fiber intact in a sagittal slice seems possible, accessing the CSFcNs with electrophysiological methods would still be a challenge.

We have now added a sentence in the Discussion, lines 561 to 564, where we discuss that CSFcN excitability is modulated by the Reissner fiber and that it remains to be explored whether in rodents the gating of PKD2L1 channels is modulated by the Reissner fibre, as has been shown in zebrafish.

eLife Assessment

This important study reports that neural activity in the auditory cortex (field L) of singing male zebra finches can be modulated by the presence of a female conspecific. These findings extend recent work showing that the activity of dopaminergic neurons in songbirds is also affected by an audience. Solid evidence is presented for the importance of singing context in modulating auditory processing during vocal production, but the study does not yet fully establish that these effects arise specifically from audience-dependent modulation of auditory feedback, as opposed to possible acoustic, temporal, or recording-related confounds. This work should be of interest to researchers studying the context dependence of sensory processing, the role of auditory feedback, and vocal communication during courtship behavior.

Reviewer #2 (Public review):

This study asks whether auditory responses in the songbird auditory pallium/field L during singing are modulated by social context. Specifically, the authors examine neural responses to delayed auditory feedback during male zebra finch song produced either alone or in the presence of a female. This is an interesting and important question because the evaluation of self-generated vocal output may differ when the song has a dedicated social function.

The main strength of the work is that it addresses auditory feedback processing during natural vocal behavior and does so across two naturalistic contexts. The revised manuscript is strengthened by additional analyses of spike waveform similarity, response significance, response latency stability, and exclusion of motifs overlapping with female calls. These additions make the reported context-dependent response differences more credible and help address some concerns about recording stability and contamination by female vocalizations.

The results show that some auditory pallium neurons respond differently to feedback perturbations during directed and undirected song. This finding is potentially significant because it suggests that auditory processing during vocal production is not rigid but could subserve a social function that depends on the listener. If robust, this would add an important dimension to models of song monitoring and sensorimotor control.

However, the strength of evidence remains moderate rather than conclusive. Several alternative explanations are not fully ruled out. Directed and undirected songs may differ acoustically in ways that could influence neural responses, and it is not yet clear that relevant song features were directly compared or controlled across contexts. The experimental sequence also appears to be ordered, with undirected song recorded before directed song, which makes it difficult to fully separate social-context effects from time-dependent changes in recording quality or neural responsiveness. The added waveform analysis is useful, but does not completely establish continuous unit stability across long recording sessions. In addition, possible song changes around the delayed-feedback target point, including compensatory modifications before or after feedback, remain an important potential confound. Finally, clarification of the time-warping and spike-alignment procedures is important because condition-specific alignment could affect comparisons between directed and undirected song.

Overall, the data support context-dependent differences in neural responses in some neurons, but do not yet fully establish that these differences arise specifically from audience-dependent modulation of auditory feedback processing rather than from acoustic, temporal, or recording-related confounds. The work is likely to be useful to researchers interested in vocal communication, auditory feedback, and social modulation of sensorimotor processing, particularly as a foundation for future experiments using counterbalanced designs and more direct controls of song structure across contexts.

Reviewer #3 (Public review):

In this study, Jones et al. examine how neural activity in auditory regions (the auditory pallium) of singing male songbirds is modulated by the presence or absence of an audience (a female conspecific). They test whether activity in auditory pallium differs between conditions in which the male is singing to a female (directed song) or alone (undirected song) and whether response to distortions of auditory feedback (DAF) differ between these conditions. Previous work has shown that in other parts of the songbird brain, sensory-motor activity can differ between directed and undirected song, and that responses to DAF are attenuated when males sing directed song versus undirected song. These prior results raise the interesting question of the extent to which such modulations of activity by the presence of an audience are already present in primarily auditory areas within the pallium. This possibility is also motivated by prior work that has shown that activity in the auditory pallium is not exclusively explained by auditory input, but can also be modulated by the bird's state - whether it is singing or not.

Against this background, the questions asked here are of interest for two inter-related reasons:

(1) The authors address whether the presence of an audience (a female conspecific) alters activity in an auditory region during singing. Primary songbird auditory areas such as Field L, and analogous mammalian thalamo-recipient cortical regions such as A1, are often thought of as responding very specifically to the features of sensory stimuli, but are also understood to be modulated by a variety of factors including the attentional and behavioral state of the animal. For audition, such modulation includes whether or not animals are vocalizing and listening to themselves or listening to playback of their own vocalizations. Cited works from Keller (2009) as well as Eliades and Wang (2008) have indicated that the act of vocalizing can modulate auditory responses to self-generated feedback in primary auditory areas relative to those arising from playback of the same sounds. Here, the question is whether responses to self-generated feedback differ between conditions of singing alone versus singing to a female audience. A demonstration that the presence of an audience matters to responses in auditory pallium would add to a general understanding of how it is that non-auditory factors can modulate activity within regions that are considered primarily sensory.

(2) The authors address the possible source of an audience-dependent modulation of responses to feedback perturbation in the VTA previously reported by Goldberg and colleagues (2023). In the VTA, responses to perturbations during singing are consistently attenuated when males are singing to females versus when they are singing alone, but the underlying mechanisms of this modulation are unknown. Here, the authors test the possibility that such modulation by an audience is already present at the level of auditory pallium. The previously reported attenuation in VTA is a nice example of how neural processing can differ with varying behavioral priorities. Understanding whether this modulation of responses to DAF arises already in auditory areas would further a mechanistic understanding of an intriguing example of state-dependent modulation of sensory processing and behavior and lend broad insight into related phenomena.

The authors report 1) that activity in the auditory pallium differs between directed and undirected singing at many individual recording sites, but that these changes are heterogeneous, with both increases and decreases in activity, so that there is no consistent change across the population and 2) that modulation of activity by DAF can differ between directed and undirected song, but that there is no consistent attenuation of response (as observed in the VTA) and instead heterogeneous increases and decreases in response to DAF so that there is no net change at the population level.

These findings are important and of general interest; while they do not readily explain the source of the audience-dependent attenuation of auditory responses to DAF in the VTA, the demonstration of audience-dependent modulation of self-generated feedback and its disruption in the auditory pallium provides an opportunity for further investigation of how changes in social context influence brain and behavior.

Additional comments and suggestions:

The authors have done a good job of addressing many of the issues that were raised in the initial round of reviews. There is additional analysis that strengthens the study, including 1) applying a stability criterion to assess the quality of unit isolation, 2) shifting away from a categorical identification of units as "retuning" or not, to an analysis that presents a continuum of changes to neural firing between conditions, 3) use of non-parametric statistics for the assessment of significance of differences in response measures between conditions and 4) exclusion from analysis data from motifs during which females were observed to be vocalizing.

The authors also have added to the text several important clarifications, and modified language in several ways that improve the presentation and interpretation of results. This includes 1) noting that differences between neural activity during singing with and without DAF does not necessarily reflect "error detection" but could instead reflect how neurons with fixed auditory receptive fields might respond differently to the distinct auditory inputs present between these conditions, 2) clarifying that the recordings were not specifically restricted to Field L, but were distributed more broadly across the auditory pallium, and 3) discussing some of the mechanisms whereby tuning might change due to various sensory, motor and internal factors associated with differences between singing alone and singing to a female.

I only have a couple of areas of remaining concern that I think could be addressed with further analysis, or some additional discussion, according to the authors' preferences.

(1) Stationarity of neural response

My main residual concern relates to the issue raised in the previous round of review of how much of the observed difference in activity between morning sessions when the male is alone and later sessions when the male is singing to a female could reflect changes in neural response properties (non-stationarity) due to the passage of time (sometimes at least several hours) rather than specifically due to the presence or absence of an audience.<br /> The authors restriction of data to recordings that passed a stability criterion for unit waveforms is helpful in addressing whether the same units are 'held' over the course of the experiment. However, even with well isolated units, the response properties or tuning of units can change over time due to a variety of factors that include changes in internal state, circuit excitability, up and down states, neural plasticity, etc.

The previous review noted several examples of data from the manuscript that illustrated this concern - instances where response properties of neurons appeared to change over time within a given condition. Any such changes in response properties that occur in the absence of a change in audience would tend to contribute to the reported "retuning" of responses.

One thing that the authors could do to address this issue would be to discuss potential contributions of non-stationarity of responses over time as a potential confounding variable and then editorialize about why they think this seems unlikely to explain many cases in which response properties change between conditions. See comments to authors for one specific suggestions along these lines.

Alternatively, the authors could carry out additional analyses to evaluate this issue more quantitatively. For example, by measuring the magnitude of "spontaneous" changes in responsiveness observed within conditions (such as by comparing the motif aligned activity for the first n examples within a condition against the activity during the last n examples) and comparing that with the magnitude of changes observed across conditions.

Another approach would be to carry out some sort of "change point analysis" on the motif-related activity for each experiment in order to establish how often the most abrupt changes in activity occur at the transition between conditions versus spontaneously at other times.

Lastly, while the experimental design didn't specifically include interleaved blocks of undirected (alone) singing and female directed singing, the methods indicate that the female directed singing data were collected by repeatedly introducing females for 10 minutes at a time. If there are even a couple of cases where the males produced song alone in the periods between female presentation, it would be worth testing whether modulation of neural firing tracked these interleaved conditions.

Previously published work such as the interleaved recordings of Hessler and Doupe indicate a close and reversible tracking between modulation of neural activity in sensorimotor song system nuclei and switches between singing alone and singing to a female. With respect to the possibility raised in the rebuttal of whether males continue to sing 'female directed song' even after the removal of a female, these and other published data suggest that this is not likely to be the case. But if this were a concern in interpreting any data, the authors could directly assess the male's song for previously described changes in acoustic variability that also track changes in the presence of an audience.

(2) Further discussion of how an audience might influence responses.

With respect to the mechanisms whereby an audience might modulate neural responses, a somewhat expanded discussion of possibilities with reference to relevant literature would be helpful. This could include reference to evidence for various neuromodulatory systems participating in modulating singing related activity in song system nuclei based on presence or absence of a female - do these neuromodulatory systems project to the relevant regions of the auditory pallium or its lower-level inputs within the ascending auditory pathway such that they could concurrently act on auditory circuitry?

In addition to possibility that the presence or absence of an audience affects auditory circuitry via a change in attention, alertness, or motivation, might efference signals associated with singing or locomotion/dancing reach and influence auditory pathways? Given that premotor activity and acoustic structure of song differ between conditions, might any singing-related efference copy activity that reached auditory regions also differ between conditions? A related interesting possibility that could be worth noting is that the presence of a female generally elicits increased locomotion and dancing on the part of the male that accompanies female directed song. Several studies have noted that general forms of locomotion can also result in efference copy signals reaching and influencing auditory regions (e.g. see Schneider and Mooney, Annual Review, 2018; Han et al. "Locomotion-induced neural activity independent of auditory feedback in the mouse inferior colliculus" iScience 2026 - the latter reference is interesting in that it appears to indicate bi-directional modulation of neural activity as observed across units in the current study).

Minor:

(1) The authors describe some units as showing "Activation by the absence of DAF." Because the birds in the study have extensive experience with DAF on a subset of trials, it is possible that the increased responses in the absence of DAF reflect a positive deviation from expectation of distortion (as seems to be the case for VTA neurons in previous work). But it is also possible that the broadband DAF stimulus drives inhibition of auditory responses in some cases, and the greater responses in the interleaved trials with normal feedback simply reflect the absence of that inhibition (rather than a positive deviation from a learned expectation). In keeping with the authors shift away from the use of "error detection" elsewhere in the manuscript, it might also be good to use less interpretive language here instead of "activation by absence of DAF".

(2) At the authors discretion, it would be interesting to know if there is any relationship between the way in which changes in audience affect activity with normal auditory feedback versus with DAF. For example, if normal singing responses are attenuated in the female directed condition, are the responses to DAF also attenuated?

(3) In figure 2, the vertical dashed lines associated with the rasters indicate the onset and offset of motifs. For several of the figure panels, the spectrograms show motifs that are not aligned with these onsets and offsets. Please clarify or modify (are the rasters from time-warped data but the spectrograms are not -time warped?).

(4) The authors equate peaks in activity before the onsets of motifs with premotor activity: ["A previous study recording from Field L in zebra finches reported neural activations prior to the onset of singing, consistent with premotor signaling (Keller and Hahnloser, 2009). We tested for context dependent changes in premotor activity by examining peaks in neural activity aligned to motif onsets. Across the population neurons did not exhibit significant changes in the timing of motif onset-aligned activity (Figure S4)."]<br /> However, the spectrograms as shown in Figures 1 and 2 indicate that each motif is often preceded immediately by other song syllables such as introductory notes or syllables from the end of the preceding motif. Further analysis would be required in the current study to demonstrate that the activity present before motif onsets reflects premotor activity rather than auditory responses to the proceeding syllables. Please soften the claim that this reflects premotor activity or provide additional analysis or argument.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This study examines the context-dependent modulation of auditory cortical neurons in response to expected sensory input, either self-generated sounds or expected perturbations of self-generated sounds. Specifically, using songbirds, the authors ask whether social context (the presence of a female conspecific) affects 1) the response of auditory cortical neurons to the bird's own song when he is singing; and 2) the response of neurons to perturbations of auditory feedback that the bird has been trained to expect.

Strengths:

First, the authors report that across the population, the responses of the neurons does not differ when a male bird sings alone or if he sings to a female. A fraction of auditory cortical neurons, however, do show significant differences in the firing rate, precision, and/or degree of burst firing when males sing alone vs. when they sing to females. This finding is broadly consistent with the literature showing that sensory neurons (visual, auditory, somatosensory, etc.) can be rapidly reconfigured into different "information processing modes" depending on behavioral state (e.g., quiescence vs. vigilance).

For the perturbation experiments, the authors trained birds to expect distorted auditory feedback during a particular syllable. They found that some neurons showed greater responses during perturbation when a female was present (compared to when males were alone) while other neurons had smaller responses during perturbation when a female was present. In addition, the response of a small number of auditory cortical neurons were not affected by behavioral state. These results contrast with their prior report that the responses of midbrain dopaminergic neurons that project to the basal ganglia are "uniformly reduced" in the presence of a female, raising a question of how an evaluation signal is transformed in the circuit from the primary sensory region to the midbrain.

Weaknesses:

While the experiments and analysis are solid, the finding that social context can alter responses of auditory cortical neurons in a multitude of ways (increase, decrease or no change) raises several questions that can be examined with additional analysis. For example, do context-dependent differences in auditory responses derive from context-dependent differences in the songs? Are context-dependent differences present in all classes of neurons and throughout the auditory system?

The observed heterogeneity in the firing properties of auditory cortical neurons, both in response to self-generated sounds and during perturbations of auditory feedback, raises the question of which neurons are sensitive to social context (which likely can be addressed by the authors in a revision). The authors should provide additional details about the recordings:

(a) What are the locations of the recording sites? Prior work has shown that there is an organized map of spectrotemporal features of sounds in the auditory cortex of songbirds; spectral tuning widths change along the medial-lateral axis and temporal tuning widths differ between the input and output layers of Field L. Were the recordings primarily in Field L2 (thalamo-recipient region), L1 or L3? Were some recordings lateral to Field L in secondary auditory regions? Were the neurons that showed context-dependent changes in firing properties localized or distributed throughout Field L (i.e., were the context-dependent differences in neural responses truly brain-wide)? At a minimum, the authors should include a schematic showing the different regions of Field L and a summary of the location of the recording sites. Images of the processed tissue with electrolytic lesions would also be helpful.

We agree that the anatomical targeting and limits of localization should be made explicit. In the original manuscript, we referred broadly to recordings from "Field L" and described targeting coordinates in the Methods. In the revised manuscript, we have softened the anatomical claim from "Field L" to "auditory pallium" where appropriate, while explicitly stating that electrodes were aimed at Field L. We also added anatomical caveats and a new supplemental figure.

The revised title and abstract now reflect this more conservative anatomical framing. For example, the abstract now states: "Here we recorded neural activity from the auditory pallium in zebra finches practicing singing alone and directing courtship songs to females." In the Introduction, we now explicitly state both the intended target and the limitation: "We targeted our recording electrodes to Field L, a primary auditory pallial area that projects into multiple higher auditory areas that, in turn, project to VTA."

We then added the caveat: "Field L is composed of multiple subdivisions and surrounds the interfacial nucleus and because the implanted wire bundles spread in a small radius of up to ~0.5 mm, our recordings likely included large territories of the auditory pallium (Figure S1)."

We also added mechanistic/anatomical context for why these recordings may reflect activity shaped by broader auditory forebrain circuitry: "Although Field L is classically described as a primary auditory thalamorecipient region, its activity may also be shaped by contextual signals related to the courtship context, potentially via recurrent interactions with higher-order auditory forebrain regions such as the caudal mesopallium (CM) and caudomedial nidopallium (NCM) (Bauer et al., 2008; Figure S1C)."

Changes made in revision: We added Figure S1, which includes anatomical subdivisions, an example histological slice showing the area where the cannula was implanted, and auditory pathway connectivity. We also revised the wording throughout the manuscript from "Field L neurons" to more conservative phrasing such as "auditory pallium neurons" or "pallial auditory neurons" when appropriate. We did not claim layer-specific localization, because the revised manuscript explicitly states that we cannot make such claims.

(b) Was the context-dependent modulation limited to a particular class of neurons (distinguished by spike waveform shape, spontaneous firing rate, or other feature)?

We agree that identifying whether context-dependent modulation is associated with specific neuronal classes is important. In the revision, we added analyses examining relationships between spike width and various firing characteristics. We also looked for potential relationships between mean rate and DAF response, IMCC and DAF response, and found no clear trend. Overall, we did not observe any clear relationship between DAF response, DAF response modulation, and metrics like spike width or mean firing rate.

The revised manuscript states: "Action potential width of individual neurons has previously been used to classify putative interneurons or putative principal cells in the zebra finch auditory pallium (Calabrese and Woolley, 2015)."

We then describe the new analysis: "We tested if DAF-response scores, mean firing rates during singing, burst fraction, IMCC, and the change in all of these between undirected and directed singing was correlated with spike half width (spike half-width measured as peak-to-trough time; Figure S5)."

The revised result is: "DAF response in either condition, the change in DAF response across conditions, and the change in firing rate, burst fraction, and IMCC were not significantly correlated with spike width (Figure S5A-B)."

We also report that some general firing properties did correlate with spike width: "Consistent with previous literature, firing rate was significantly correlated with spike width (Pearson's correlation, p=0.003; Figure S5C). Interestingly, burst fraction (p=8.3x10-4) and IMCC (p=5.4x10-5) were also significantly correlated with spike width (Figure S5C)."

Changes made in revision: We added Figure S5 and associated text analyzing whether context-dependent changes in DAF response, firing rate, burst fraction, and IMCC were correlated with spike half-width. These analyses did not support the conclusion that context-dependent DAF modulation was restricted to a waveform-defined neuronal class.

(a) Prior work has shown that songs of zebra finches differ slightly when males sing alone compared to when they sing to females: songs are faster; pitch is less variable; and the number of introductory elements is greater when males sing to females. Do some of the observed social context-dependent differences in the responses of auditory neurons reflect differences in the songs in the two conditions? Did the authors of this study also find premotor activity in Field L, and if so, did it differ between the two social contexts? Might differences in Field L responses reflect motor/song differences?

The revised manuscript now addresses the issues of context-dependent changes in song in several ways. First, we explain why motif-aligned comparisons are meaningful: "The acoustic structure of undirected and directed motifs is highly similar in adult finches, enabling singing-related neural activity to be precisely aligned and compared across contexts."

Second, we added analysis and discussion of premotor-related activity. Changes made in revision: New results paragraph and new Fig S4. "A previous study recording from Field L in zebra finches reported neural activations prior to the onset of singing, consistent with premotor signaling (Keller and Hahnloser, 2009). We tested for context-dependent changes in premotor activity by examining peaks in neural activity aligned to motif onsets. Across the population neurons did not exhibit significant changes in the timing of motif onset-aligned activity (Figure S4)."

(b) For the perturbation experiments, this raises a question of whether perturbation amplitude is different when a male is alone and when a female is present. It would be useful to know if (and how much) perturbation amplitude varied depending on the location inside the cage as well as whether the sound pressure level of the underlying song was higher (e.g., Lombard effect).

We previously calibrated the perturbation amplitude in Roeser et al., 2023, in an identical recording setup. Two speakers deliver the feedback on either side of the bird's home cage. We acknowledge the possibility that the position and orientation of the bird can affect the way the sound hits either of the bird's eardrums and thus potentially affect a neural response. However, neural activations following the absence of distortion playbacks were a major feature of the dataset and were context-dependent in some cases.

The Methods state: "DAF was implemented with a custom LabVIEW acquisition program that analyzed song syllables in real-time and delivered syllable-targeted feedback." and "DAF (50 ms broadband noise bandpass filtered at 1.5-8 kHz to match frequency range of zebra finch song) was played over speakers in the recording chamber on top of a specific target syllable randomly on 50% of motif renditions."

The revised manuscript also makes clear that experiments occurred in the bird's home cage: "Experiments were carried out in the male's home cage, which was inside a sound isolation chamber."

Importantly, the revised Results show that not all DAF-related responses were simple activations to additional sound. Some neurons were activated by the absence of distortion: "Unexpectedly, some neurons were not activated by the song distortion but rather by the lack of target syllable distortion." and "These activations following undistorted renditions could also depend on the courtship context."

Changes made in revision: We clarified the DAF stimulus and recording setup in Methods and added Figure 3 showing neurons activated following undistorted renditions. These data argue that context-dependent responses are not simply explained by DAF sound amplitude, although we do not claim that position-dependent acoustic variation was fully eliminated.

Finally, it would be helpful if the authors could include a model and/or more discussion of how the uniform attenuation in midbrain dopaminergic neurons may arise given the heterogeneous responses in Field L.

The revised manuscript provides evidence for context-dependent retuning upstream of VTA, but does not offer a direct mechanistic explanation for the uniform attenuation seen in dopaminergic neurons. The revised Discussion states: "Because the main goal of this study was to test if courtship-associated reduction in DAF signaling, recently observed in VTA DA neurons (Roeser et al., 2023), resulted from a local process in VTA or reflected a retuning of auditory responsiveness, we explicitly tested for changes in DAF responsiveness between alone and female-directed singing."

It then explicitly contrasts auditory pallium and VTA: "Surprisingly, we discovered that Field L neurons could retune at the transition from lone to courtship singing in diverse ways, consistent with a more widespread process in the brain that does not fully explain the uniform DAF-signal attenuation observed in VTA."

Changes made in revision: We expanded the Discussion to explicitly state that auditory pallium retuning is heterogeneous and therefore does not fully explain the uniform attenuation observed in VTA. We do not present a formal circuit model, but we now more clearly frame the result as evidence for broader sensory retuning that is likely transformed downstream.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Jones and Goldberg study auditory cortex in male zebra finches. They explore song-related responses in two different contexts, when the male is either alone or in the presence of a female. They find a heterogeneity of responses, in line with auditory cortical neurons computing the social modulation of responses found in VTA.

Weaknesses:

Stability of responses has not been studied: some neurons seem to have responses that slowly drift in time, which could lead to observed differences between alone and with-female conditions. Also, possible motor confounds and sound-of-audience confounds should be addressed. The language is often imprecise.

Stability and Reversal: It is a bit unfortunate that stability of effects seemingly has not been studied by reversing experimental conditions. The work would be much stronger if authors could show that audience-dependent tuning is robust in individual cells. Did they record from some neurons during reversal back to the alone condition?

We agree that recording stability is essential. A reversal experiment was not feasible for this dataset, as it is difficult to confirm whether song motifs produced immediately following female presence represent undirected singing or are directed to an unseen but recently present female. Instead, the revised manuscript adds a strict unit-stability criterion based on waveform similarity across conditions.

The revised Results state: "Importantly, because these neural recordings were performed over long time courses (~2-8 hours), a strict threshold for stability was imposed." The exact criterion is: "A Pearson's correlation coefficient of at least 0.99 between the average neural waveform during undirected and directed singing was required for a unit to be considered stable (Dickey et al., 2009; Figure S2)."

Changes made in revision: The strict waveform-stability inclusion criterion and new Figure S2 directly showcase unit stability across the time course of the experiments.

Motor responses: Does DAF playback change song? If so, especially if it applies only in one of the two conditions (audience/no audience), then the observed response differences could be motor-related rather than auditory responses.

We agree that motor confounds must be minimized. We previously found that DAF did not affect the acoustics of the subsequent syllable (Gadagkar et al., 2016). The revised manuscript clarifies that DAF and undistorted trials were randomly interleaved and analyzed by comparing matched renditions within conditions. Importantly, we only analyzed motif-aligned activity, ensuring that all syllables within the song motif are the same.

Changes made in revision: We clarified the DAF analysis framework and added a more conservative permutation-based analysis comparing distorted and undistorted trials within each context, then comparing those DAF-response vectors across contexts. We do not claim that all possible motor consequences of DAF are eliminated, but the analysis directly tests neural responses to randomly interleaved distorted versus undistorted renditions.

Similarly, motif-aligned spiking activity was time warped to the median duration of undirected or directed motifs. Could the shorter motifs during directed song lead to alignment differences that would account for the different error responses in alone/with-female conditions?

We agree this is an important technical point. The time-warping we conducted, standard in the field, compensates for the tempo differences between directed and undirected song. Importantly, our main analysis of change in error response no longer uses a 100 ms response window, but rather includes all windows in the motif.

Changes made in revision: We clarified that the revised DAF response analysis uses motif-aligned, time-warped spike trains. Importantly, the revised analysis moves away from relying on a single scalar response window and uses bin-wise permutation tests with family-wise error correction.

Audience versus sound of audience: Is it truly the audience that causes the difference in error responses or is it the sounds the audience makes?

We agree that the sensory cues defining "audience" cannot be fully separated in this experiment. The reviewer raises an important point that female zebra finches occasionally call at the male. We have excluded all song motifs from analyses that include an overlapping female call.

The revised Methods now explicitly state that motifs overlapping with female calls were excluded: "Any motifs that had overlapping time with a female call in directed motifs was excluded from analysis."

We also revised the Discussion to treat the mechanism by which auditory pallium receives information about the female as an open question: "An open question is how auditory pallium receives information about whether a female is present, and how this information influences neural activity."

Changes made in revision: We excluded motifs overlapping with female calls and added discussion explicitly acknowledging that how female presence is represented in auditory pallium remains unresolved. We do not claim to distinguish visual, auditory, social, or motivational components of the female-present condition.

Reviewer #3 (Public Review):

Summary:

In this study, Jones et al. examine how neural activity in a primary auditory area (field L) of singing male songbirds is modulated by the presence or absence of an audience (a female conspecific). Prior work has demonstrated that the presence of an audience attenuates the responses of dopaminergic neurons to distortions of auditory feedback (DAF). Here the authors report that even in a region that is primarily considered sensory, responses to DAF are also modulated by the audience, although in a heterogeneous manner. However, to be fully persuasive, additional analyses will be required to address how much of the apparent modulation by audience may be explained by other factors such as changes in recorded neurons or their properties over time.

(1) A central concern relates to whether the main reported effects associated with differences in singing directed versus undirected song reflect only those changes in conditions, versus contributions from changes in unit isolation or response properties over time.

We completely agree that unit stability is critically important in this study. To address this concern, we now quantify stability and apply strict inclusion criteria adopted from a study that assessed unit stability over days (Dickey et al., 2009). Additionally, we now include average waveform overlays for all example units across conditions as supplemental Figure S2.

Changes made in revision: We added: "Importantly, because these neural recordings were performed over long time courses (~2-8 hours), a strict threshold for stability was imposed." and "A Pearson's correlation coefficient of at least 0.99 between the average neural waveform during undirected and directed singing was required for a unit to be considered stable (Dickey et al., 2009; Figure S2)."

(2) A second concern has to do with the categorical definition of 'error neurons'. The authors define a subset of neurons as error responsive only if their responses to DAF exceed a specific threshold (2.5 standard deviations). The problem is that for some neurons categorically defined as being responsive to DAF in only one condition, there is almost certainly not a significant difference in the actual responses to DAF between conditions.

We overhauled our analyses characterizing DAF responses. Rather than relying only on a 2.5 z-score threshold, we now use a more conservative permutation-based approach that directly tests DAF responsiveness and context-dependent changes in DAF responsiveness.

The revised Results state: "Statistical tests defining auditory neurons as DAF-responsive or not in a binary fashion may not be suitable if the underlying population of DAF-related responses exist on a continuum from responsive to non-responsive."

The updated result is: "This more conservative approach identified 48/147 neurons as DAF-responsive in at least one condition, with 13 of those neurons exhibiting a significant modulation in their DAF response between undirected and female-directed singing."

(3a) Some discussion of what is already known about the auditory tuning of Field L, and the extent to which responses associated with distortion of feedback may reflect the frequency tuning of Field L neurons versus something that might be construed as more specifically as detecting an error in perceived feedback.

We agree that DAF-related changes in firing do not necessarily imply that neurons are explicitly detecting an "error" between predicted and actual feedback. Field L neurons can have spectrotemporal receptive fields and frequency tuning such that a broadband DAF stimulus could drive excitation or inhibition simply because the stimulus overlaps with excitatory or inhibitory regions of a neuron's receptive field. We therefore revised the manuscript to use more cautious language and to describe these responses as DAF-related or feedback-related signals rather than categorically as "error responses".

Changes made in revision: The title was changed from "Auditory cortical error signals retune during songbird courtship" to "Auditory cortical feedback signals are modulated during songbird courtship". We also added a sentence to the Discussion: "However, it is important to note that DAF-related changes in firing in auditory neurons do not necessarily imply that neurons compute sensory prediction errors. DAF-related responses could arise from ordinary auditory tuning to the broadband distortion stimulus."

(3b) It would also be useful to discuss further previous work on differences in auditory tuning or responses between conditions when subjects are vocalizing, versus when vocalizations are played back, and to what extent efference copy signals might contribute to the processing of feedback distortions.

We agree these are important points. Our experimental design did not include sufficient passive bird-own-song (BOS) playback trials to permit quantitative comparisons with vocalizing conditions, and we therefore cannot draw firm conclusions about the contribution of efference copy signals to the DAF responses described here. We did observe robust motif onset-associated neural activations, including some activity preceding motif onset, which were present across both social contexts (see new Figure S4). These observations are consistent with prior reports of premotor-related signals in Field L (Keller and Hahnloser, 2009), but whether such signals contribute differentially to DAF processing across contexts remains an open question that we now acknowledge in the Discussion.

(3c) To what extent did the current study control for any vocalizations or other sounds produced by females during the directed singing, and could this have contributed to differences in Field L activity between conditions?

Please see response R2.4 above, in which we describe the exclusion of all song motifs that overlapped in time with a female call. This exclusion criterion was applied throughout all analyses of directed singing.

Figure 1D: In the directed condition there are no spikes at all following the first handful of motif renditions. Were the directed and undirected recordings interleaved here?

Undirected and directed trials were not interleaved. The raster plots are presented in chronological order; however, for each behavioral condition, rows are sorted with the earliest renditions at the bottom and the most recent at the top. We have clarified this in the figure legend.

A minor issue: the raw example trace with male alone does not seem to have a corresponding set of points in the raster plot. For panel E, I also cannot find rasters that correspond to the example recordings shown at top.

In the original version, we randomly downsampled the condition with more trials to equalize trial counts across conditions in the example rasters, while performing all analyses on the full set of recorded trials. As a result, the example spike shown in the raw trace was drawn from one of the downsampled trials not displayed in the raster.

Changes made in revision: For greater transparency, we now include all trials from both conditions for each example neuron in Figure 1.

Figure 2A also shows a neuron that looks like it has non-stationarity; for the alone condition without altered feedback, the main peak has no spikes for the bottom half of the rasters.

In the original version, example neurons were selected to illustrate the DAF-response scoring method, which in some cases highlighted neurons with less stable response profiles. In the revised manuscript, we have replaced this example with neurons that exhibit more robust and stable DAF-related responses, and we now provide a broader set of example neurons illustrating both increases and decreases in DAF responsiveness across conditions.

Other figures show firing rate distributions that appear to be very non-Gaussian, with some motifs during which there is a lot of activity, and others in which there is little activity. Please consider applying non-parametric tests as appropriate.

We agree. In general, some neurons exhibited non-uniform firing rate distributions across trials. All of our main analyses are now conducted using non-parametric permutation tests, which do not assume a Gaussian distribution of trial-by-trial firing rates.

Approaches to addressing the non-stationarity issue could include more specifically indicating examples in which recordings from the alone condition and directed condition are interleaved and exhibit reversible changes in the pattern of responses.

Unfortunately, nearly all of our undirected and directed recording periods were not interleaved, as the experimental design required a block of undirected singing followed by directed singing with female presence. We find it informative, however, that DAF-response modulation was observed in both directions, with some neurons losing DAF responsiveness during directed song and others gaining it, a pattern that is difficult to attribute to a simple unidirectional drift in recording quality. We now provide additional examples illustrating both directions of modulation in Figures 2 and 3.

The methods and/or raster plots should include some further explanation of the time periods over which recordings were made in the alone versus directed conditions, and the extent to which they are interleaved or not.

We have clarified this in the revised Methods. In brief, recording began when the home cage lights came on each day, with the male left to sing alone until at least 40 undirected song motifs were collected. A female was then introduced in approximately 10-minute intervals until at least 40 directed song motifs were collected. The total recording duration on a given day ranged from 0.56 to 10.27 hours, reflecting variability across birds in the time required to elicit sufficient singing in each context. We have added this information to both the Methods and relevant figure legends.

It would be most helpful to assess the stability of waveforms and unit isolation across time.

We now apply strict inclusion criteria based on waveform stability, as described in R3.1 above. SNR was quantified as Vpp/(2*sigma_noise), where Vpp was the peak-to-peak amplitude of each filtered spike waveform and sigma_noise was estimated from the median absolute deviation of the filtered voltage trace. This combines the peak-to-peak normalization used by Nordhausen et al. (1996) with the robust noise estimator described by Rey et al. (2015). Waveform overlays for all included example units are provided in Figure S2.

It would be reassuring to see that significant differences between conditions are equally or more prevalent under the conditions of greatest unit isolation and recording stability.

The average SNR of neurons ultimately included in the analysis was 9.47 +/- 3.57, with a minimum of 4.69. Neurons that exhibited significant DAF-response modulation did not have a significantly different SNR than neurons that did not exhibit significant modulation (Wilcoxon rank-sum test, p=0.38). The mean SNR for significantly modulated neurons was 8.70, compared to 9.5 for non-modulated neurons, indicating that the detection of context-dependent modulation was not systematically biased toward neurons with lower recording quality.

One other way that the authors might be able to address the main concern would be to look at the stability of firing patterns within conditions.

We agree that stability of firing patterns within conditions is an important consideration, and this concern directly motivated the adoption of the permutation-based analysis described above. In this framework, the observed DAF-response difference between conditions is compared to a null distribution generated by shuffling condition labels across trials. This approach inherently accounts for within-condition trial-by-trial variability and does not assume stationarity of firing rates.

It would be helpful to have additional explanations of the criteria used for counting spikes, and assessing stability of recordings.

Spike waveforms were visually inspected for consistency using our custom MATLAB GUI on a 12-second file basis. Interspike interval violations below 1 ms were explicitly checked as an indicator of multi-unit contamination. Detection thresholds were manually set, and each recording file included in the analysis was independently inspected. We have added a more explicit description of these procedures to the Methods section.

For the specific examples shown in figures, it would be useful to indicate by small tick marks or otherwise which spikes were counted as single units.

We appreciate this suggestion. In the revised figures, we have improved the clarity of the example raw voltage traces by annotating the detection threshold and, where multiple units were present on a channel, indicating the waveform amplitude range corresponding to the isolated single unit. We believe this provides sufficient transparency regarding spike identity without requiring tick marks on every individual spike, which would substantially reduce legibility of the example traces.

What were the criteria for determining multi-unit versus single-unit activity?

In the context of this manuscript, "multi-unit activity" refers to channels on which no single neuron could be reliably distinguished from others based on waveform shape and amplitude. Units ultimately included in the study were those for which a single, consistent waveform cluster could be identified and isolated in the custom GUI. In cases where a second distinguishable unit was present on the same channel, it was manually excluded from the sorted single-unit record. We have clarified this distinction in the Methods.

Categorical scores: This definition results in cases where responses of 2.45 vs 2.55 are described as 'retuned', even if these responses are not significantly different. Retuning would be more persuasively demonstrated if the authors could provide a test of whether or not the responses for individual neurons differ significantly between conditions.

We completely agree, and thank the reviewer for motivating us to develop a more rigorous statistical approach. Our revised analysis uses a non-parametric permutation test that explicitly tests for significantly different DAF responses between undirected and directed singing conditions, with correction for multiple comparisons. This replaces the previous threshold-based categorical classification and directly addresses the concern that neurons near the threshold boundary were being treated as categorically different.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Minor comments:

(1) Please include a schematic of the brain, including the different subregions of Field L and the connections between auditory regions and the midbrain.

Done. Figure S1 has been added, including a schematic of Field L subdivisions and auditory pathway connectivity.

(2) The authors should include some additional information about the recordings, such as the proportion of Field L neurons that exhibited singing-related changes in firing rate. It would be helpful to include some examples of spontaneous activity when the bird is quiescent in Figs. 1-2, especially for cells that do not show firing locked to song.

We appreciate this suggestion. Given the scope of the current revision and the primary focus on DAF-response modulation, we have elected not to add spontaneous activity examples to Figures 1-2 at this time. We agree this would be a valuable addition in future work and have noted it as a limitation in the Discussion.

(3) Methods, p. 10: Surgery and awake-behaving electrophysiology: "The of the cannula" - this is the only mention of a cannula. Do the authors mean the ends of the probes?

Cannula placement and wire bundle extension from the end of the cannula has been clarified in the Methods.

(4) Bottom of p. 10: Fix reference for biorxiv paper: "ref andreas paper"

Fixed.

(5) Methods, p. 12: Redundant sentences regarding significant error response criteria.

Fixed. The redundant sentences have been removed.

Reviewer #2 (Recommendations For The Authors):

(1) The abstract is too vaguely formulated. Authors should try to quantify the statements already in the abstract.

We have reworded the abstract to align with the revision's more conservative claims regarding social context modulation of auditory feedback, and have added specific quantitative statements where possible.

(2) Authors repeatedly refer to 'perceived song errors' without performing experiments or reporting on behavioral readouts of how birds perceive the jamming sounds. The wording should be changed to something more neutral, e.g. 'DAF responses'.

We revised the manuscript throughout to use more neutral language centred on "DAF-related" or "feedback-related" responses rather than "perceived errors" or "mistakes". The title was changed from "Auditory cortical error signals retune during songbird courtship" to "Auditory cortical feedback signals are modulated during songbird courtship". We similarly revised the abstract and all relevant passages in the Results and Discussion.

(3) Authors write that 33 neurons were DAF responsive in both conditions. How should we interpret this overlap relative to independence and identity assumptions?

We agree that the original presentation made the interpretation of overlap across conditions unclear. The observed overlap is greater than expected under a strict independence assumption but smaller than expected if responsiveness were identical across conditions, consistent with partial but incomplete sharing of DAF responsiveness across social contexts. In the revised manuscript, however, we have moved away from this binary classification framework because DAF responsiveness appears to vary continuously across neurons. The permutation-based analysis now directly tests for changes in DAF responsiveness across contexts without requiring categorical assignment.

(4) Only 10 neurons were not affected by courtship state or only 10 error responsive neurons were not affected? I suggest authors do a multivariate analysis or use a mixed effect model and summarize the result as a table.

We agree that the categorical accounting of neurons across conditions was difficult to follow in the original manuscript. In the revised manuscript, we clarified the distinction between neurons responsive to DAF within a condition and neurons exhibiting significant modulation of DAF responsiveness across conditions. We now explicitly report: "This analysis identified 71/147 neurons as DAF responsive in at least one behavioral condition, whereas 76/147 were not responsive in either condition." and "This more conservative approach identified 48/147 neurons as DAF responsive in at least one condition, with 13 of those neurons exhibiting a significant modulation in their DAF response between undirected and female-directed singing."

(5) It would help if authors could define 'z-scored difference'. Better known is d prime, is this the same?

For each neuron, the z-scored DAF response was computed as the z-scored firing rate difference between distorted and undistorted trials. Importantly, our revised main analysis avoids any normalization such as z-scoring, and instead uses a permutation-based approach applied directly to spike counts.

(6) Is the 'retuning' assessment a bit conservative? Neurons could also retune by showing error scores greater than 2.5 in both conditions but a shifted response time.

We agree that neurons could retune by shifting the latency of DAF responses. Although potential latency shifts are beyond the scope of the current study, we did observe suggestive evidence of possible latency changes in some example neurons across conditions. We have noted this as an interesting direction for future analysis.

(7) Could the stability of DAF response across trials be described? E.g. as the ratio between intra versus inter condition variability?

We agree that stability of DAF responses across trials is an important concern. In addition to imposing strict waveform stability requirements, our permutation-based statistical test explicitly accounts for trial-by-trial variability by constructing null distributions from within-condition trial shuffles. We have also replaced the previously shown unstable example neuron with neurons that exhibit more consistent DAF-related responses across trials, and provide additional examples in Figures 2 and 3.

Minor:

(8) 'significant increase in burst fraction': specify effect size of t test in results section.

We now specify in the main text: "A small but significant increase in burst fraction was observed (paired t-test, p=9.3x10-6, n=138 neurons, mean +/- SEM: 0.11 +/- 0.006 vs 0.15 +/- 0.007, Figure 1J)."

(9) The IMCC parameter should be specified in the main text.

The Gaussian smoothing parameter (20 ms) has now been specified in the main text.

(10) Fig. 2: indicate the windows within which error scores are computed.

This is no longer applicable, as the revised permutation-based analysis does not rely on scoring error responses within a fixed window.

(11) In Fig. 2A, the neuron has an error score of -2.54 (significant), but the red and blue curves look almost the same.

We agree that the previous error score quantification did not always capture firing rate differences in an intuitive way. This example neuron has been replaced in the revised manuscript, and the new analysis avoids scalar error scores in favor of the permutation-based approach.

Reviewer #3 (Recommendations For The Authors):

Minor points:

(1) "(ref andreas paper)." Add reference here?

Fixed.

(2) Hessler and Doupe 1999 is a good reference for premotor signal re-tuning during courtship.

We agree. The reference has been included in the revised manuscript.

(3) Page 5: "discharge depended on courtship state, using" - should this be "depending"?

The original wording was intentional: "we tested how discharge depended on courtship state." We have verified this reads correctly in context and made no change.

(4) Page 9: "consistent with a brainwide process" - what is meant here?

We have revised this wording. The revised manuscript replaces "brainwide process" with clearer language describing a distributed modulation of auditory responsiveness that is not confined to a single nucleus.

eLife Assessment

In this fundamental manuscript, Richter et al. present a thorough anatomical characterization of the Drosophila melanogaster larval pharyngeal sensory system, which is involved in taste-guided behaviors. This study fills a major gap in the larval sensory map, providing a compelling neuroanatomical foundation for future investigations into sensory circuits and behavior. The exceptional data will significantly enrich the field of Drosophila neurobiology.

Reviewer #2 (Public review):

Summary:

The authors wanted to achieve a detailed ultrastructural reconstruction of the gustatory sensory organs in the Drosophila pharynx. Using serial EM and the associated bioinformatics tools they have achieved their goal.

Strengths:

Given the dataset, finding presented are solid and will be an important work of reference for the future.

Comments on revised version.

The authors have well responded to my previous comments and added text and figure material.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors provide a detailed ultrastructural analysis of the larval pharyngeal sensory organs, including the dorsal pharyngeal sensilla, dorsal pharyngeal organ, ventral pharyngeal sensilla, and posterior pharyngeal sensilla. Using electron microscopy and 3D reconstruction, Richter et al., present a comprehensive mapping and classification of pharyngeal sensory structures, defining the morphological type of pharyngeal sensilla based on ultrastructure and generating a neuron-to-sensillum map. These findings significantly advance our understanding of internal larval sensory systems and establish a robust framework for future functional studies in coordination with external sensory systems.

Strengths:

The application of high-resolution electron microscopy and 3D imaging analysis successfully overcomes technical challenges associated with visualizing deep internal structures. This enables an unprecedented level of anatomical detail of the larval pharyngeal sensory system. Thus, the study complements and completes existing maps of larval sensory circuits, contributing a comprehensive neuroanatomical characterization of larval sensory input pathways. These insights will inform future studies on larval behavior, sensory processing, and may also have applied relevance for insect control strategies.

Weaknesses:

While the manuscript is concise, clearly written, and methodologically rigorous, it primarily addresses a specialized readership with expertise in insect neuroanatomy.

We thank the reviewer for the positive assessment of our study and for the helpful suggestions. In response, we have clarified the visual presentation of the pharyngeal sense organs in Figure 1, expanded the discussion of adult pharyngeal sensory systems, briefly broadened the comparison to other insect species, checked and corrected the scale bars, and added further methodological detail where appropriate.

Reviewer #2 (Public review):

Summary:

This manuscript documents the structure of the pharyngeal nervous system of the Drosophila larva. The authors wanted to achieve a detailed ultrastructural reconstruction of the gustatory sensory organs in the Drosophila pharynx. Using serial EM and the associated bioinformatics tools, they have achieved their goal. The paper is written clearly and illustrated beautifully with 3D models and annotated sections. The data will significantly enrich the field of Drosophila neurobiology.

Strengths:

Given the dataset, the findings presented are solid and will be an important work of reference for the future.

Weaknesses:

Previous work, including EM, on the pharyngeal sensory organ is not sufficiently referenced and used for comparison with the data presented in this study.

We are grateful for the reviewer’s thoughtful comments and for the suggestion to strengthen the historical and comparative context of the work. We have revised the introduction to better acknowledge and discuss the relevant previous EM-based literature on adult and larval internal gustatory sensilla, clarified the organization of the shared pore structure in T1–T3, highlighted the DPO multidendritic neurons more explicitly, and added a comparison that emphasizes the added value of the complete serial EM dataset.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) For improved clarity, highlight the pharyngeal sense organs in Figure 1B. Consider using the color schemes to differentiate between peripheral and internal sensory organs.

We thank the reviewer for this helpful suggestion. We have revised Figure 1 to more clearly separate the pharyngeal sense organs from the external sense organs in the head region. This revision improves visual clarity and accessibility for readers.

(2) In reference to lines 80-84, expand the discussion to address how future studies could explore the conserved morphological and functional characterization of the adult pharyngeal sensory system.

We appreciate this suggestion and have expanded the discussion accordingly. We now briefly address how future work could compare the larval and adult pharyngeal sensory systems to examine conserved morphological and functional features.

(3) To broaden the manuscript's appeal and emphasize its relevance beyond Drosophila, briefly discuss similarities, differences, or conserved roles of pharyngeal sensory systems in other insect species.

Thank you for this valuable recommendation. We have added a paragraph placing the Drosophila pharyngeal sensory system in a broader insect context, including similarities, differences and potential conservation across species.

(4) Recheck the scale bars in all figures, including the supplemental material.

We thank the reviewer for pointing this out. We carefully rechecked all scale bars across the main and supplemental figures and corrected the missing ones.

(5) Consider including additional details on image processing or provide appropriate citations for further reading.

We appreciate this suggestion. We have expanded the methods section to include additional information on technical details and provide the relevant reference for further reading.

Reviewer #2 (Recommendations for the authors):

(1) Line 57ff: The previous literature describes internal gustatory sensilla in considerable detail.

(a) Adult: These sensilla form three complexes, the labral sensory organ, and the ventral and dorsal cibarial sensory organ (Nayak & Singh, 1983, 1985; Singh, 1997; Stocker & Schorderet, 1981; Kendroud et al., 2017). The work by Nayak and Sing includes TEM and presents detailed EM-based schematics. This should be referenced and discussed.

(b) Larva: Gendre et al. 2004, describes the internal gustatory organs and relates them to their adult counterparts:

- Dorsal pharyngeal sense organ (DPS) and dorsal pharyngeal organ DPO) are the forerunners of adult labral and ventral cibarial sensory organs

- Posterior pharyngeal sensory organ (PPS) is the forerunner of the adult dorsal cibarial sensory organ

- Ventral pharyngeal sensory organ (VPS), derived from the labial segment, undergoes apoptosis during metamorphosis

This work, connecting larva and adult (and containing detailed diagrams comparing adult and larval pharyngeal sensilla) should be presented in the introduction.

We thank the reviewer for this important comment. We have revised the introduction to better cite and discuss previous EM-based studies of internal gustatory sensilla in both adult and larval stages, and we now place our findings more explicitly in the context of this prior work.

(2) Line 180: the relationship between the ending of T1-T3 in one shared pore, and the individually wrapped sensilla should be explained; maybe a simple diagram would help. I did not understand how it works. Normally, in a gustatory sensillum, you have one or more sensory neurons, surrounded by thecogen, trichogen, and tormogen cells. The trichogen generates the shaft with the pore at its tip. Now here, in T1-T3, you have three sets of thecogen/trichogen/tormogen. Do all three trichogen cells somehow participate in the shaft with the common pore? Or only a single one, and the other two generate no shaft? It is possible this cannot be resolved, but the authors should address the problem and suggest a possible scenario.

We appreciate the reviewer’s concern and agree that this point required clarification. We have revised the relevant text to better explain the organization of T1-T3 and their shared pore and the organization of the support cells.

(3) Line 205: the DPO multidendritic neurons with dendrites into the hemolymph should be shown; in Figure S4G, I could see only cell bodies. These MD neurons in the gustatory system are, I believe, a true novelty and should be emphasized more if the material allows (text figure!)

Thank you for highlighting this point. We have revised the results and supplementary material to show these neurons more clearly and to emphasize their novelty and potential relevance to the pharyngeal sensory system.

(4) A somewhat detailed comparison between the ultrastructure of the DPS as extracted from the serial EM stack of this study, and the conclusions of Nayak and Singh 1983 as depicted in their diagram Figure 7a would be productive. The idea being: what additional details can (only) a complete EM stack provide, compared to conventional EM.

We appreciate this suggestion. Rather than directly comparing larval and adult structures in detail, we now emphasize what the complete serial EM dataset adds beyond conventional single-section EM, namely a more comprehensive and complete reconstruction of the sensory organs and associated cell types (multidendritic neurons, papilla sensilla, and chordotonal organs that were not described before, organization of support cells)

(5) To round off the work and connect it to the previously published analysis of gustatory terminal arborizations and connectivity in the brain (Miroschnikow et al.,2018), it would be helpful to add an analysis of the distribution of axons from the different sensilla in the nerves. Miroschnikow analyzes the central terminations of the same sense for which the peripheral structure is described here, only that in their L1 connectome, the periphery was cut off. Do the findings of the current study match their predictions, as to the number of sensory neurons, etc? It should be possible to follow, even at the lower resolution of the dataset presented here, to follow axons of sensory neurons through the nerves to the neuropil entry, and thereby make the connection. I consider this to be of great importance for the field, for authors who want to use the data of this study, and the Miroschnikow et al analysis, for their own studies.

We thank the reviewer for this thoughtful and constructive suggestion. We fully agree that linking the peripheral sensory anatomy described in this study to the central projections analyzed by Miroschnikow et al. would be highly valuable and of broad interest. However, a systematic analysis of axon distributions from the different sensilla through the nerves to their neuropil entry points is beyond the scope of the present work. Owing especially to the dataset’s resolution and inherent limitations, tracing the connections from sensory organs through the nerves to their projections in the brain is technically highly challenging and extremely time-consuming, since much of the process would need to be performed manually. We therefore do not include a detailed comparison with the predictions from Miroschnikow et al. in this manuscript. Nevertheless, we appreciate that such an analysis would be an important next step for the field and a useful resource for future studies.

We are grateful for the reviewers’ thoughtful feedback, which has helped us improve the manuscript substantially. We hope that the revised version addresses the concerns raised and better conveys the significance of our work.

No liability when: 1. Duly authorized 2. Adds words to his signature indicating that he signs as an agent 3. He discloses his principal

Mere signing does not complete the contracdt on the instrument. There must be intention and delivery to give effect to the instrument.

There must be actual or constructive transfer of posession.

Immediate parties: those in direct relationship with previous owner(s).

*Conditional delivery allowed

If incomplete and undelivered, it will become a real defense. Can be a defense even against a holder in due course.

Requisites: 1. Instrument is complete and regular upon its face 2. Holder before it was overdue, and no notice that it had been previously dishonored 3. Good faith and for value 4. At the time it was negotiated to him, he had no notice of defects

Cases where date may be inserted:

1. When instrument was issued at a fixed period but undated. (after date)
2. Acceptance is undated and is material to maturity (after sight)

Briefing Document : Violences dans le Périscolaire et Traitement Judiciaire

Résumé Exécutif

Ce document synthétise l'audition de Madame Marie-Suzanne Le Quéau, procureure générale près la cour d'appel de Paris, devant la mission d'information du Sénat dotée des pouvoirs d'une commission d'enquête. L'audition porte sur le traitement judiciaire des violences, notamment sexuelles, commises sur des mineurs dans le cadre périscolaire.

Les points clés à retenir sont les suivants :

• Urgence et volume : La situation à Paris est jugée « vertigineuse » avec 235 enquêtes pénales en cours concernant le milieu scolaire et périscolaire. La Brigade de protection des mineurs (BPM) fait face à une surcharge massive, avec environ 2 900 dossiers en stock.- Priorité pénale : Les infractions sexuelles sur mineurs de moins de 15 ans et les violences périscolaires sont érigées en priorités nationales de politique pénale.- Tensions systémiques : Un fossé subsiste entre l'exigence de célérité des familles et les contraintes judiciaires (secret de l'enquête, présomption d'innocence, recueil complexe de la parole de l'enfant).- Réformes de coordination : Suite à l'affaire « Liana », de nouvelles directives de coordination entre parquets ont été instaurées, privilégiant le lieu de commission des faits comme critère de compétence.- Pistes d'amélioration : La généralisation des Unités d’accueil pédiatrique enfant en danger (UAPED) et un meilleur accompagnement des familles via des associations spécialisées sont identifiés comme des leviers essentiels.

I. État des Lieux et Priorités de la Politique Pénale

La procureure générale souligne que son rôle consiste à décliner régionalement la politique pénale fixée par le gouvernement. Actuellement, trois priorités majeures occupent le ministère public :

• La lutte contre les violences intrafamiliales.- Les infractions sexuelles commises sur les mineurs de moins de 15 ans.- Les violences dans le champ périscolaire.

Données chiffrées (Ressort de Paris)

Le tableau suivant présente les données critiques identifiées lors de l'audition :

| Indicateur | Valeur/Détails | | --- | --- | | Enquêtes en cours à Paris | 235 procédures (milieux scolaire et périscolaire) | | Stock total de la BPM (Paris) | ~ 2 900 affaires au moment de l'audition | | Procédures en Seine-Saint-Denis | 5 procédures identifiées (à date) | | Profil des victimes | Jeunes enfants (2 à 5 ans) majoritairement |

L'ampleur du contentieux parisien interroge sur l'existence d'une « faille systémique », bien que la procureure générale suggère que la densité et l'organisation du système périscolaire parisien puissent également expliquer ce volume élevé de signalements.

II. Les Défis du Recueil de la Preuve et de la Parole

Le traitement des dossiers impliquant de très jeunes enfants (maternelles) présente des difficultés techniques majeures qui peuvent affaiblir l'action publique.

La fragilité de la parole

La parole de l'enfant est décrite comme « déterminante mais extrêmement fragile ». Plusieurs facteurs entravent la condamnation :

• Délais de prise en charge : La surcharge des services (BPM) entraîne des auditions tardives. Or, chez un enfant de 2 à 5 ans, les souvenirs s'estompent ou se diffusent en quelques semaines.- Complexité de la qualification : La frontière entre un comportement « inapproprié » ou « inadapté » et une agression sexuelle caractérisée est parfois ténue, rendant les décisions de poursuite complexes pour les magistrats.- L'exigence de preuve : Pour condamner, le magistrat doit disposer d'éléments faisant pencher la balance au-delà du doute. Un état de « 50/50 » conduit inévitablement au classement sans suite ou à la relaxe.

III. Gestion des Victimes : Entre Secret de l'Enquête et Besoin d'Information

Une tension forte existe entre les familles, qui dénoncent une forme d'« omerta » et un sentiment d'abandon, et l'institution judiciaire, tenue par des règles strictes.

Obstacles à la communication

• Secret de l'enquête : Nécessaire pour ne pas compromettre les investigations, il limite l'information des parents durant la phase de flagrance ou d'enquête préliminaire.- Présomption d'innocence : Ce principe impose une prudence extrême dans la communication et la suspension des agents mis en cause, afin d'éviter des préjudices irréparables en cas de relaxe ultérieure.- Déficit pédagogique : La procureure reconnaît que l'absence d'information sur l'état d'avancement des dossiers nourrit la défiance des citoyens envers la justice.

Pistes d'accompagnement

La procureure suggère d'impliquer davantage les associations d'aide aux victimes et des avocats spécialisés dès la phase d'enquête pour expliquer la procédure aux familles et rompre leur isolement.

IV. Coordination Inter-Juridictionnelle et Enseignements Post-Liana

L'audition a mis en lumière la nécessité d'une meilleure circulation de l'information entre les différents parquets pour éviter que des agresseurs potentiels ne « disparaissent dans la nature » en changeant de département.

Nouvelles règles de compétence

Suite aux défaillances identifiées dans l'affaire « Liana », la Direction des affaires criminelles et des grâces (DACG) a précisé les règles :

• Critère prioritaire : Le lieu de commission de l'infraction.- Critère subsidiaire : Le lieu de domicile de l'auteur (soumis à un accord entre parquets).- Échanges techniques : Un groupe de travail a été mis en place à la cour d'appel de Paris pour harmoniser les procédures de dessaisissement (utilisation du mail, téléphone, fiches pratiques).

Outils de suivi

La justice dispose du bureau d'ordre national Cassiopée, qui permet de vérifier si un individu fait l'objet de plusieurs enquêtes sur le territoire national, à condition que ces affaires aient été portées à la connaissance de la justice et ne soient pas bloquées dans les stocks de la police ou de la gendarmerie.

V. Vers une Procédure Idéale

En conclusion, l'audition dessine les contours d'une prise en charge plus efficace et humaine des violences périscolaires.

• Généralisation des UAPED : Créer des lieux uniques où l'enfant est entendu par des services spécialisés, avec la présence de psychiatres, psychologues et assistantes sociales.- Célérité accrue : Réduire le délai entre le signalement et la première audition pour préserver la mémoire de l'enfant.- Transparence du Ministère Public : Améliorer la motivation des classements sans suite et envisager, malgré les contraintes d'effectifs, des rendez-vous physiques pour expliquer les décisions judiciaires aux familles.- Décloisonnement : Renforcer la transmission d'informations entre la justice, l'Éducation nationale et les municipalités (employeurs), tout en respectant le cadre légal de la présomption d'innocence.

Prévention et traitement des violences dans le périscolaire : Synthèse des auditions des élus locaux au Sénat

Résumé exécutif

Cette synthèse détaille les travaux de la mission d'information du Sénat portant sur les violences commises par des animateurs en milieu périscolaire. L'analyse met en lumière un système de protection de l'enfance gravement défaillant, marqué par un fonctionnement "en silos" entre l'Éducation nationale, les municipalités et l'institution judiciaire.

Les points de rupture identifiés sont les suivants :

• Isolement institutionnel des maires : Les élus se retrouvent en première ligne face à l'émotion des familles tout en étant tenus à l'écart des informations judiciaires par le secret de l'enquête.- Failles du recrutement : L'accès limité aux fichiers de police (TAJ) et l'effacement légal des casiers judiciaires (B2) après quelques années créent des "angles morts" permettant à des prédateurs de réintégrer le circuit.- Déni structurel : Une culture du "pas de vague" a longtemps prévalu, entravant la remontée des signalements et le traitement efficace des suspicions d'agression.- Urgence d'une réforme nationale : Les élus appellent à une uniformisation des protocoles, à une formation obligatoire à la détection des "signaux faibles" et à la création d'un fichier national des prédateurs sexuels accessible aux employeurs.

1. Les failles systémiques du recrutement et du contrôle

Les auditions révèlent que les mécanismes actuels de vérification des antécédents sont insuffisants pour garantir la sécurité des enfants.

L'illusion du Casier Judiciaire (Bulletin B2)

• Effacement légal : Les maires soulignent qu'en France, les peines pénales s'effacent automatiquement du B2 après 5 à 10 ans. Un animateur condamné pour agression sexuelle par le passé peut ainsi présenter un casier vierge à 40 ans.- Accès restreint au TAJ : Contrairement aux forces de l'ordre, les mairies n'ont pas accès au Traitement des Antécédents Judiciaires (TAJ), qui conserve pourtant la mémoire des faits, même après effacement du casier.- Le cas des mineurs : À leur majorité, les condamnations prononcées à l'encontre d'agresseurs mineurs sont souvent annulées, leur permettant de postuler à des postes d'animateurs avec une attestation d'honorabilité vierge.

Une filière sous tension

• Pénurie et précarité : La filière de l'animation est décrite comme "tendue" (notamment à Vincennes ou Charly), avec des horaires hachés et des salaires bas, ce qui nuit à la qualité du recrutement.- Recrutements dans l'urgence : Le cas de Créteil illustre les dérives possibles : une journaliste a pu se faire recruter en 8 minutes sans vérification de casier, l'agent municipal ayant privilégié le respect des taux d'encadrement sur la sécurité du recrutement.- Limites du BAFA : Le brevet d'aptitude aux fonctions d'animateur (BAFA) n'est pas considéré comme un "certificat de non-agression". Il manque de modules approfondis sur la protection de l'enfance.

2. La rupture de communication entre la Justice et les Collectivités

Le "tunnel judiciaire" est identifié comme l'un des obstacles majeurs à la gestion des crises par les élus locaux.

Le silence opposé aux maires

• Absence de retour automatique : Si un parent dépose plainte directement au commissariat sans passer par la mairie, l'élu (employeur) n'en est pas informé. Il ne peut donc pas suspendre l'agent suspecté immédiatement.- Déphasage des temporalités : La suspension administrative est limitée dans le temps, tandis que le temps de la justice est long. Des maires se retrouvent à devoir réintégrer des agents dont l'enquête est toujours en cours ou classée "sans suite" sans en connaître les raisons.- Injonctions de non-communication : Les procureurs demandent souvent aux maires de ne pas communiquer avec les parents pour ne pas entraver l'enquête, ce qui est perçu par les familles comme une volonté de dissimulation (omertà).

Les "auditions sauvages"

• Des incidents graves ont été rapportés (Vincennes, Colombe) où la brigade des mineurs est intervenue dans des classes pour entendre des dizaines d'enfants sans que les parents ou le maire n'aient été prévenus au préalable. Ce mode opératoire est dénoncé comme une "seconde victimisation" traumatisante pour les mineurs.

3. Le traumatisme des familles et la gestion de la parole

La prise en charge de la parole de l'enfant reste le maillon faible de l'organisation périscolaire.

• Le mépris de la parole de l'enfant : Les maires constatent un déni structurel : tant qu'il n'y a pas de signalement explicite, on considère qu'il n'y a pas de problème. Or, un enfant ne parle pas "sur commande" et exprime souvent son traumatisme par des comportements (signaux faibles) plutôt que par des mots.- Accompagnement vs Communication : Le maire de Colombe distingue l'accompagnement (soutien psychologique, réunions) de la communication (faits de l'enquête). Le manque d'information transforme les réunions publiques en "tribunaux populaires" où les élus sont pris pour cibles.- Le rôle pivot des parents : Ce sont presque exclusivement les familles qui font remonter les informations. Les institutions (école, mairie) échouent souvent à détecter les faits en interne.

4. Recommandations et axes de réforme

Les élus proposent plusieurs leviers pour transformer durablement la protection de l'enfance en milieu périscolaire.

| Axe d'intervention | Propositions concrètes | | --- | --- | | Arsenal Législatif | Création d'un "Fichier S" des prédateurs sexuels incluant les mis en cause et les classements sans suite. | | | Reconnaissance d'un statut sanctuarisé et protégé pour l'enfant. | | | Rendre les crimes sexuels sur mineurs imprescriptibles. | | Partage d'Information | Mise en place d'un "secret partagé" entre le procureur, le préfet et le maire. | | | Ritualisation des échanges au sein des CLSPDR (Conseils Locaux de Sécurité et de Prévention de la Délinquance). | | Formation & Protocoles | Rendre la formation à la détection des signaux faibles obligatoire pour tous les agents. | | | Intégrer un module "Protection de l'enfance" au BAFA ou créer un diplôme d'État. | | | Généraliser les boîtes aux lettres (type association Les Papillons) pour libérer la parole. | | Organisation | Mettre fin à la gestion en silos : unification de l'autorité scolaire et périscolaire. | | | Création de référents protection de l'enfance clairement identifiés dans chaque structure. |

Citations clés

"La présomption d'innocence ne doit pas se traduire par un silence opposé aux familles." — Agnès Évren, Rapporteur.

"Un enfant ne vit pas en silo. [...] La protection de l'enfance ne peut pas être organisée en fonction de nos frontières administratives, elle doit être organisée autour de l'enfant." — Olivier Arojot, Maire de Charly.

"On mesure la grandeur d'une nation [...] à la manière avec laquelle on protège nos femmes et nos enfants." — Karim Bouamrane, Maire de Saint-Ouen.

"Un classement sans suite n’est pas un certificat d’innocence. Cela ne veut pas dire que les faits n’ont pas eu lieu." — Agnès Évren, Rapporteur.

"Le sentiment d'abandon institutionnel des élus locaux est immense lorsqu'une procédure judiciaire commence." — Olivier Arojot, Maire de Charly.

Synthèse de l'audition : Prévention et traitement des violences dans le milieu périscolaire

Résumé exécutif

L'audition de Laure Beccuau, procureure de la République de Paris, et de son adjoint Laurent Guy, révèle une crise profonde au sein du milieu périscolaire et scolaire, marquée par une « massification » sans précédent des signalements de violences physiques et sexuelles sur mineurs. À Paris, plus de 235 enquêtes pénales sont en cours pour des faits commis dans ce cadre, un chiffre qui s'inscrit dans un volume global alarmant de 2 900 enquêtes pour violences sexuelles sur mineurs traitées par le parquet.

Les principaux constats mettent en lumière une rupture de confiance entre les familles et les institutions (justice, école, mairies). Cette défiance est alimentée par des délais de procédure jugés incompatibles avec la psychologie de l'enfant, un manque criant de communication envers les victimes, et une « omerta » institutionnelle persistante. Pour répondre à cet « électrochoc », le parquet de Paris déploie un plan d'action visant à lutter contre la victimisation secondaire, à renforcer la formation des intervenants et à généraliser des dispositifs d'accueil spécialisés (Salles Mélanie). L'enjeu majeur reste la conciliation entre l'exigence de qualité de la preuve pénale et l'urgence de protéger les enfants dans un système aux moyens contraints.

I. État des lieux et données chiffrées

L'ampleur du phénomène à Paris, bien que perçue comme exceptionnelle, reflète une réalité nationale touchant près de 70 départements. Le périscolaire est désormais qualifié d'« angle mort » de la protection de l'enfance.

Statistiques clés du Parquet de Paris

| Catégorie | Données chiffrées | | --- | --- | | Enquêtes périscolaires/scolaires en cours | 235 enquêtes pénales | | Établissements concernés (mai 2024) | 84 écoles maternelles, ~20 élémentaires, ~10 crèches | | Volume global violences sexuelles mineurs | 2 900 enquêtes actives (le périscolaire représente ~10%) | | Évolution des signalements | Passage de 5-7 par mois à une vingtaine depuis fin 2023 | | Origine des procédures | 60% de signalements administratifs / 40% de plaintes initiales |

La « massification » observée s'explique par une libération de la parole suite à la médiatisation de certaines affaires et au rôle croissant des réseaux sociaux entre parents.

II. Les défis majeurs du traitement judiciaire

L'institution judiciaire fait face à une confrontation entre deux exigences contradictoires : la rapidité nécessaire au temps de l'enfant et la rigueur de la preuve pénale.

1. La fragilité de la parole de l'enfant

• Temporalité : Chez les enfants de 3 à 5 ans, la mémoire s'efface ou se fragilise en quelques semaines. Or, les délais d'audition peuvent atteindre plusieurs mois.- Suggestibilité : Les enfants en bas âge sont très sensibles aux questions suggestives. Des interrogatoires répétés par les parents ou des services non formés peuvent induire des biais et affaiblir la valeur probatoire du témoignage.- Conditions d'audition : Le manque de salles spécialisées (Salles Mélanie) a conduit à des auditions traumatisantes en commissariat par des policiers en uniforme, ce que le parquet cherche désormais à proscrire.

2. Les délais et la chaîne de la preuve

• Durée des enquêtes : Une enquête standard dure entre 4 et 5 mois, mais peut s'étendre à 10 mois ou plusieurs années (instruction) en cas de multiplicité de victimes ou d'auteurs.- Complexité de l'étayage : La parole seule est rarement suffisante pour obtenir une condamnation. Elle doit être corroborée par des expertises médico-psychologiques, des enquêtes d'environnement et des vérifications matérielles.

III. Dysfonctionnements institutionnels et « Victimisation Secondaire »

Les sénateurs et la procureure soulignent plusieurs points de friction majeurs qui aggravent le traumatisme des familles.

Le silence et l'absence d'information

Le secret de l'enquête est trop souvent opposé aux parents comme un « silence de mépris ».

• Classements sans suite : Beaucoup de parents découvrent par hasard le classement de leur plainte. Le parquet reconnaît que l'absence d'explication personnalisée est une « seconde violence » pour les familles.- Sentiment d'abandon : La longueur des procédures, sans nouvelles du parquet ou de la police, brise la confiance dans l'institution judiciaire.

Obstacles opérationnels et juridiques

• Refus de plainte : Des cas de refus de prise de plainte en commissariat ont été signalés. La procureure qualifie ces faits d'« inadmissibles » et contraires au code de procédure pénale.- Incohérence des délais de suspension : Il existe une déconnexion entre le temps judiciaire et le temps administratif. Les suspensions conservatoires d'agents (souvent 4 mois renouvelables) sont fréquemment plus courtes que les délais d'enquête, créant des situations où des mis en cause pourraient reprendre leur poste avant la fin des investigations.

IV. Stratégies de réforme et axes d'amélioration

Le parquet de Paris a initié une réorganisation profonde pour traiter cette crise.

1. Plan d'action contre la victimisation secondaire

Depuis le 1er septembre, un plan de 27 mesures est déployé, incluant :

• L'individualisation systématique des avis de classement sans suite.- Une meilleure information des plaignants tout au long de la chaîne pénale.- La création de groupes de travail incluant des collectifs de victimes pour ajuster les réponses institutionnelles.

2. Renforcement des moyens techniques et humains

• Salles Mélanie : Signature d'une convention avec la Ville de Paris pour généraliser ces salles dans tous les hôpitaux pédiatriques et les Unités Médico-Judiciaires (UMJ).- Spécialisation : Désignation d'un magistrat dédié par semaine au parquet des mineurs pour suivre les dossiers de violences sexuelles.- Formation : Nécessité de former les policiers, gendarmes, magistrats et professionnels du périscolaire au psychotrauma et au recueil de la parole.

3. Coopération institutionnelle et prévention

• Article 40 : Rappel ferme du devoir des fonctionnaires (Éducation nationale, mairies) de signaler systématiquement les faits dont ils ont connaissance.- Révision des protocoles : Mise à jour des conventions de 2012 avec la Mairie de Paris et l'Éducation nationale pour fluidifier les échanges d'informations sur le statut des mis en cause.- Prévention primaire : Apprendre aux enfants à « savoir dire non » et former les professionnels à « écouter » plutôt qu'à simplement « entendre » les alertes.

Citations clés

« Les violences périscolaires, c'est à peu près 10 % des dossiers que nous avons à traiter sur les 2 900 enquêtes en cours pour violences sexuelles sur mineurs. » — Laure Beccuau

« Le secret de l'enquête ne peut pas devenir le silence opposé aux victimes. » — Agnès Canayer (Rapporteur)

« Les refus de dépôt de plainte, c’est inadmissible. Cela ne doit pas avoir lieu. C’est une obligation légale. » — Laure Beccuau

« On n'a pas cru mon enfant : c'est la phrase terrible qui revient dans chaque témoignage. » — Agnès Canayer (Rapporteur)

Audition de la Brigade de Protection des Mineurs de Paris : Synthèse des Enjeux et des Procédures

Résumé Exécutif

Ce document de synthèse relate les points clés de l'audition de la Brigade de Protection des Mineurs (BPM) de Paris devant une commission d'information du Sénat. L'audition se concentre sur le traitement des violences sexuelles commises sur mineurs dans le cadre périscolaire.

Les points majeurs identifiés sont :

• Une charge de travail massive : 235 procédures sont actuellement en cours à Paris dans le seul domaine périscolaire, s'inscrivant dans un volume global de 2 900 signalements annuels pour infractions sexuelles sur mineurs.- La primauté du protocole NICHT : Méthode de référence pour l'audition des enfants de 2 à 12 ans, ce protocole privilégie la "pureté" du témoignage sans influence extérieure (parents ou avocats), malgré les critiques des familles sur la froideur apparente du processus.- Une déconnexion entre attentes et procédures : Un décalage significatif existe entre le ressenti des parents (délais longs, manque d'empathie, sentiment de "planning contraint") et les exigences de la preuve pénale, qui nécessite une parole non polluée pour être recevable en justice.- Le cloisonnement institutionnel : Le fonctionnement dit en "tuyaux d'orgue" entre les services sociaux, l'Éducation nationale et la police nuit à l'efficacité des enquêtes et peut engendrer une victimisation secondaire pour l'enfant.- Des enjeux matériels critiques : La disponibilité des "Salles Mélanie" et le temps nécessaire à la retranscription exhaustive des auditions (verbal et non-verbal) constituent des goulots d'étranglement opérationnels.

1. Le Recueil de la Parole : Le Protocole NICHT

Le recueil de la parole de l'enfant est l'élément central de l'enquête, car les preuves physiques font souvent défaut dans les affaires de violences sexuelles. La BPM utilise le protocole NICHT (spécifique aux enfants de 2 à 12 ans).

Les phases de l'audition

Contrairement à la perception des parents, l'audition ne se limite pas au temps passé dans la salle d'enregistrement.

• La mise en confiance : L'enfant est accueilli dans un environnement adapté (jouets, dessins). L'enquêteur discute avec lui, lui fait visiter les locaux pour établir un lien et évaluer ses capacités cognitives (mémoire, temporalité).- L'audition proprement dite : Elle se déroule en "Salle Mélanie". Elle peut être courte en raison de la faible capacité de concentration des très jeunes enfants.- La retranscription : Phase extrêmement lourde où l'enquêteur retranscrit le dit et le non-verbal (gestes, comportement).

L'exclusion des tiers

La présence des parents ou d'avocats lors de l'audition est proscrite par le protocole pour plusieurs raisons :

• Éviter l'influence : L'enfant pourrait chercher à faire plaisir à son parent ou craindre de l'attrister.- Garantir la recevabilité pénale : Un témoignage perçu comme "pollué" ou influencé est immédiatement fragilisé par la défense devant un tribunal ou une cour d'assises.- Neutralité des questions : Les policiers privilégient les questions ouvertes pour ne pas orienter les réponses, contrairement aux familles ou à d'autres intervenants qui posent souvent des questions fermées ("Est-ce que c'est Monsieur X ?").

2. Tension entre les Familles et l'Institution

Le rapporteur du Sénat souligne un "désarroi" et une "colère" des familles face à l'accueil de la BPM.

| Perception des Parents | Réponse de la BPM | | --- | --- | | Manque d'empathie | Les enquêteurs sont investis mais doivent rester neutres pour la procédure pénale. | | Auditions trop courtes (20 min) | La concentration d'un enfant est limitée ; le travail préparatoire n'est pas toujours perçu par les parents. | | Délais d'attente (semaines/mois) | Dus à la surcharge (ex: 44 enfants à entendre dans un seul dossier) et à la disponibilité des experts/psychologues. | | Sentiment d'être "puni" | Les enquêteurs travaillent en civil dans des locaux décorés pour atténuer l'aspect policier. |

3. Contraintes Opérationnelles et Logistiques

Capacité d'accueil et salles spécialisées

La BPM dispose de salles aménagées appelées Salles Mélanie. Bien qu'une seule salle soit actuellement pleinement utilisée à la BPM (l'autre servant de bureau aux renforts), de nouvelles salles doivent ouvrir dans les Unités Médico-Judiciaires (UMJ) de Necker, Trousseau et Robert Debré.

Personnel et expertise

• La BPM est composée de 80 % de femmes.- Le manque d'enquêteurs formés n'est pas le frein principal ; la difficulté réside dans la coordination avec les experts (psychologues, médecins légistes des UMJ) et la charge de retranscription.

Priorisation des dossiers

Devant l'afflux d'affaires (périscolaire, inceste, proxénétisme de mineurs, pédopornographie), la BPM priorise selon :

• Le risque immédiat pour l'enfant.- La réitération des faits.- Le profil et les antécédents de la personne mise en cause (criblage).

4. La Problématique des "Tuyaux d'Orgue"

Le manque de communication entre les différents acteurs de la protection de l'enfance est vivement critiqué.

• Victimisation secondaire : Lorsque l'enfant est entendu par l'école, les services sociaux, puis la police, il revit son traumatisme à chaque fois. Cela modifie son discours et fragilise la preuve pénale.- Absence de partage d'information : La BPM déplore ne pas savoir systématiquement ce que font les services sociaux en parallèle.- Information des employeurs : Il n'existe pas de procédure systématique pour informer tous les employeurs (actuels ou passés) d'un suspect mis en cause dans une structure. La BPM est liée par le secret de l'enquête, et cette responsabilité incombe au procureur.

5. Préservation des Preuves et Enquêtes Numériques

Une préoccupation majeure concerne la disparition des preuves numériques (photos, fichiers) avant l'interpellation d'un suspect, notamment lorsque celui-ci est suspendu administrativement par son employeur avant l'action policière.

• Méthodes d'investigation : La BPM affirme disposer de capacités techniques pour vérifier certains éléments sans saisie immédiate du matériel.- Exploitation technique : Un groupe dédié au sein de la police judiciaire réalise des extractions de données même si le suspect a tenté de supprimer des fichiers.- Perquisitions : Elles ont généralement lieu au moment de l'interpellation et du placement en garde à vue pour maximiser les chances de saisie.

Citations Clés

"L'audition nich n'est pas une audition d'adulte c'est une audition qui fait appel à tout le reste [...] elle est filmée également de manière à pouvoir justement voir ce mouvement." — Ton Nicolas, Directeur adjoint de la PJ.

"Quand vous ne pouvez pas traiter suffisamment vite, qui en paye le prix de ce retard ? Ce ne sont pas les institutions, ce sont les enfants traumatisés." — Agnès Evren, Rapporteur.

"Nous ne sommes pas en capacité de pouvoir traiter avec suffisamment de diligence les très nombreux dossiers relatifs au périscolaire." — Extrait du questionnaire de la BPM cité lors de l'audition.

"Le but d'une affaire pénale c'est d'arriver finalement justice et que ça puisse être tranchée c'est l'audition qui semblera la plus naturelle la moins finalement compromise." — Vincent Cosserov, Chef de la BPM.

Analyse du Déclin du Niveau Scolaire en France : Enjeux, Causes et Perspectives

Résumé Exécutif

Les récents résultats du classement PISA révèlent une situation alarmante pour le système éducatif français, marquée par une chute historique du niveau des élèves, particulièrement en mathématiques et en compréhension de l'écrit. En dix ans, la France a perdu l'équivalent de près de deux années d'apprentissage. Ce déclin, bien que s'inscrivant dans une tendance mondiale à la baisse, place désormais la France dans la moyenne basse des pays de l'OCDE.

Le débat sur le redressement du niveau scolaire oppose deux visions : d'un côté, une analyse pointant la responsabilité des réformes structurelles des dix dernières années et le manque d'attractivité du métier d'enseignant ; de l'autre, une lecture soulignant l'impact délétère de la surexposition aux écrans et des mutations sociétales. Trois leviers majeurs de remédiation émergent : l'exploitation du "dividende démographique" pour réduire les effectifs par classe, la revalorisation de la condition enseignante (salaire, formation, mobilité) et la régulation stricte de l'usage des technologies numériques.

Le Constat : Une Chute Historique des Performances

Le classement PISA, enquête internationale de l'OCDE menée tous les trois ans, sert de baromètre mondial pour évaluer les compétences des élèves de 15 ans. Les derniers résultats indiquent un décrochage massif de la France.

Indicateurs de Performance (Évolution sur 10 ans)

| Domaine de compétence | Perte de points | Équivalence pédagogique | | --- | --- | --- | | Lecture / Compréhension de l'écrit | \- 18 points | Environ 1 an d'apprentissage | | Mathématiques | \- 35 points | Près de 2 ans d'apprentissage |

Observations clés :

• Positionnement : La France est passée de la moyenne haute à la moyenne basse du classement.- Effet Post-Covid : Contrairement aux espoirs d'un rebond après la crise sanitaire, la "dégringolade" s'est accentuée, prouvant que la fermeture des classes n'est pas l'unique facteur de baisse.- Inégalités : Le système français reste marqué par des inégalités extrêmes, avec un tiers des élèves en très grande difficulté, notamment dans les lycées professionnels.

Les Facteurs de Déclin : Une Multicausalité en Débat

L'analyse des causes du décrochage scolaire identifie des facteurs internes au système éducatif et des influences sociétales externes.

1. L'Impact des Écrans et du Numérique

Le ministère de l'Éducation nationale pointe une "rupture éducative" liée à la consommation massive des réseaux sociaux :

• Délégation du savoir : Une tendance à déléguer l'éducation aux plateformes numériques et aux réseaux sociaux.- Érosion de l'attention : Le format "court" et "accéléré" des contenus (type TikTok) réduit le temps d'attention des élèves, passant de 8 minutes il y a dix ans à environ 3 minutes aujourd'hui.- Abandon de la lecture : Un déclin massif de la lecture au profit d'une surexposition aux écrans, entraînant une perte de vocabulaire et de syntaxe.

2. La Condition Enseignante et l'Attractivité

Le manque d'attractivité du métier est identifié comme un frein majeur :

• Rémunération : En début de carrière, un enseignant français gagne 50 % de moins que son homologue allemand.- Mobilité et Affectation : Les contraintes géographiques et les premières affectations souvent éloignées et dans des quartiers difficiles découragent les vocations ou provoquent des démissions.- Formation : Un paradoxe est relevé : les professionnels chargés de former sont ceux qui reçoivent le moins de formation continue tout au long de leur carrière.

3. Les Rythmes et l'Organisation Scolaire

• Rythmes scolaires : Les journées françaises (souvent de 8h à 18h) sont jugées trop denses par rapport aux pays en tête du classement PISA, qui privilégient des semaines plus légères étalées sur plus de semaines dans l'année.- Effectifs : Bien que des efforts de dédoublement aient été faits (touchant 20 % d'une classe d'âge en REP/REP+), la France conserve des effectifs globaux plus élevés que la moyenne des pays performants.

Analyse des Politiques Éducatives et Controverses

Le bilan des politiques menées sous la présidence d'Emmanuel Macron fait l'objet de vifs échanges.

Le Bilan du "Macronisme Scolaire"

• Défenseurs du bilan : Ils soutiennent que les réformes (dédoublement des CP/CE1, nouveaux programmes) visent le temps long. Les élèves testés par PISA en 2022 (nés en 2010) n'ont pas encore bénéficié de ces mesures, qui ont commencé en 2011 pour une partie de la population.- Critiques : Les opposants estiment que dix ans au pouvoir permettent de juger une politique. Ils soulignent une "humiliation" nationale face aux résultats et dénoncent une tendance du gouvernement à se défausser sur les parents ou les réseaux sociaux pour éviter d'assumer la responsabilité de l'école.

La Relation Parents-École

L'enquête PISA révèle une distension du lien éducatif :

• Seuls 63 % des jeunes de 15 ans déclarent que leurs parents s'intéressent à leur scolarité au moins une fois par semaine, contre 74 % trois ans plus tôt.- L'usage d'outils comme Pronote est paradoxalement critiqué : en permettant un suivi numérique des notes, il tuerait le dialogue direct entre parents et enfants sur les apprentissages.

Perspectives de Redressement : Pistes et Solutions

1. Exploiter le "Dividende Démographique"

D'ici 2035, la France comptera 1,7 million d'élèves en moins. Ce recul doit être vu comme une opportunité historique :

• Objectif : Ne pas réduire les budgets mais utiliser cette baisse pour diminuer drastiquement le nombre d'élèves par classe (viser 20 élèves par classe).- Maintien du service public : Assurer la présence de l'école dans les territoires ruraux malgré la baisse des effectifs.

2. Revalorisation et Recrutement

• Salaire : Des propositions visent une augmentation socle de 10 % à 20 % selon le déroulement de la carrière.- Concours : La possibilité de passer le concours à Bac+3 (au lieu de Bac+5) est avancée pour remplir les concours de recrutement qui sont actuellement déficitaires.- Autonomie : Donner plus d'agilité aux établissements pour gérer les remplacements et les recrutements sur "postes à profil".

3. Évolutions Pédagogiques et Technologiques

• Pédagogie Explicite : S'inspirer du modèle anglais (10ème au classement PISA) qui privilégie une pédagogie énonçant clairement les objectifs, plus efficace pour les élèves en difficulté que les méthodes de construction autonome du savoir.- Sanctuarisation de l'école : Interdiction stricte du téléphone portable au collège et au lycée pour créer des "sas" d'attention.- Régulation des Algorithmes : Agir au niveau politique pour imposer des contenus éducatifs sur les plateformes (à l'instar du modèle chinois de TikTok) et protéger les mineurs des contenus violents.- IA Générative : Former les enseignants à l'usage de l'intelligence artificielle pour personnaliser les apprentissages et l'aide aux devoirs.

eLife Assessment

This important study provides mechanistic evidence for how the tea-adapted Kanzawa spider mite, Tetranychus kanzawai, overcomes the catechin-based defenses of green tea plants. The work identifies the horizontally transferred dioxygenase DOG15 as a key contributor to host adaptation and supports a two-step model involving evolutionary modification of enzyme activity together with strong inducible upregulation upon feeding on tea. The evidence is convincing because comparative behavioral and toxicological assays, transcriptomic and proteomic analyses, RNAi-mediated functional validation, and recombinant enzyme assays converge to link DOG15 activity and expression with improved performance on tea. The revised manuscript appropriately acknowledges that the products of catechin cleavage have not yet been characterized and that additional detoxification pathways may contribute to tea adaptation, providing a balanced interpretation of the otherwise strong mechanistic evidence.

Reviewer #1 (Public review):

Summary:

This study investigates the molecular mechanisms allowing the KSM mite to infest tea plants, a host that is toxic to the closely related TSSM mite due to high concentrations of phenolic catechins. The authors utilize a comparative approach involving tea-adapted KSM, non-adapted KSM, and TSSM to assess behavioral avoidance and physiological tolerance to catechins. The main finding is that tea-adapted KSM possesses a specific detoxification mechanism mediated by an enzyme, TkDOG15, which was acquired via horizontal gene transfer. The study demonstrates that adaptation is a two-step process: (1) structural refinement of the TkDOG15 enzyme through amino acid substitutions that enhance enzymatic efficiency against catechins, and (2) significant transcriptional upregulation of this gene in response to tea feeding. This enzymatic adaptation allows the mites to cleave and detoxify tea catechins, enabling survival on a toxic host plant.

Strengths:

A multiomics approach (transcriptomics and proteomics) provided a compelling cross-validation of its findings. Functional bioassays, such as RNAi and recombinant enzyme assays, demonstrated that the adapted mite has higher activity against catechins via TkDOG15. Other methodologies, like feeding assay using a parafilm-covered leaf disc, were effective in avoiding contact chemosensation.

Comments on revised version.

The authors have satisfied all previous concerns through necessary text revisions and clarified discussions. The manuscript is now well-balanced and scientifically sound.

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Comments on revised version.

I believe the manuscript has been significantly refined since the initial draft was submitted.

Author response:

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

eLife Assessment:

This important study provides mechanistic evidence that tea-adapted two-spotted spider mite overcomes green tea catechin defenses via the horizontally transferred dioxygenase TkDOG15, supporting a two-step adaptation model, combining enzyme refinement and inducible upregulation. The evidence is convincing because multi-omics signals converge with functional validation (RNAi knockdown and recombinant enzyme assays) and well-controlled behavioral/toxicity assays to link TkDOG15 activity and expression to survival and feeding on tea.

We thank the editors and reviewers for this positive assessment of the importance of our study and the strength of the evidence. We would like to point out one factual correction. The assessment describes the tea-adapted mite as the "two-spotted spider mite" (TSSM, Tetranychus urticae), but the species adapted to tea in this study is the Kanzawa spider mite (KSM, Tetranychus kanzawai). We would suggest revising "tea-adapted two-spotted spider mite" to "tea-adapted spider mite" or "tea-adapted Kanzawa spider mite" accordingly.

Reviewer #1 (Public review):

Summary:

This study investigates the molecular mechanisms allowing the KSM mite to infest tea plants, a host that is toxic to the closely related TSSM mite due to high concentrations of phenolic catechins. The authors utilize a comparative approach involving tea-adapted KSM, non-adapted KSM, and TSSM to assess behavioral avoidance and physiological tolerance to catechins. The main finding is that tea-adapted KSM possesses a specific detoxification mechanism mediated by an enzyme, TkDOG15, which was acquired via horizontal gene transfer. The study demonstrates that adaptation is a two-step process: (1) structural refinement of the TkDOG15 enzyme through amino acid substitutions that enhance enzymatic efficiency against catechins, and (2) significant transcriptional upregulation of this gene in response to tea feeding. This enzymatic adaptation allows the mites to cleave and detoxify tea catechins, enabling survival on a toxic host plant.

Strengths:

A multiomics approach (transcriptomics and proteomics) provided a compelling crossvalidation of its findings. Functional bioassays, such as RNAi and recombinant enzyme assays, demonstrated that the adapted mite has higher activity against catechins via TkDOG15. Other methodologies, like feeding assay using a parafilm-covered leaf disc, were effective in avoiding contact chemosensation.

Weaknesses:

Although TkDOG15 is assumed to "detoxify" catechins by ring cleavage, the study doesn't identify or characterize the breakdown metabolic products. If the metabolites are indeed non-toxic compared to the parent catechins, that would strengthen the detoxification hypothesis. Also, the transcriptomic and proteomic analyses identified other potential detoxification enzymes, such as CCEs, UGTs, and ABC (Supplementary Tables 3-1 & 3-2), which were also upregulated. The manuscript focuses almost exclusively on TkDOG15, potentially overlooking a multigenic adaptation mechanism, where these other enzymes might play synergistic roles, although it was mentioned in the discussion section.

Reviewer #1 (Recommendations for the authors):

There is no need for additional experiments, but I suggest revising the discussion section to mention the weaknesses pointed out above.

We thank the reviewer for the positive assessment and helpful suggestions. We have revised the Discussion (L276-283) to address both points as limitations. First, we now note that we did not characterize the products of TkDOG15-mediated catechin cleavage, and that confirming their reduced toxicity relative to the parent catechins would further support its detoxification role. We note this as a direction for future work. Second, we note that DOG15 in KSM on tea was the only enzyme upregulated at both the mRNA and protein levels, whereas the CCEs, UGTs, ABC transporter, and other DOGs were enriched in only one dataset. We now state that tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15 and warranting functional validation.

Minor corrections below:

(1) Figure 1a: For better readability, I recommend adding "KSM" and "TSSM" to the two pictures, respectively.

Done.

(2) L165: tetur20g01790 refers to a TSSM gene, while TkDOG15 refers to a TSM protein. Revise it accordingly. (Same for L442 and L481).

The reviewer is correct that tetur20g01790 is the TSSM gene ID. As the KSM genome is not yet available, we identified the TkDOG15 gene, the KSM ortholog of tetur20g01790, by de novo assembly of our RNA-seq reads. We have revised L169 and L455 accordingly.

(3) L264: Supplemental Table 3-2.

Done (L271).

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Weaknesses:

While transcriptome/proteome analyses reported changes in the expression of other detoxification-related enzymes, including CCEs, UGTs, ABC transporters, DOG1, DOG4, and DOG7, it is regrettable that the contribution of each enzyme, including its interaction with TkDOG15 and the functional analysis of each enzyme within the overall catechin detoxification system, was not investigated.

We thank the reviewer for the encouraging assessment and this comment. We agree that the contributions of the other detoxification-related enzymes, including their interaction with DOG15, remain to be investigated. As this point overlaps with a comment from Reviewer 1, we have revised the Discussion (L276-283) to note that DOG15 was the only enzyme upregulated at both the mRNA and protein levels, whereas the CCEs, UGTs, ABC transporter, and other DOGs were enriched in only one dataset. We now state that tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15, and that the functional analysis of their individual and combined contributions warrants future work.

Reviewer #2 (Recommendations for the authors):

The manuscript titled "Adaptation of an Herbivorous Arthropod to Green Tea Plants by Overcoming Catechin Defenses" presents a well-designed, mechanistically insightful study that advances our understanding of herbivore adaptation to plant chemical defenses. The work is scientifically sound and of potential interest to a broad readership in chemical ecology and evolutionary biology.

However, before the manuscript can be considered for acceptance, the authors must adequately address the comments outlined below regarding clarity, presentation, and interpretation across the manuscript.

We thank the reviewer for the positive evaluation of our study. We have carefully addressed each of the specific comments below regarding clarity, presentation, and interpretation, and we believe these revisions have substantially improved the manuscript.

Specific comments on each section:

(1) Abstract

(a) The authors are encouraged to add a concise concluding sentence summarizing the broader significance of the study and indicating potential future research directions or limitations, which would strengthen the impact of the abstract.

We have added a concluding sentence to the Abstract summarizing the broader significance of the study and indicating future directions (L38-40).

(b) The authors may consider adding representative quantitative results to the abstract, as this would enhance clarity and increase the impact and interpretability of the study for readers.

We have added representative quantitative results to the Abstract. Specifically, we now state that the mRNA and protein levels of DOG15 in tea-adapted T. kanzawai are up to 31.6 and 12.1 times higher, respectively, than in T. urticae fed on tea plants (L30-32). For consistency, we now refer to the gene as "DOG15" throughout the Abstract (L29, L30, and L36).

(2) Introduction

(a) While the paragraph is informative, it reads more like a summary of the main results than a statement of study objectives. The authors are encouraged to reframe this section to explicitly define the study's aims and hypotheses.

We have reframed the final paragraph of the Introduction to explicitly state the study's aims and hypotheses rather than to summarize the results (L73-81).

(b) The authors should avoid excessive citation of multiple references for a single thematic statement when one key reference is sufficient. Where appropriate, inclusion of more recent literature is encouraged.

We have reduced multiple citations for single statements to the most representative references: Cabrera et al. (2006) for the health benefits of catechins (L46) and Grbić et al. (2011) and Dermauw et al. (2013) for the DOG gene count (L66-67).

(3) Materials and Methods

(a) The Materials and Methods section is comprehensive and technically sound; however, its length and density reduce overall clarity. The authors are encouraged to streamline descriptions of standard or well-established protocols and rely on appropriate citations where possible.

We agree that clarity can be improved by removing redundancy. The Materials and Methods are intentionally detailed to allow independent replication of our protocols, so we have retained this detail and instead removed the overlapping methodological descriptions from the figure captions, where the same information was repeated (see our response to comment 6a).

(b) Greater consistency is needed in reporting biological and technical replicates across different experiments (e.g., performance assays, transcriptomics, proteomics, and enzymatic activity assays) to enhance reproducibility.

We have standardized the reporting of replicates across all experiments to the format "x independent experimental runs (n = y per run)." Throughout the manuscript, "independent experimental runs" denotes biological replicates, with technical replicates specified separately where applicable (three technical replicates for qRT-PCR).

(c) The authors should provide brief justification for key methodological parameters, such as catechin concentrations, exclusion criteria in behavioral assays, and thresholds used for defining DEGs and DEPs, to improve transparency and interoperability.

We have added brief justifications for the three parameters. 1) The catechin concentration range was chosen to encompass the individual catechin levels measured in fresh tea leaves (L340-341). 2) In the behavioral assays, inactive mites were excluded because their movement was insufficient to determine chemo-orientation behavior, and escaped mites were excluded because they did not complete the assay (L365-367). 3) The thresholds for DEGs and DEPs follow criteria commonly applied in mite transcriptomic studies (Vidal-Quist et al., 2025, newly added to the references) (L414-416) and are consistent with our previous spider mite proteomic analysis (Arai et al., 2025) (L444-445).

(4) Results

(a) While significant differences in survival and fecundity are reported, briefly indicating the magnitude of these differences (e.g., percentage or fold change) would improve clarity and strengthen the presentation (Lines 91-96).

We have added the magnitude of the differences (L94-97). The revised text now states that after 10 days, almost 90% of tea-adapted KSM survived, compared with about 5% of non-adapted KSM and 33% of TSSM, and that tea-adapted KSM laid up to about 2 eggs/surviving female daily, whereas the other two populations laid almost no eggs.

(b) The final sentences include interpretative and concluding statements regarding catechins as key metabolites and mite adaptation. These statements would be more appropriate for the Discussion section rather than the Results (Lines 127-130). Follow the same for the rest of the Results section also.

Following the reviewer's suggestion, we have removed the interpretive and concluding statements from the end of the Results section, so that it now reports only the observations (L129-130). The interpretation regarding the multiple modes of action of catechins and the insensitivity of tea-adapted KSM is already presented in the Discussion (L206-213 and Conclusions), so we did not duplicate it there. We also reviewed the remaining Results subsections and confirmed that they report the experimental observations and their direct conclusions without broader interpretation.

(c) The comparison among catechin classes is clear; however, briefly listing the mean concentrations of each catechin (as shown in Figure 2a) in the text would improve readability without duplicating the figure (Lines 135-139).

We have added the approximate mean concentration of each catechin to the text (L136-137).

(d) Please clarify in the Results whether the same exposure concentration and duration were applied for all catechins and mite species, or explicitly direct readers to the Methods section (Lines 141-142).

We have clarified in the Results section that all four catechins were tested at the same concentration series (0, 10, 10², 10³, 10⁴, and 10⁵ ppm) and the same exposure duration (24 h) for both mite populations (L143).

(e) The phrase "lower sensitivity" should be explicitly linked to LC₅₀ estimates to ensure that the basis of comparison is immediately clear to readers (Lines 143-144).

Following the reviewer's suggestion, we have linked the sensitivity comparison to the LC₅₀ values (L143-147). The comparison is now stated relative to TSSM based on the LC₅₀ estimates, and for ECg and EC we note that the LC₅₀ of tea-adapted KSM exceeded the highest concentration tested.

(f) This section clearly identifies TkDOG15 as a key gene underlying tea adaptation in KSM; however, the authors are encouraged to briefly clarify the criteria used to define "highly enriched" mRNAs and proteins (e.g., fold-change and statistical thresholds) in the Results text or by explicitly directing readers to the Methods. This would improve transparency and facilitate interpretation of the multi-omics comparisons (Lines 147-173).

We have added the criteria used to define the enriched mRNAs and proteins (log2 fold change ≥ 1 with adjusted p-value < 0.05 for mRNA and p-value < 0.05 for protein) and referred readers to the Materials and Methods (L159-160).

(g) The enzymatic comparison between TkDOG15 and TuDOG15 is well presented; however, the authors are encouraged to briefly discuss whether the two amino acid substitutions (Q127A and T203A) were individually or jointly responsible for the increased catalytic efficiency, or to acknowledge this as a limitation and potential direction for future functional studies (Lines 176-190).

We have added a brief discussion of whether the two substitutions (Q127A and T203A) act individually or jointly (L254-257). We note that T203A is adjacent to the active-site residue Y202 and may contribute more directly to catalytic efficiency, and we acknowledge that dissecting their individual contributions by site-directed mutagenesis is a direction for future work.

(5) Discussion

(a) The authors appropriately acknowledge that the molecular basis of chemosensory insensitivity and the contribution of additional detoxification enzymes remain unresolved. To further improve clarity, these statements could be explicitly framed as hypotheses or future research directions to clearly distinguish them from experimentally supported mechanisms (Lines 205-208; 266-270).

We have reframed the statements on chemosensory insensitivity (L209-213) and the contribution of additional detoxification enzymes (L272-274) as hypotheses and future directions, distinguishing them from the experimentally supported mechanisms.

(b) While DOG15 is convincingly identified as a key contributor to tea adaptation, a brief clarification of its relative importance compared with other upregulated detoxification enzymes would strengthen interpretative balance, even if the roles of these enzymes remain unresolved (Lines 259-265).

DOG15 was the only enzyme upregulated at both the mRNA and protein levels (Figure 3d,e), and the only enzyme functionally validated in this study, by RNAi silencing (Figure 3f) and recombinant enzyme assays (Figure 4c). We have established that DOG15 contributes to tea adaptation, but because the other upregulated enzymes were not functionally tested, their relative contributions cannot be determined at this stage. As we note in the Discussion, tea adaptation in KSM may be multigenic, with these enzymes potentially acting synergistically with DOG15 (L277-283). We therefore did not add further text, to avoid duplication.

(c) The discussion linking host plant adaptation to reproductive isolation and ecological speciation is interesting and well contextualized; however, these evolutionary implications should be slightly tempered or explicitly framed as potential long-term outcomes beyond the immediate scope of the present study (Lines 271-281).

We have tempered the evolutionary implications (L292-294). The revised sentence now frames the link to reproductive isolation and ecological speciation as a potential outcome over longer evolutionary timescales rather than a direct finding of the present study.

(6) Figure captions

(a) The figure captions (Figures 1-4) are exceptionally detailed and, in several places, repeat methodological information already described in the Materials and Methods. The authors are encouraged to shorten the captions by retaining only information necessary to interpret the figures, while referring readers to the Methods for experimental details.

We have shortened the figure captions (Figures 1-4) by removing methodological details that are described in the Materials and Methods, retaining only the information needed to interpret each figure. Where appropriate, readers are now referred to the Materials and Methods or to Supplemental Figure 1-2 for the full experimental procedures.

(b) Several captions contain long, multi-sentence descriptions that may hinder readability. The authors may consider simplifying the wording, grouping related panels more concisely, and removing procedural details (e.g., extraction conditions, exposure durations, and instrument settings) to improve clarity and visual accessibility.

As described in our response to comment 6a, we have simplified the figure captions by removing procedural details such as extraction conditions, exposure durations, and instrument settings, and by grouping related panels more concisely. These details are retained in the Materials and Methods.

(c) In Figure 1, the panel labels (a-h) do not appear in a clear sequential order. For consistency with the other figures and to improve readability, the authors should ensure that panel lettering is arranged in a logical, sequential order throughout the manuscript.

We appreciate the reviewer's attention to panel ordering. In the current layout, the panel lettering follows the order in which the panels are first cited in the text. Arranging the panels in a strict left-to-right, top-to-bottom sequence would require reducing the size of several panels, including the HPLC chromatogram in panel (e) and the survival and fecundity time courses in panels (c) and (d), which would compromise their readability. We have therefore retained the current arrangement, in which related panels are grouped together and the larger panels are kept at a legible size. We hope the reviewer finds this acceptable.

DOI: 10.64898/2026.07.15.738595

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_32941

Curator: @maulamb

SciCrunch record: RRID:BDSC_32941

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_34967

Curator: @maulamb

SciCrunch record: RRID:BDSC_34967

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_32882

Curator: @maulamb

SciCrunch record: RRID:BDSC_32882

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_30504

Curator: @maulamb

SciCrunch record: RRID:BDSC_30504

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_35695

Curator: @maulamb

SciCrunch record: RRID:BDSC_35695

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_32468

Curator: @maulamb

SciCrunch record: RRID:BDSC_32468

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_34710

Curator: @maulamb

SciCrunch record: RRID:BDSC_34710

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_28315

Curator: @maulamb

SciCrunch record: RRID:BDSC_28315

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_32391

Curator: @maulamb

SciCrunch record: RRID:BDSC_32391

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_60110

Curator: @maulamb

SciCrunch record: RRID:BDSC_60110

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_32942

Curator: @maulamb

SciCrunch record: RRID:BDSC_32942

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_62328

Curator: @maulamb

SciCrunch record: RRID:BDSC_62328

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_34580

Curator: @maulamb

SciCrunch record: RRID:BDSC_34580

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_67275

Curator: @maulamb

SciCrunch record: RRID:BDSC_67275

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_77374

Curator: @maulamb

SciCrunch record: RRID:BDSC_77374

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_58290

Curator: @maulamb

SciCrunch record: RRID:BDSC_58290

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_28562

Curator: @maulamb

SciCrunch record: RRID:BDSC_28562

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_65219

Curator: @maulamb

SciCrunch record: RRID:BDSC_65219

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_51758

Curator: @maulamb

SciCrunch record: RRID:BDSC_51758

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_57426

Curator: @maulamb

SciCrunch record: RRID:BDSC_57426

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_66002

Curator: @maulamb

SciCrunch record: RRID:BDSC_66002

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_55375

Curator: @maulamb

SciCrunch record: RRID:BDSC_55375

What is this?

DOI: 10.64898/2026.04.28.721503

Resource: RRID:BDSC_33897

Curator: @maulamb

SciCrunch record: RRID:BDSC_33897

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248926

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248926

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248925

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248925

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_257992

Curator: @sonofthor

SciCrunch record: RRID:Addgene_257992

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248924

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248924

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248923

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248923

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248922

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248922

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_257989

Curator: @sonofthor

SciCrunch record: RRID:Addgene_257989

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248930

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248930

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248929

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248929

What is this?

DOI: 10.64898/2026.04.07.716332

Resource: RRID:Addgene_248920

Curator: @sonofthor

SciCrunch record: RRID:Addgene_248920

What is this?

DOI: 10.21203/rs.3.rs-7072571

Resource: Rat Resource and Research Center (RRID:SCR_002044)

Curator: @Apiekniewska

SciCrunch record: RRID:SCR_002044

What is this?

DOI: 10.21203/rs.3.rs-7072571

Resource: RRID:RRRC_00699

Curator: @bandrow

SciCrunch record: RRID:RRRC_00699

What is this?

DOI: 10.14232/abs.2026.1.34-39

Resource: RRID:BDSC_4937

Curator: @maulamb

SciCrunch record: RRID:BDSC_4937

What is this?

DOI: 10.1126/sciadv.aef8132

Resource: Salk Institute Next Generation Sequencing Core (NGS) (RRID:SCR_014846)

Curator: @dhovakimyan1

SciCrunch record: RRID:SCR_014846

What is this?

DOI: 10.1126/sciadv.aef8132

Resource: RRID:IMSR_JAX:000664

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1038/s44319-026-00920-3

Resource: (DSMZ Cat# ACC-305, RRID:CVCL_0045)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0045

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_17438

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_17438

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_40619

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_40619

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_21471

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_21471

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_50547

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_50547

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_123299

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_123299

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: (RRID:CVCL_0042)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_0042

What is this?

DOI: 10.1038/s44318-026-00891-0

Resource: RRID:Addgene_16331

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_16331

What is this?

DOI: 10.1016/j.virusres.2026.199795

Resource: (CCLV Cat# CCLV-RIE 1035, RRID:CVCL_0023)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0023

What is this?

DOI: 10.1016/j.virusres.2026.199795

Resource: (ATCC Cat# HTB-55, RRID:CVCL_0609)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0609

What is this?

DOI: 10.1016/j.scr.2026.104095

Resource: Samtools (RRID:SCR_002105)

Curator: @nmaralla

SciCrunch record: RRID:SCR_002105

What is this?

DOI: 10.1016/j.neuron.2026.08.007

Resource: (MMRRC Cat# 031168-UCD,RRID:MMRRC_031168-UCD)

Curator: @nmaralla

SciCrunch record: RRID:MMRRC_031168-UCD

What is this?

DOI: 10.1016/j.isci.2026.117487

Resource: RRID:IMSR_JAX:008863

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:008863

What is this?

DOI: 10.1016/j.isci.2026.117466

Resource: R Project for Statistical Computing (RRID:SCR_001905)

Curator: @nmaralla

SciCrunch record: RRID:SCR_001905

What is this?

DOI: 10.1016/j.isci.2026.117461

Resource: (ATCC Cat# CRL-4000, RRID:CVCL_4388)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_4388

What is this?

DOI: 10.1016/j.isci.2026.117461

Resource: (TKG Cat# TKG 0331, RRID:CVCL_0030)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0030

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: (Cell Signaling Technology Cat# 92345, RRID:AB_2800183)

Curator: @nmaralla

SciCrunch record: RRID:AB_2800183

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: (RRID:CVCL_0063)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0063

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: RRID:AB_3739973

Curator: @nmaralla

SciCrunch record: RRID:AB_3739973

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: (Cell Signaling Technology Cat# 11998, RRID:AB_2750900)

Curator: @nmaralla

SciCrunch record: RRID:AB_2750900

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: RRID:AB_3662816

Curator: @nmaralla

SciCrunch record: RRID:AB_3662816

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: (Cell Signaling Technology Cat# 8165, RRID:AB_11217436)

Curator: @nmaralla

SciCrunch record: RRID:AB_11217436

What is this?

DOI: 10.1016/j.isci.2026.116105

Resource: (Cell Signaling Technology Cat# 11940, RRID:AB_2637071)

Curator: @nmaralla

SciCrunch record: RRID:AB_2637071

What is this?

DOI: 10.1016/j.envint.2026.110506

Resource: (NCI-DTP Cat# MCF7, RRID:CVCL_0031)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0031

What is this?

DOI: 10.1016/j.cels.2026.101734

Resource: (RCB Cat# RCB0988, RRID:CVCL_0025)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0025

What is this?

DOI: 10.1016/j.celrep.2026.117962

Resource: RRID:Addgene_12260

Curator: @nmaralla

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1016/j.celrep.2026.117962

Resource: RRID:Addgene_8454

Curator: @nmaralla

SciCrunch record: RRID:Addgene_8454

What is this?

DOI: 10.1016/j.celrep.2026.117962

Resource: RRID:Addgene_74061

Curator: @nmaralla

SciCrunch record: RRID:Addgene_74061

What is this?

DOI: 10.1016/j.celrep.2026.117962

Resource: RRID:Addgene_117721

Curator: @nmaralla

SciCrunch record: RRID:Addgene_117721

What is this?

DOI: 10.1016/j.ccell.2026.07.010

Resource: (RCB Cat# RCB3676, RRID:CVCL_0126)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0126

What is this?

DOI: 10.1016/j.ccell.2026.07.010

Resource: (CCLV Cat# CCLV-RIE 1035, RRID:CVCL_0023)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0023

What is this?

DOI: 10.1016/j.ccell.2026.07.010

Resource: (RRID:CVCL_0062)

Curator: @nmaralla

SciCrunch record: RRID:CVCL_0062

What is this?

DOI: 10.1016/j.bioorg.2026.110509

Resource: RRID:SCR_027240

Curator: @nmaralla

SciCrunch record: RRID:SCR_027240

What is this?