Author Response

Reviewer #1 (Public Review):

In Figure 1A, the authors should show TEM images of control mock treated samples to show the difference between infected and healthy tissue. Based on the data shown in Figure 1B-E that the overexpression of GFP-P in N. benthamiana leads to formation of liquid-like granules. Does this occur during virus infection? Since authors have infectious clones, can it be used to show that the virally encoded P protein in infected cells does indeed exist as liquid-like granules? If the fusion of GFP to P protein affects its function, the authors could fuse just the spGFP11 and co-infiltrate with p35S-spGFP1-10. These experiments will show that the P protein when delivered from virus does indeed form liquid-like granules in plants cells. Authors should include controls in Figure 1H to show that the interaction between P protein and ER is specific.

We agree with the reviewer and appreciate the helpful suggestion. As suggested, we added TEM images of control mock treated barley leaves. We also carried out immune-electron microscope to show the presence of BYSMV P protein in the viroplasms. Please see Figure 1–Figure supplement 1.

BYSMV is a negative-stranded RNA virus, and is strictly dependent on insect vector transmission for infecting barley plants. We have tried to fuse GFP to BYSMV P in the full-length infectious clones. Unfortunately, we could not rescue BYSMV-GFP-P into barley plants through insect transmission.

In Figure 1H, we used a PM localized membrane protein LRR84A as a negative control to show LRR84A-GS and BYSMV P could not form granules although they might associate at molecular distances. Therefore, the P granules were formed and tethered to the ER tubules. Please see Figure 1–Figure supplement 4

Data shown in Figure 2 do demonstrate that the purified P protein could undergo phase separation. Furthermore, it can recruit viral N protein and part of viral genomic RNA to P protein induced granules in vitro.

Because the full-length BYSMV RNA has 12,706 nt and is difficult to be transcribed in vitro, we cannot show whether the BYSMV genome is recruited into the droplets. We have softened the claim and state that the P-N droplets can recruit 5′ trailer of BYSMV genome as shown in Figure 3B. Please see line 22, 177 and 190.

Based on the data shown in Figure 4 using phospho-null and phospho-mimetic mutants of P protein, the authors conclude that phosphorylation inhibits P protein phase separation. It is unclear based on the experiments, why endogenous NbCK1 fails to phosphorylate GFP-P-WT and inhibit formation of liquid-like granules similar to that of GFP-P-S5D mutant? Is this due to overexpression of GFP-P-WT? To overcome this, the authors should perform these experiments as suggested above using infectious clones and these P protein mutants.

As we known, phosphorylation and dephosphorylation are reversible processes in eukaryotic cells. Therefore, as shown in Figure 5B and 6B, the GFP-PWT protein have two bands, corresponding to P74 and P72, which represent hyperphosphorylation and hypophosphorylated forms, respectively. Only overexpression of NbCK1 induced high ratio of P74 to P72 in vivo, and then abolished phase separation of BYSMV.

In Figure 5, the authors overexpress NbCK1 in N. benthamiana or use an in vitro co-purification scheme to show that NbCK1 inhibits phase separation properties of P protein. These results show that overexpression of both GFP-P and NbCK1 proteins is required to induce liquid-like granules. Does this occur during normal virus infection? During normal virus infection, P protein is produced in the plant cells and the endogenous NbCK1 will regulate the phosphorylation state of P protein. These are reasons for authors to perform some of the experiments using infectious clones. Furthermore, the authors have antibodies to P protein and this could be used to show the level of P protein that is produced during the normal infection process.

We detected the P protein existed as two phosphorylation forms in BYSMV-infected barley leaves, and λPPase treatment decreased the P44 phosphorylation form. Therefore, these results indicate that endogenous CK1 cannot phosphorylate BYSMV P completely.

Based on the data shown in Figure 6, the authors conclude that phase separated P protein state promotes replication but inhibits transcription by overexpressing P-S5A and P-S5D mutants. To directly show that the NbCK1 controlled phosphorylation state of P regulates this process, authors should knockdown/knockout NbCK1 and see if it increases P protein condensates and promote recruitment of viral proteins and genomic RNA to increase viral replication.

In our previous studies, BLAST searches showed that the N. benthamiana and barley genomes encode 14 CK1 orthologs, most of which can phosphorylated the SR region of BYSMV P. Therefore, it is difficult to make knockdown/knockout lines of all the CK1 orthologues. Accordingly, we generated a point mutant (K38R and D128N) in HvCK1.2, in which the kinase activity was abolished. Overexpression of HvCK1.2DN inhibit endogenous CK1-mediated phosphorylation of BYSMV P, indicating that HvCK1.2DN is a dominant-negative mutant.

It is important to note that both replication and transcription are required for efficient infection of negative-stranded RNA viruses. Therefore, our previous studies have revealed that both PS5A and PS5D are required for BYSMV infection. Therefore, expression of HvCK1.2DN in BYSMV vector inhibit virus infection by impairing the balance of endogenous CK1-mediated phosphorylation in BYSMV P.

Reviewer #2 (Public Review):

The manuscript by Fang et al. details the ability of the P protein from Barley yellow striate mosaic virus (BYSMV) to form phase-separated droplets both in vitro and in vivo. The authors demonstrate P droplet formation using recombinant proteins and confocal microscopy, FRAP to demonstrate fluidity, and observed droplet fusion. The authors also used an elaborate split-GFP system to demonstrate that P droplets associate with the tubulur ER network. Next, the authors demonstrate that the N protein and a short fragment of viral RNA can also partition into P droplets. Since Rhabdovirus P proteins have been shown to phase separate and form "virus factories" (see https://doi.org/10.1038/s41467-017-00102-9), the novelty from this work is the rigorous and conclusive demonstration that the P droplets only exist in the unphosphorylated form. The authors identify 5 critical serine residues in IDR2 of P protein that when hyper-phosphorylated /cannot form droplets. Next, the authors conclusively demonstrate that the host kinase CK1 is responsible for P phosphorylation using both transient assays in N. benthamiana and a co-expression assay in E. coli. These findings will likely lead to future studies identifying cellular kinases that affect phase separation of viral and cellular proteins and increases our understanding of regulation of condensate formation. Next, the authors investigated whether P droplets regulated virus replication and transcription using a minireplicon system. The minireplicon system needs to be better described as the results were seemingly conflicting. The authors also used a full-length GFP-reporter virus to test whether phase separation was critical for virus fitness in both barley and the insect vector. The authors used 1, 6-hexanediol which broadly suppresses liquid-liquid phase separation and concluded that phase separation is required for virus fitness (based on reduced virus accumulation with 1,6 HD). However, this conclusion is flawed since 1,6-hexanediol is known to cause cell toxicity and likely created a less favorable environment for virus replication, independent of P protein phase separation. These with other issues are detailed below:

1. In Figure 3B, the authors display three types of P-N droplets including uniform, N hollow, and P-N hollow droplets. The authors do not state the proportion of droplets observed or any potential significance of the three types. Finally, as "hollow" droplets are not typically observed, is there a possibility that a contaminating protein (not fluorescent) from E. coli is a resident client protein in these droplets? The protein purity was not >95% based on the SDS-PAGE gels presented in the supplementary figures. Do these abnormalities arise from the droplets being imaged in different focal planes? Unless some explanation is given for these observations, this reviewer does not see any significance in the findings pertaining to "hollow" droplets.

Thanks for your constructive suggestions. We removed the "hollow" droplets as suggested. We think that the hollow droplets might be an intermediate form of LLPS. Please see PAGE 7 and 8 of revised manuscript.

1. Pertaining to the sorting of "genomic" RNA into the P-N droplets, it is unlikely that RNA sorting is specific for BYSMV RNA. In other words, if you incubate a non-viral RNA with P-N droplets, is it sorted? The authors conclusion that genomic RNA is incorporated into droplets is misleading in a sense that a very small fragment of RNA was used. Cy5 can be incorporated into full-length genomic RNAs during in vitro transcription and would be a more suitable approach for the conclusions reached.

Thanks for your constructive suggestions. Unfortunately, we could not obtain the in vitro transcripts of the full-length genomic RNAs (12706 nucleotides). We have softened the claim and state that the P-N droplets can recruit the 5′ trailer of BYSMV genome as shown in Figure 3B. Please see line 22, 177 and 190.

According to previous studies (Ivanov, et al., 2011), the Rhabdovirus P protein can bind to nascent N moleculaes, forming a soluble N/P complex, to prevent from encapsidating cellular RNAs. Therefore, we suppose that the P-N droplets can incorporate viral genomic RNA specifically.

Reference: Ivanov I, Yabukarski F, Ruigrok RW, Jamin M. 2011. Structural insights into the rhabdovirus transcription/ replication complex. Virus Research 162:126–137. DOI: https://doi.org/10.1016/j.virusres.2011.09.025

1. In Figure 4C, it is unclear how the "views" were selected for granule counting. The methods should be better described as this reviewer would find it difficult to select fields of view in an unbiased manner. This is especially true as expression via agroinfiltration can vary between cells in agroinfiltrated regions. The methods described for granule counting and granule sizes are not suitable for publication. These should be expanded (i.e. what ImageJ tools were used?).

We agree with the reviewer that it is important to select fields of view in an unbiased manner. We selected the representative views and provided large views in the new Supplement Figures. In addition, we added new detail methods in revision. Please see Figure 4–Figure supplement 1, Figure 5–Figure supplement 1, and method (line 489-498).

1. In Figure 4F, the authors state that they expected P-S5A to only be present in the pellet fraction since it existed in the condensed state. However, WT P also forms condensates and was not found in the pellet, but rather exclusively in the supernatant. Therefore, the assumption of condensed droplets only being found in the pellet appears to be incorrect.

Many thanks for pointing this out. This method is based on a previous study (Hubstenberger et al., 2017). The centrifugation method might efficiently precipitate large granules more than small granules. As shown in Figure 4B, GFP-PS5A formed large granules, therefore GFP-PS5A mainly existed in the pellet. In contrast, GFP-PWT only existed in small granule and fusion state, thus most of GFP-PWT protein was existed in supernatant, and only little GFP-PWT protein in the pellet. These results also indicate the increased phase separation activity of GFP-PS5A compared with GFP-PWT. Please see the new Figure 4F.

Reference: Hubstenberger A, Courel M, Benard M, Souquere S, Ernoult-Lange M, Chouaib R, Yi Z, Morlot JB, Munier A, Fradet M, et al. 2017. P-Body Purification Reveals the Condensation of Repressed mRNA Regulons. Molecular Cell 68(1): 144-157 e145.

1. The authors conclude that P-S5A has enhanced phase separation based on confocal microscopy data (Fig S6A). The data presented is not convincing. Microscopy alone is difficult for comparing phase separation between two proteins. Quantitative data should be collected in the form of turbidity assays (a common assay for phase separation). If P-S5A has enhanced phase separation compared to WT, then S5A should have increased turbidity (OD600) under identical phase separation conditions. The microscopy data presented was not quantified in any way and the authors could have picked fields of view in a biased manner.

Thanks for your constructive suggestions. As suggested, turbidity assays were performed to show both GFP-PWT and GFP-PS5A had increased turbidity (OD600) compared with GFP. Please see Figure 4–Figure supplement 3.

1. The authors constructed minireplicons to determine whether mutant P proteins influence RNA replication using trans N and L proteins. However, this reviewer finds the minireplicon design confusing. How is DsRFP translated from the replicon? If a frameshift mutation was introduced into RsGFP, wouldn't this block DsRFP translation as well? Or is start/stop transcription used? Second, the use of the 2x35S promoter makes it difficult to differentiate between 35S-driven transcription and replication by L. How do you know the increased DsRFP observed with P5A is not due to increased transcription from the 35S promoter? The RT-qPCR data is also very confusing. It is not clear that panel D is only examining the transcription of RFP (I assume via start/stop transcription) whereas panel C is targeting the minireplicon.

Thank you for your questions and we are sorry for the lack of clarity regarding to the mini-replicon vectors. Here, we updated the Figure supplement 14 to show replication and transcription of BYSMV minireplicon, a negative-stranded RNA virus derivative. In addition, we insert an A after the start codon to abolish the translation of GFP mRNA, which allow us to observe phase separation of GFP-PWT, GFP-PS5A, and GFP-PS5D during virus replication. Use this system, we wanted to show the localization and phase separation of GFP-PWT, GFP-PS5A, and GFP-PS5D during replication and transcription of BYS-agMR. Please see Figure 6–Figure supplement 1.

1. Pertaining to the replication assay in Fig. 6, transcription of RFP mRNA was reduced by S5A and increased by S5D. However, the RFP translation (via Panel A microscopy) is reversed. How do you explain increased RFP mRNA transcription by S5D but very low RFP fluorescence? The data between Panels A, C, and D do not support one another.

Many thanks for pointing this out! We also noticed the interesting results that have been repeated independently. As shown the illustration of BYSMV-agMR system in Figure 6–Figure supplement 1, the relative transcriptional activities of different GFP-P mutants were calculated from the normalized RFP transcript levels relative to the gMR replicate template (RFP mRNA/gMR), because replicating minigenomes are templates for viral transcription.

Since GFP-PS5D supported decreased replication, the ratio of RFP mRNA/gMR increased although the RFP mRNA of GFP-PS5D is not increased. In addition, the foci number of GFP-PS5D is much less than GFP-PWT and GFP-PS5A, indicating mRNAs in GFP-PS5D samples may contain aberrant transcripts those cannot be translated the RFP protein. In contrast, mRNAs in GFP-PS5A samples are translated efficiently. These results were in consistent with our previous studies using the free PWT, PS5A, and PS5D.

Reference: Gao Q, et al. 2020. Casein kinase 1 regulates cytorhabdovirus replication and transcription by phosphorylating a phosphoprotein serine-rich motif. The Plant Cell 32(9): 2878-2897.

1. The authors relied on 1,6-hexanediol to suppress phase separation in both insect vectors and barley. However, the authors disregarded several publications demonstrating cellular toxicity by 1,6-hexanediol and a report that 1,6-HD impairs kinase and phosphatase activities (see below). doi: 10.1016/j.jbc.2021.100260,

We agree with the reviewer that 1, 6-hexanediol induced cellular toxicity. Therefore, we removed these results, which does not affect the main conclusion of our results.

1. The authors state that reduced accumulation of BYSMV-GFP in insects and barley under HEX treatment "indicate that phase separation is important for cross-kingdom infection of BYSMV in insect vectors and host plants." The above statement is confounded by many factors, the most obvious being that HEX treatment is most likely toxic to cells and as a result cannot support efficient virus accumulation. Also, since HEX treatment interferes with phosphorylation (see REF above) its use here should be avoided since P phase separation is regulated by phosphorylation.

We agree with the reviewer that 1, 6-hexanediol induced cellular toxicity and hereby affected infections of BYSMV and other viruses. In addition, 1, 6-hexanediol would inhibit LLPS of cellular membraneless organelles, such as P-bodies, stress granules, cajal bodies, and the nucleolus, which also affect different virus infections directly or indirectly. Therefore, we removed these results, which does not affect the main conclusion of our results.

Reviewer #3 (Public Review):

Membrane-less organelles formed through liquid-liquid phase separation (LLPS) provide spatiotemporal control of host immunity responses and other cellular processes. Viruses are obligate pathogens proliferating in host cells which lead their RNAs and proteins are more likely to be targeted by immune-related membrane-less organelles. To successfully infect and proliferate in host cells, virus need to efficiently suppressing the immune function of those immune-related membrane-less organelles. Moreover, viruses also generate exogenous membrane-less organelles/RNA granules to facilitate their proliferation. Accordingly, host cells also need to target and suppress the functions of exogenous membrane-less organelles/RNA granules generated by viruses, the underlying mechanisms of which are still mysterious.

In this study, Fang et al. investigated how plant kinase confers resistance against viruses via modulating the phosphorylation and phase separation of BYSMV P protein. They firstly characterized the phase separation feature of BYSMV P protein. They also discovered that droplets formed by P protein recruit viral RNA and other viral protein in vivo. The phase separation activity of P protein is inhibited by the phosphorylation on its intrinsically disordered region. Combined with their previous study, this study demonstrated that host casein kinase (CK1) decreases the phase separation of P protein via increasing the phosphorylation of P protein. Finally, the author claimed that the phase separation of P protein facilitates BYSMV replication but decreases its transcription. Taking together, this study uncovered the molecular mechanism of plant regulating viral proliferation via decreasing the formation of exogenous RNA granules/membraneless organelles. Overall, this paper tells an interesting story about the host immunity targeting viruses via modulating the dynamics of exogenous membraneless organelles, and uncovers the modulation of viral protein phase separation by host protein, which is a hotspot in plant immunity, and the writing is logical.

Thanks for your positive comment on our studies.

Author Response:

Reviewer #1 (Public Review):

Here the authors use a variety of sophisticated approaches to assess the contribution of synaptic parameters to dendritic integration across neuronal maturation. They provide high-quality data identifying cellular parameters that underlie differences in AMPAR-mediated synaptic currents measured between adolescent and adult cerebellar stellate cells, and conclude that differences are attributed to an increase in the complexity of the dendritic arbor. This conclusion relies primarily on the ability of a previously described model for adult stellate cells to recapitulate the age-dependent changes in EPSCs by a change in dendritic branching with no change in synapse density. These rigorous results have implications for understanding how changing structure during neuronal development affects integration of AMPR-mediated synaptic responses.

The data showing that younger SCs have smaller dendritic arbors but similar synapse density is well-documented and provides compelling evidence that these structural changes affect dendritic integration. But the main conclusion also relies on the assumption that the biophysical model built for adult SCs applies to adolescent SCs, and there are additional relevant variables related to synaptic function that have not been fully assessed. Thus, the main conclusions would be strengthened and broadened by additional experimental validation.

We thank the reviewer for the positive assessment of the quality and importance of our manuscript. Below we address the reviewer’s comments directly but would like to stress that the goal of the manuscript was to understand the cellular mechanisms underlying developmental slowing of mEPSCs in SCs and the consequent implication for developmental changes in dendritic integration, which have rarely been examined to date, and not to establish a detailed biophysical model of cerebellar SCs. The latter would require dual-electrode recordings (one on 0.5 um dendrites), detailed description of the expression, dendritic localization of the gap junction protein connexin 36 (as done in Szoboszlay neuron 2016), and a detailed description prameter variability across the SC population (e.g. variations in AMPAR content at synapses, Rm, and dendritic morphology). Such experiments are well beyond the scope of the manuscript. Here we use biophysical simulations to support conclusions derived from specific experiments, more as a proof of principle rather than a strict quantitative prediction.

Nevertheless, we would like to clarify our selection of parameters for the biophysical models for immature and adult SCs. We did not simply “assume” that the biophysical models were the same at the two developmental stages. We either used evidence from the literature or our own measured parameters to establish an immature SC model. As compared to adult SCs, we found that immature SCs had 1) an identical membrane time constant, 2) an only slightly larger dendrite diameter, 3) decreased dendritic branching and maximum lengths, 4) a comparable synapse density, and 5) a homogeneous synapse distribution. Taken together, we concluded that increased dendritic branching during SC maturation resulted in a larger fraction of synapses at longer electrotonic distances in adult SCs. These experimental findings were incorporated into two distinct biophysical models representing immature and adult SCs. Evidence from the literature suggests that voltage-gated channels expression is not altered between the two developmental stages studied here. Therefore, like the adult SC model, we considered only the passive membrane properties and the dendritic morphology. The simulation results supported our conclusion that the increased apparent dendritic filtering of mEPSCs resulted from a change in the distribution of synapse distance to the soma rather than cable properties. Some of the measured parameters (e.g., membrane time constant) were not clearly stated manuscript, which we have corrected in the revised manuscript.

We are not sure what the reviewer meant by suggesting that we did not examine “other relevant variables related to synaptic function.” Later, the reviewer refers to alterations in AMPAR subunit composition or changes in cleft glutamate concentration (low-affinity AMPAR antagonist experiments). We performed experiments to directly examine both possible contributions by comparing qEPSC kinetics and performing low-affinity antagonist experiments, respectively, but we found that neither mechanism could account for the developmental slowing of mEPSCs. We, therefore, did not explore further possible developmental changes AMPAR subunits. See below for a more specific response and above for newly added text.

While many exciting questions could be examined in the future, we do not think the present study requires additional experiments. Nevertheless, we recognize that perhaps we can improve the description of the results to justify our conclusions better (see specifics below).

Reviewer #2 (Public Review):

This manuscript investigates the cellular mechanisms underlying the maturation of synaptic integration in molecular layer interneurons in the cerebellar cortex. The authors use an impressive combination of techniques to address this question: patch-clamp recordings, 2-photon and electron microscopy, and compartmental modelling. The study builds conceptually and technically on previous work by these authors (Abrahamsson et al. 2012) and extends the principles described in that paper to investigate how developmental changes in dendritic morphology, synapse distribution and strength combine to determine the impact of synaptic inputs at the soma.

1) Models are constructed to confirm the interpretation of experimental results, mostly repeating the simulations from Abrahamsson et al. (2012) using 3D reconstructed morphologies. The results are as expected from cable theory, given the (passive) model assumptions. While this confirmation is welcome and important, it is disappointing to see the opportunity missed to explore the implications of the experimental findings in greater detail. For instance, with the observed distributions of synapses, are there more segregated subunits available for computation in adult vs immature neurons?

As described in our response to reviewer 1, this manuscript intends to identify the cellular mechanisms accounting developmental slowing of mEPSCs and its implication for dendritic integration. The modeling was designed to support the most plausible explanation that increased branching resulted in more synapses at longer electrotonic distances. This finding is novel and merits more in-depth examination at a computation level in future studies.

Quantifying dendritic segregation is non-trivial due to dendritic nonlinearities and the difficulties in setting criteria for electrical “isolation” of inputs. However, because the space constant does not change with development, while both dendrite length and branching increase, it is rather logical to conclude qualitatively that the number of computational segments increases with development.

We have added the following sentence to the Discussion (line 579):

“Moreover, since the space constant does not change significantly with development and the dendritic tree complexity increases, the number of computational segments is expected to increase with development.”

How do SCs respond at different developmental stages with in vivo-like patterns of input, rather than isolated activation of synapses? Answering these sorts of questions would provide quantitative support for the conclusion that computational properties evolve with development.

While this is indeed a vital question, the in vivo patterns of synaptic activity are not known, so it is difficult to devise experiments to arrive at definitive conclusions.

2) From a technical perspective, the modeling appears to be well-executed, though more methodological detail is required for it to be reproducible. The AMPA receptor model and reversal potential are unspecified, as is the procedure for fitting the kinetics to data.

We did not use an explicit channel model to generate synaptic conductances. We simply used the default multiexponential function of Neuron (single exponential rise and single exponential decay) and adjusted the parameters tauRise and tauDecay such that simulated EPSCs matched somatic quantal EPSC amplitude, rise time and τdecay (Figure 4).

We added the following text to the methods (line 708):

“The peak and kinetics of the AMPAR-mediated synaptic conductance waveforms (gsyn) were set to simulate qEPSCs that matched the amplitude and kinetics of experimental somatic quantal EPSCs and evoked EPSCs. Immature quantal gsyn had an peak amplitude of 0.00175 μS, a 10-90 % RT of 0.0748 ms and a half-width of 0.36 ms (NEURON synaptic conductance parameter Tau0 = 0.073 ms, Tau1 = 0.26 ms and Gmax = 0.004 μS) while mature quantal gsyn had an peak amplitude of 0.00133 μS, a 10-90 % RT of 0.072 ms and a half-width of 0.341 ms (NEURON synaptic conductance parameters Tau0 = 0.072 ms, Tau1 = 0.24 ms and Gmax = 0.0032 μS). For all simulations, the reversal potential was set to 0 mV and the holing membrane potential was to – 70 mV. Experimental somatic PPR for EPSCs were reproduced with a gsyn 2/ gsyn 1 of 2.25.”

Were simulations performed at resting potential, and if yes, what was the value?

The membrane potential was set at – 70 mV to match that of experimental recordings and has been updated in the Methods section.

How was the quality of the morphological reconstructions assessed? Accurate measurement of dendritic diameters is crucial to the simulations in this study, so providing additional morphometrics would be helpful for assessing the results. Will the models and morphologies be deposited in ModelDB or similar?

For the two reconstructions imported into NEURON for simulations, we manually curated the dendritic diameters to verify a matching of the estimated diameter to that of the fluorescence image using NeuroStudio, which uses a robust subpixel estimation algorithm (Rayburst diameter, Rodriguez et al. 2008). The reconstructions include all variations in diameter throughout the dendritic tree (see as a example the the result of the reconstruction on the image below for the immature SC presented in the Figure 2D). The mean diameter across the entire dendritic tree of the reconstructed immature and adult SC was 0.42 and 0.36 μm, respectively, similar to the ratio of measured diameters estimated using confocal microscopy.

We have updated the methods section to include how reconstructions were curated and analyzed (line 693).

“An immature (P16) and adult SC (P42) were patch loaded with 30 μM Alexa 594 in the pipette and imaged using 2PLSM. Both cells were reconstructed in 3D using NeuronStudio in a semiautomatic mode which uses a robust subpixel estimation algorithm (calculation of Rayburst diameter (Rodriguez et al., 2008)). We manually curated the diameters to verify that it matched the fluorescence image to faithfully account for all variations in diameter throughout the dendritic tree. The measured diameter across the entire dendritic tree of the reconstructed immature and adult SCs was 0.42 and 0.36 μm, respectively. The 16% smaller diameter in adult was similar to the 13% obtained from confocal image analysis from many SCs (see Figure 2B).”

We agree with the reviewer that accurate measurements of dendritic diameters are crucial for the simulations. We did not rely soley on the reconstructed SCs, but we also performed highresolution confocal microscopy analysis of 16 different dye-filled SCs. We examined differences in the FWHM of intensity line profiles drawn perpendicular to the dendrite between immature and adult SCs. The FWHM is a good approximation of dendritic diameter and was performed similarly to adult SCs (Abrahamsson et al., 2012) to allow direct assessment of possible developmental differences. We confirmed that 98% of the estimated diameters are larger than the imaging resolution (0.27 μm). We observed only a small developmental difference in the mean FWHM (0.41 vs. 0.47 μm, 13% reduction) using this approach. Because the dendritic filtering is similar for diameters ranging from 0.3 to 0.6 μm (Figure 4G and 4H, Abrahamsson et al. 2012), we concluded that developmental changes in dendritic diameter cannot account for for developmental differences in mEPSC time course.

We added the following text to the methods (line 777):

“The imaging resolution within the molecular layer was estimated from the width of intensity line profiles of SC axons. The FWHM was 0.30 +/- 0.01 μm (n = 57 measurements over 16 axons) and a mean of 0.27 +/- 0.01 μm (n = 16) when taking into account the thinnest section for each axon. Only 2% of all dendritic measurements are less than 270 nm, suggesting that the dendritic diameter estimation is hardly affected by the resolution of our microscope”

Regarding additional morphometrics:

1) We added two panels (H and I) to Figure 6 showing the number of primary dendrites and branch points for immature and adult using the same estimation criteria as Myoga et al;, 2009. We have updated the Results section (line 389). “Thus, the larger number of puncta located further from the soma in adult SCs is not due to increased puncta density with distance, but a larger dendritic lengths (Figure 6E and 6F) and many more distal dendritic branches (Figure 6G, Sholl analysis) due to a larger number of branch points (Figure 6H), but not a larger number of primary dendrites (Figure 6I). The similarity between the shapes of synapse (Figure 6B) and dentric segment (Figure 6C) distributions was captured by a similarity in their skewness (0.38 vs. 0.32 for both distributions in immature and -0.10 and -0.08 for adult distributions). These data demonstrate that increased dendritic complexity during SC maturation is responsible for a prominent shift toward distal synapses in adult SCs.

2) As suggested by the reviewer, we estimated the dendritic width as a function branch order and observed a small reduction of dendritic segments as a function of distance from the soma that does not significantly alter the dendritic filtering (0.35 to 0.6 μm): there is a tendency to observe smaller diameter for more distal segments.

3) We also show the variability in dendritic diameter within single SCs and between different SCs, which can be very large. These results have been added to Figure 2B. See also point one below in response to “comment to authors.”

We will upload the two SC reconstructions to ModelDB.

3) The Discussion should justify the assumption of AMPA-only synapses in the model (by citing available experimental data) as well as the limitations of this assumption in the case of different spatiotemporal patterns of parallel fiber activation.

NMDARs are extrasynaptic in immature and adult SCs. Therefore they do not contribute to postsynaptic strength in response to low-frequency synaptic activation. We therefore do not consider their contribution to synaptic integration in this study. Please see also out detailed response to reviewer’s point 4. We have updated the Results accordingly.

4) What is the likely influence of gap junction coupling between SCs on the results presented here, and on synaptic integration in SCs more generally - and how does it change during development? This should also be discussed.

Please see a detailed response to Editor’s point 2. In brief, all recordings were performed without perturbing gap junction coupling between cells, which have been shown to affect axial resistance and membrane capacitance in other cell types (Szoboszlay et al., 2016). While our simulations do not explicitly include gap junctions, their effect on passive membrane properties is implicitly included because we matched the simulated membrane time constant to experimental values. Moreover, gap junctions are more prominent in cerebellar basket cells than SCs in both p18 to p21 animals (Rieubland 2015) and adult mice (Hoehne et al., 2020). Ultimately, the impact of gap junctions also depends on their distance from the activated synapses (Szoboszlay et al., 2016). Unfortunately, the distribution of gap junctions in SCs and their conductance is not known at this time. We, therefore, did not explicitly consider gap junction in this study.

Nevertheless, we have added a section in the Discussion (line 552):

“We cannot rule out that developmental changes in gap junction expression could contribute to the maturation of SC dendritic integration, since they are thought to contribute to the axial resistivity and capacitance of neurons (Szoboszlay et al., 2016). All the recordings were made with gap junctions intact, including for membrane time constant measurements. However, their expression in SCs is likely to be lower than their basket cell counterparts (Hoehne et al., 2020; Rieubland et al., 2014).”

5) All experiments and all simulations in the manuscript were done in voltage clamp (the Methods section should give further details, including the series resistance). What is the significance of the key results of the manuscript on synapse distribution and branching pattern of postsynaptic dendrites in immature and adult SCs for the typical mode of synaptic integration in vivo, i.e. in current clamp? What is their significance for neuronal output, considering that SCs are spontaneously active?

It should be noted that not all simulations were done in voltage-clamp, see figure 8.

Nevertheless, we have given additional details about the following experimental and simulation parameters:

1) Description of the whole-cell voltage-clamp procedure.

2) Series resistance values of experiments and used for simulations.

Initial simulations with the idealized SC model were performed with a Rs of 20 MOhm. In the reconstructed model Rs was set at 16 mOhm to match more precisely the experimental values obtained for the mEPSC experiments. We verified that there were no statistical difference in Rs between Immature and adult recordings.

Reviewer #3 (Public Review):

1) Although the authors were thorough in their efforts to find the mechanism underlying the differences in the young and adult SC synaptic event time course, the authors should consider the possibility of inherently different glutamate receptors, either by alterations in the subunit composition or by an additional modulatory subunit. The literature actually suggests that this might be the case, as several publications described altered AMPA receptor properties (not just density) during development in stellate cells (Bureau, Mulle 2004; Sun, Liu 2007; Liu, Cull-Candy 2002). The authors need to address these possibilities, as modulatory subunits are known to alter receptor kinetics and conductance as well.

Properties of synaptic AMPAR in SCs are known to change during development and in an activity-dependent manner. EPSCs in immature SC have been shown to be mediated by calcium permeable AMPARs, predominantly containing GluR3 subunits that are associated with TARP γ2 and γ7 (Soto et al. 2007; Bats et al., 2012). During development GluR2 subunits are inserted to the synaptic AMPAR in an activity-dependent manner (Liu et al, 2000), affecting the receptors’ calcium permeability (Liu et al., 2002). However, those developmental changes do not appear to affect EPSC kinetics (Liu et al., 2002) and have very little impact on AMPAR conductance (Soto et al., 2007). When we compare qEPSC kinetics for somatic synapses between immature and adult SC, we did not observe changes in EPSC decay. In the light of this observation and also consistent with the studies cited above, we concluded that differences in AMPAR composition could not contribute to kinetics differences observed in the developmental changes in mEPSC properties.

We have modified the manuscript to make this point clearer (see section starting line 332) :

“This reduction in synaptic conductance could be due to a reduction in the number of synaptic AMPARs activated and/or a developmental change in AMPAR subunits. SC synaptic AMPARs are composed of GluA2 and GluA3 subunits associated with TARP γ2 and γ7 (Bats et al., 2012; Liu and Cull-Candy, 2000; Soto et al., 2007; Yamazaki et al., 2015). During development, GluR2 subunits are inserted to the synaptic AMPAR in an activity-dependent manner (Liu and Cull-Candy, 2002), affecting receptors calcium permeability (Liu and Cull-Candy, 2000). However, those developmental changes have little impact on AMPAR conductance (Soto et al., 2007), nor do they appear to affect EPSC kinetics (Liu and Cull-Candy, 2002); the latter is consistent with our findings. Therefore the developmental reduction in postsynaptic strength most likely results from fewer AMPARs activated by the release of glutamate from the fusion of a single vesicle. “

The authors correctly identify the relationship between local dendritic resistance and the reduction of driving force, but they assume the same relationship for young SCs as well in their model. This assumption is not supported by recordings, and as there are several publications about the disparity of input impedance for young versus adult cells (Schmidt-Hieber, Bischoffberger 2007).

The input resistance of the dendrite will indeed determine local depolarization and loss of driving force. However, its impact on dendritic integration depends on it precise value, and perhaps the reviewer thought we “assumed” that the input resistance to be the same between immature and adult SCs. This was not the case, and we have since clarified this in the manuscript. We performed three important measurements that support a loss of driving force in immature SCs (for reference, the input resistance for an infinite cable is described by the following equation (Rn= sqrt(RmRi/2)/(2pi*r^(3/2)), where r is the dendrite radius):

1) The input resistance is inversely proportional to the dendritic diameter, which we measured to be only slightly larger in immature SCs (0.47 versus 0.41 μm). This result is described in Figure 2.

2) We measured the membrane time constant, which provides an estimate of the total membrane conductance multiplied by the total capacitance. The values between the two ages were similar, suggesting a slightly larger membrane resistance to compensate the smaller total membrane capacitance of the immature SCs. This was explicitly accounted for when performing the simulations using reconstructed immature and adult SCs (Figure 2 and 7 and 8) by adjusting the specific membrane resistance until the simulated membrane time constant matched experimental values. These values were not clearly mentioned and are now included on line 233 in the Results and 704 in the Methods.

3) We directly examined paired-pulse facilitation of synapses onto immature SC dendrites versus that for somatic synapses. We previously showed in adult SCs that sublinear summation of synaptic responses, due to loss of synaptic current driving force (Tran- Van-Minh et al. 2016), manifests in decreased facilitation for dendritic synapses (Abrahamsson et al. 2012). Figure 8A shows that indeed dendritic facilitation was less than observed in the soma. We have now modified Figure 8 to include the results of the simulations showing that the biophysical model could reproduce this difference in shortterm plasticity (Figure 8B).

Together, we believe these measurements support the presence of similar sublinear summation mechanisms in immature SCs.

2) The authors use extracellular stimulation of parallel fibers. The authors note that due to the orientation of the PF, and the slicing angle, they can restrict the spatial extent of the stimuli. However, this method does not guarantee that the stimulated fibers will all connect to the same dendritic branch. Whether two stimulated synapses connect to the same dendrite or not can heavily influence summation. This is especially a great concern for these cells as the Scholl analysis showed that young and adult SC cells have different amount of distal dendrites. Therefore, if the stimulated axons connect to several different neighboring dendrites instead of the one or two in case of young SC cells, then the model calculations and the conclusions about the summation rules may be erroneous.

We selected isolated dendrites and delivered voltage stimuli using small diameter glass electrodes (~ 1 μm) 10 - 15 V above threshold to stimulate single dendrites. This procedure excites GC axons in brain slices made from adult mice within less than 10 μm from the tip (Figure 2C, Tran-Van-Minh et al. 2016). It produces large dendritic depolarizations that are sufficient to decrease synaptic current driving force (Figure 1, Tran-Van-Minh et al. 2016). When we reproduced the conductance ratio using uncaging of single dendrites, we observed paired-pulse facilitation in the dendrites – suggesting that electrical stimulation activated synapses on common dendritic branches, or at least within close electrotonic distance to cause large dendritic depolarizations (Figure 7, Abrahamsson et al. 2012). Finally, we expect that the decreased branching in immature SCs further ensures that a majority of recorded synapses are contacting a common dendritic segment. We cannot rule out that occasionally some synaptic responses recorded at the soma are from synapses on different dendritic branches, but we do not see how this would alter our results and change our principal conclusions, particularly since this possible error only effects the interpretation of how many synapses are activated in paired-pulse experiments. The majority of the conclusions arise from the stimulation of single vesicle release events, and given the strikingly perpendicular orientation of GC axons, a 10 μm error in synapse location along a dendrite when we stimulated in the outthird would not alter our interpretations of the data.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study by Teplenin and coworkers assesses the combined effects of localized depolarization and excitatory electrical stimulation in myocardial monolayers. They study the electrophysiological behaviour of cultured neonatal rat ventricular cardiomyocytes expressing the light-gated cation channel Cheriff, allowing them to induce local depolarization of varying area and amplitude, the latter titrated by the applied light intensity. In addition, they used computational modeling to screen for critical parameters determining state transitions and to dissect the underlying mechanisms. Two stable states, thus bistability, could be induced upon local depolarization and electrical stimulation, one state characterized by a constant membrane voltage and a second, spontaneously firing, thus oscillatory state. The resulting 'state' of the monolayer was dependent on the duration and frequency of electrical stimuli, as well as the size of the illuminated area and the applied light intensity, determining the degree of depolarization as well as the steepness of the local voltage gradient. In addition to the induction of oscillatory behaviour, they also tested frequency-dependent termination of induced oscillations.

Strengths:

The data from optogenetic experiments and computational modelling provide quantitative insights into the parameter space determining the induction of spontaneous excitation in the monolayer. The most important findings can also be reproduced using a strongly reduced computational model, suggesting that the observed phenomena might be more generally applicable.

Weaknesses:

While the study is thoroughly performed and provides interesting mechanistic insights into scenarios of ventricular arrhythmogenesis in the presence of localized depolarized tissue areas, the translational perspective of the study remains relatively vague. In addition, the chosen theoretical approach and the way the data are presented might make it difficult for the wider community of cardiac researchers to understand the significance of the study.

Reviewer #2 (Public review):

In the presented manuscript, Teplenin and colleagues use both electrical pacing and optogenetic stimulation to create a reproducible, controllable source of ectopy in cardiomyocyte monolayers. To accomplish this, they use a careful calibration of electrical pacing characteristics (i.e., frequency, number of pulses) and illumination characteristics (i.e., light intensity, surface area) to show that there exists a "sweet spot" where oscillatory excitations can emerge proximal to the optogenetically depolarized region following electrical pacing cessation, akin to pacemaker cells. Furthermore, the authors demonstrate that a high-frequency electrical wave-train can be used to terminate these oscillatory excitations. The authors observed this oscillatory phenomenon both in vitro (using neonatal rat ventricular cardiomyocyte monolayers) and in silico (using a computational action potential model of the same cell type). These are surprising findings and provide a novel approach for studying triggered activity in cardiac tissue.

The study is extremely thorough and one of the more memorable and grounded applications of cardiac optogenetics in the past decade. One of the benefits of the authors' "two-prong" approach of experimental preps and computational models is that they could probe the number of potential variable combinations much deeper than through in vitro experiments alone. The strong similarities between the real-life and computational findings suggest that these oscillatory excitations are consistent, reproducible, and controllable.

Triggered activity, which can lead to ventricular arrhythmias and cardiac sudden death, has been largely attributed to sub-cellular phenomena, such as early or delayed afterdepolarizations, and thus to date has largely been studied in isolated single cardiomyocytes. However, these findings have been difficult to translate to tissue and organ-scale experiments, as well-coupled cardiac tissue has notably different electrical properties. This underscores the significance of the study's methodological advances: the use of a constant depolarizing current in a subset of (illuminated) cells to reliably result in triggered activity could facilitate the more consistent evaluation of triggered activity at various scales. An experimental prep that is both repeatable and controllable (i.e., both initiated and terminated through the same means).

The authors also substantially explored phase space and single-cell analyses to document how this "hidden" bi-stable phenomenon can be uncovered during emergent collective tissue behavior. Calibration and testing of different aspects (e.g., light intensity, illuminated surface area, electrical pulse frequency, electrical pulse count) and other deeper analyses, as illustrated in Appendix 2, Figures 3-8, are significant and commendable.

Given that the study is computational, it is surprising that the authors did not replicate their findings using well-validated adult ventricular cardiomyocyte action potential models, such as ten Tusscher 2006 or O'Hara 2011. This may have felt out of scope, given the nice alignment of rat cardiomyocyte data between in vitro and in silico experiments. However, it would have been helpful peace-of-mind validation, given the significant ionic current differences between neonatal rat and adult ventricular tissue. It is not fully clear whether the pulse trains could have resulted in the same bi-stable oscillatory behavior, given the longer APD of humans relative to rats. The observed phenomenon certainly would be frequency-dependent and would have required tedious calibration for a new cell type, albeit partially mitigated by the relative ease of in silico experiments.

For all its strengths, there are likely significant mechanistic differences between this optogenetically tied oscillatory behavior and triggered activity observed in other studies. This is because the constant light-elicited depolarizing current is disrupting the typical resting cardiomyocyte state, thereby altering the balance between depolarizing ionic currents (such as Na+ and Ca2+) and repolarizing ionic currents (such as K+ and Ca2+). The oscillatory excitations appear to later emerge at the border of the illuminated region and non-stimulated surrounding tissue, which is likely an area of high source-sink mismatch. The authors appear to acknowledge differences in this oscillatory behavior and previous sub-cellular triggered activity research in their discussion of ectopic pacemaker activity, which is canonically expected more so from genetic or pathological conditions. Regardless, it is exciting to see new ground being broken in this difficult-to-characterize experimental space, even if the method illustrated here may not necessarily be broadly applicable.

We thank the reviewers for their thoughtful and constructive feedback, as well as for recognizing the conceptual and technical strengths of our work. We are especially pleased that our integrated use of optogenetics, electrical pacing, and computational modelling was seen as a rigorous and innovative approach to investigating spontaneous excitability in cardiac tissue.

At the core of our study was the decision to focus exclusively on neonatal rat ventricular cardiomyocytes. This ensured a tightly controlled and consistent environment across experimental and computational settings, allowing for direct comparison and deeper mechanistic insight. While extending our findings to adult or human cardiomyocytes would enhance translational relevance, such efforts are complicated by the distinct ionic properties and action potential dynamics of these cells, as also noted by Reviewer #2. For this foundational study, we chose to prioritize depth and clarity over breadth.

Our computational domain was designed to faithfully reflect the experimental system. The strong agreement between both domains is encouraging and supports the robustness of our framework. Although some degree of theoretical abstraction was necessary (thereby sometimes making it a bit harder to read), it reflects the intrinsic complexity of the collective behaviours we aimed to capture such as emergent bi-stability. To make these ideas more accessible, we included simplified illustrations, a reduced model, and extensive supplementary material.

A key insight from our work is the emergence of oscillatory behaviour through interaction of illuminated and non-illuminated regions. Rather than replicating classical sub-cellular triggered activity, this behaviour arises from systems-level dynamics shaped by the imposed depolarizing current and surrounding electrotonic environment. By tuning illumination and local pacing parameters, we could reproducibly induce and suppress these oscillations, thereby providing a controllable platform to study ectopy as a manifestation of spatial heterogeneity and collective dynamics.

Altogether, our aim was to build a clear and versatile model system for investigating how spatial structure and pacing influence the conditions under which bistability becomes apparent in cardiac tissue. We believe this platform lays strong groundwork for future extensions into more physiologically and clinically relevant contexts.

In revising the manuscript, we carefully addressed all points raised by the reviewers. We have also responded to each of their specific comments in detail, which are provided below.

Recommendations for the Authors:

Reviewer #1 (Recommendations for the authors):

Please find my specific comments and suggestions below:

(1) Line 64: When first introduced, the concept of 'emergent bi-stability' may not be clear to the reader.

We concur that the full breadth of the concept of emergent bi-stability may not be immediately clear upon first mention. Nonetheless, its components have been introduced separately: “emergent” was linked to multicellular behaviour in line 63, while “bi-stability” was described in detail in lines 39–56. We therefore believe that readers could form an intuitive understanding of the combined term, which will be further clarified as the manuscript develops. To further ease comprehension of the reader, we have added the following clarification to line 64:

“Within this dynamic system of cardiomyocytes, we investigated emergent bi-stability (a concept that will be explained more thoroughly later on) in cell monolayers under the influence of spatial depolarization patterns.”

(2) Lines 67-80: While the introduction until line 66 is extremely well written, the introduction of both cardiac arrhythmia and cardiac optogenetics could be improved. It is especially surprising that miniSOG is first mentioned as a tool for optogenetic depolarisation of cardiomyocytes, as the authors would probably agree that Channelrhodopsins are by far the most commonly applied tools for optogenetic depolarisation (please also refer to the literature by others in this respect). In addition, miniSOG has side effects other than depolarisation, and thus cannot be the tool of choice when not directly studying the effects of oxidative stress or damage.

The reviewer is absolutely correct in noting that channelrhodopsins are the most commonly applied tools for optogenetic depolarisation. We introduced miniSOG primarily for historical context: the effects of specific depolarization patterns on collective pacemaker activity were first observed with this tool (Teplenin et al., 2018). In that paper, we also reported ultralong action potentials, occurring as a side effect of cumulative miniSOG-induced ROS damage. In the following paragraph (starting at line 81), we emphasize that membrane potential can be controlled much better using channelrhodopsins, which is why we employed them in the present study.

(3) Line 78: I appreciate the concept of 'high curvature', but please always state which parameter(s) you are referring to (membrane voltage in space/time, etc?).

We corrected our statement to include the specification of space curvature of the depolarised region:

“In such a system, it was previously observed that spatiotemporal illumination can give rise to collective behaviour and ectopic waves (Teplenin et al. (2018)) originating from illuminated/depolarised regions (with high spatial curvature).”

(4) Line 79: 'bi-stable state' - not yet properly introduced in this context.

The bi-stability mentioned here refers back to single cell bistability introduced in Teplenin et al. (2018), which we cited again for clarity.

“These waves resulted from the interplay between the diffusion current and the single cell bi-stable state (Teplenin et al. (2018)) that was induced in the illuminated region.”

(5) Line 84-85: 'these ion channels allow the cells to respond' - please describe the channel used; and please correct: the channels respond to light, not the cells. Re-ordering this paragraph may help, because first you introduce channels for depolarization, then you go back to both de- and hyperpolarization. On the same note, which channels can be used for hyperpolarization of cardiomyocytes? I am not aware of any, even WiChR shows depolarizing effects in cardiomyocytes during prolonged activation (Vierock et al. 2022). Please delete: 'through a direct pathway' (Channelrhodopsins a directly light-gated channels, there are no pathways involved).

We realised that the confusion arose from our use of incorrect terminology: we mistakenly wrote hyperpolarisation instead of repolarisation. In addition to channelrhodopsins such as WiChR, other tools can also induce a repolarising effect, including light-activatable chloride pumps (e.g., JAWS). However, to improve clarity, we recognize that repolarisation is not relevant to our manuscript and therefore decided to remove its mention (see below). Regarding the reported depolarising effects of WiChR in Vierock et al. (2022), we speculate that these may arise either from the specific phenotype of the cardiomyocytes used in the study, i.e. human induced pluripotent stem cell-derived atrial myocytes (aCMs), or from the particular ionic conditions applied during patch-clamp recordings (e.g., a bath solution containing 1 mM KCl). Notably, even after prolonged WiChR activation, the aCMs maintained a strongly negative maximum diastolic potential of approximately –55 mV.

“Although effects of illuminating miniSOG with light might lead to formation of depolarised areas, it is difficult to control the process precisely since it depolarises cardiomyocytes indirectly. Therefore, in this manuscript, we used light-sensitive ion channels to obtain more refined control over cardiomyocyte depolarisation. These ion channels allow the cells to respond to specific wavelengths of light, facilitating direct depolarisation (Ördög et al. (2021, 2023)). By inducing cardiomyocyte depolarisation only in the illuminated areas, optogenetics enables precise spatiotemporal control of cardiac excitability, an attribute we exploit in this manuscript (Appendix 2 Figure 1).”

(6) Figure 1: What would be the y-axis of the 'energy-like curves' in B? What exactly did you plot here?

The graphs in Figure 1B are schematic representations intended to clarify the phenomenon for the reader. They do not depict actual data from any simulation or experiment. We clarified this misunderstanding by specifying that Figure 1B is a schematic representation of the effects at play in this paper.

“(B) Schematic representation showing how light intensity influences collective behaviour of excitable systems, transitioning between a stationary state (STA) at low illumination intensities and an oscillatory state (OSC) at high illumination intensities. Bi-stability occurs at intermediate light intensities, where transitions between states are dependent on periodic wave train properties. TR. OSC, transient oscillations.”

To expand slightly beyond the paper: our schematic representation was inspired by a common visualization in dynamical systems used to illustrate bi-stability (for an example, see Fig. 3 in Schleimer, J. H., Hesse, J., Contreras, S. A., & Schreiber, S. (2021). Firing statistics in the bistable regime of neurons with homoclinic spike generation. Physical Review E, 103(1), 012407.). In this framework, the y-axis can indeed be interpreted as an energy landscape, which is related to a probability measure through the Boltzmann distribution: . Here, p denotes the probability of occupying a particular state (STA or OSC). This probability can be estimated from the area (BCL × number of pulses) falling within each state, as shown in Fig. 4C. Since an attractor corresponds to a high-probability state, it naturally appears as a potential well in the landscape.

(7) Lines 92-93: 'this transition resulted for the interaction of an illuminated region with depolarized CM and an external wave train' - please consider rephrasing (it is not the region interacting with depolarized CM; and the external wave train could be explained more clearly).

We rephrased our unclear sentence as follows:

“This transition resulted from the interaction of depolarized cardiomyocytes in an illuminated region with an external wave train not originating from within the illuminated region.”

(8) Figure 2 and elsewhere: When mentioning 'frequency', please state frequency values and not cycle lengths. Please also reconsider your distinction between high and low frequencies; 200 ms (5 Hz) is actually the normal heart rate for neonatal rats (300 bpm).

In the revised version, we have clarified frequency values explicitly and included them alongside period values wherever frequency is mentioned, to avoid any ambiguity. We also emphasize that our use of "high" and "low" frequency is strictly a relative distinction within the context of our data, and not meant to imply a biological interpretation.

(9) Lines 129-131: Why not record optical maps? Voltage dynamics in the transition zone between depolarised and non-depolarised regions might be especially interesting to look at?

We would like to clarify that optical maps were recorded for every experiment, and all experimental traces of cardiac monolayer activity were derived from these maps. We agree with the reviewer that the voltage dynamics in the transition zone are particularly interesting. However, we selected the data representations that, in our view, best highlight the main mechanisms. When we analysed full voltage profiles, they didn’t add extra insights to this main mechanism. As the other reviewer noted, the manuscript already presents a wide range of regimes, so we decided not to introduce further complexity.

(10) Lines 156-157: Why was the model not adapted to match the biophysical properties (e.g., kinetics, ion selectivity, light sensitivity) of Cheriff?

The model was not adapted to the biophysical properties of Cheriff, because this would entail a whole new study involving extensive patch-clamping experiments, fitting, and calibration to model the correct properties of the ion channel. Beyond considerations of time efficiency, incorporating more specific modelling parameters would not change the essence of our findings. While numeric parameter ranges might shift, the core results would remain unchanged. This is a result of our experimental design where we applied constant illumination of long duration (6s or longer), thus making a difference in kinetical properties of an optogenetic tool irrelevant. In addition, we were able to observe qualitatively similar phenomena using many other depolarising optogenetic tools (e.g. ChR2, ReaChR, CatCh and more) in our in-vitro experiments. We ended up with Cheriff as our optotool-of-choice for the practical reasons of good light-sensitivity and a non-overlapping spectrum with our fluorescent dyes.

Therefore, computationally using a more general depolarising ion channel hints at the more general applicability of the observed phenomena, supporting our claim of a universal mechanism  (demonstrated experimentally with CheRiff and computationally with ChR2).

(11) Line 158: 1.7124 mW/mm^2 - While I understand that this is the specific intensity used as input in the model, I am convinced that the model is not as accurate to predict behaviour at this specific intensity (4 digits after the comma), especially given that the model has not been adapted to Cheriff (probably more light sensitive than ChR2). Can this be rephrased?

We did not aim for quantitative correspondence between the computational model and the biological experiments, but rather for qualitative agreement and mechanistic insight (see line 157). Qualitative comparisons are computationally obtained in a whole range of different intensities, as demonstrated in the 3D diagram of Fig. 4C. We wanted to demonstrate that at one fixed light intensity (chosen to be 1.7124 mW/mm^2 for the most clear effect), it was possible for all three states (STA, OSC. TR. OSC.) to coexist depending on the number of pulses and their period. Therefore the specific intensity used in the computational model is correct, and for reproducibility, we have left it unchanged while clarifying that it refers specifically to the in silico model:

“Simulating at a fixed constant illumination of 1.7124 𝑚𝑊∕𝑚𝑚² and a fixed number of 4 pulses, frequency dependency of collective bi-stability was reproduced in Figure 4A.”

(12) Lines 160, 165, and elsewhere: 'Once again, Once more' - please delete or rephrase.

We agree that we could have written these binding words better and reformulated them to:

“Similar to the experimental observations, only intermediate electrical pacing frequencies (500-𝑚𝑠 period) caused transitions from collective stationary behaviour to collective oscillatory behaviour and ectopic pacemaker activity had periods (710 𝑚𝑠) that were different from the stimulation train period (500 𝑚𝑠). Figure 4B shows the accumulation of pulses necessary to invoke a transition from the collective stationary state to the collective oscillatory state at a fixed stimulation period (600 𝑚𝑠). Also in the in silico simulations, ectopic pacemaker activity had periods (750 𝑚𝑠) that were different from the stimulation train period (600 𝑚𝑠). Also for the transient oscillatory state, the simulations show frequency selectivity (Appendix 2 Figure 4B).”

(13) Line 171: 'illumination strength': please refer to 'light intensity'.

We have revised our formulation to now refer specifically to “light intensity”:

“We previously identified three important parameters influencing such transitions: light intensity, number of pulses, and frequency of pulses.”

(14) Lines 187-188: 'the illuminated region settles into this period of sending out pulses' - please rephrase, the meaning is not clear.

We reformulated our sentence to make its content more clear to the reader:

“For the conditions that resulted in stable oscillations, the green vertical lines in the middle and right slices represent the natural pacemaker frequency in the oscillatory state. After the transition from the stationary towards the oscillatory state, oscillatory pulses emerging from the illuminated region gradually dampen and stabilize at this period, corresponding to the natural pacemaker frequency.”

(15) Figure 7: A)- please state in the legend which parameter is plotted on the y-axis (it is included in the main text, but should be provided here as well); C) The numbers provided in brackets are confusing. Why is (4) a high pulse number and (3) a low pulse number? Why not just state the number of pulses and add alpha, beta, gamma, and delta for the panels in brackets? I suggest providing the parameters (e.g., 800 ms cycle length, 2 pulses, etc) for all combinations, but not rate them with low, high, etc. (see also comment above).

We appreciate the reviewer’s comments and have revised the caption for figure 7, which now reads as follows:

“Figure 7. Phase plane projections of pulse-dependent collective state transitions. (A) Phase space trajectories (displayed in the Voltage – x_r plane) of the NRVM computational model show a limit cycle (OSC) that is not lying around a stable fixed point (STA). (B) Parameter space slice showing the relationship between stimulation period and number of pulses for a fixed illumination intensity (1.72 𝑚𝑊 ∕𝑚𝑚2) and size of the illuminated area (67 pixels edge length). Letters correspond to the graphs shown in C. (C) Phase space trajectories for different combinations of stimulus train period and number of pulses (α: 800 ms cycle length + 2 pulses, β: 800 ms cycle length + 4 pulses, γ: 250 ms cycle length + 3 pulses, δ: 250 ms cycle length + 8 pulses). α and δ do not result in a transition from the resting state to ectopic pacemaker activity, as under these circumstances the system moves towards the stationary stable fixed point from outside and inside the stable limit cycle, respectively. However, for β and γ, the stable limit cycle is approached from outside and inside, respectively, and ectopic pacemaker activity is induced.”

(16) Line 258: 'other dimensions by the electrotonic current' - not clear, please rephrase and explain.

We realized that our explanation was somewhat convoluted and have therefore changed the text as follows:

“Rather than producing oscillations, the system returns to the stationary state along dimensions other than those shown in Figure 7C (Voltage and x_r), as evidenced by the phase space trajectory crossing itself. This return is mediated by the electrotonic current.”

(17) Line 263: ‘increased too much’ – please rephrase using scientific terminology.

We rephrased our sentence to:

“However, this is not a Hopf bifurcation, because in that case the system would not return to the stationary state when the number of pulses exceeds a critical threshold.”

(18) Line 275: 'stronger diffusion/electrotonic influence from the non-illuminated region' - not sure diffusion is the correct term here. Please explain by taking into account the membrane potential. Please make sure to use proper terminology. The same applies to lines 281-282.

We appreciate this comment, which prompted us to revisit on our text. We realised that some sections could be worded more clearly, and we also identified an error in the legend of Supplementary Figure 7. The corresponding corrections are provided below:

“However, repolarisation reserve does have an influence, prolonging the transition when it is reduced (Appendix 2 Figure 7). This effect can be observed either by moving further from the boundary of the illuminated region, where the electrotonic influence from the non-illuminated region is weaker, or by introducing ionic changes, such as a reduction in I_Ks and/or I_to. For example, because the electrotonic influence is weaker in the center of the illuminated region, the voltage there is not pulled down toward the resting membrane potential as quickly as in cells at the border of the illuminated zone.”

“To add a multicellular component to our single cell model we introduced a current that replicates the effect of cell coupling and its associated electrotonic influence.”

“Figure 7. The effect of ionic changes on the termination of pacemaker activity. The mechanism that moves the oscillating illuminated tissue back to the stationary state after high frequency pacing is dependent on the ionic properties of the tissue, i.e. lower repolarisation reserves (20% 𝐼_𝐾𝑠 + 50% 𝐼_𝑡𝑜) are associated with longer transition times.”

(19) Line 289: -58 mV (to be corrected), -20 mV, and +50 mV - please justify the selection of parameters chosen. This also applies elsewhere- the selection of parameters seems quite arbitrary, please make sure the selection process is more transparent to the reader.

Our choice of parameters was guided by the dynamical properties of the illuminated cells as well as by illustrative purposes. The value of –58 mV corresponds to the stimulation threshold of the model. The values of 50 mV and –20 mV match those used for single-cell stimulation (Figure 8C2, right panel), producing excitable and bistable dynamics, respectively. We refer to this point in line 288 with the phrase “building on this result.” To maintain conciseness, we did not elaborate on the underlying reasoning within the manuscript and instead reported only the results.

We also corrected the previously missed minus sign: -58 mV.

(20) Figure 8 and corresponding text: I don't understand what stimulation with a voltage means. Is this an externally applied electric field? Or did you inject a current necessary to change the membrane voltage by this value? Please explain.

Stimulation with a specific voltage is a standard computational technique and can be likened to performing a voltage-clamp experiment on each individual cell. In this approach, the voltage of every cell in the tissue is briefly forced to a defined value.

(21) Figure 8C- panel 2: Traces at -20 mV and + 50 mV are identical. Is this correct? Please explain.

Yes, that is correct. The cell responds similarly to a voltage stimulus of -20 mV or one of 50 mV, because both values are well above the excitation threshold of a cardiomyocyte.

(22) Line 344 and elsewhere: 'diffusion current' - This is probably not the correct terminology for gap-junction mediated currents. Please rephrase.

A diffusion current is a mathematical formulation for a gap junction mediated current here, so , depending on the background of the reader, one of the terms might be used focusing on different aspects of the results. In a mathematical modelling context one often refers to a diffusion current because cardiomyocytes monolayers and tissues can be modelled using a reaction-diffusion equation. From the context of fine-grain biological and biophysical details, one uses the term gap-junction mediated current. Our choice is motivated by the main target audience we have in mind, namely interdisciplinary researchers with a core background in the mathematics/physics/computer science fields.

However, to not exclude our secondary target audience of biological and medical readers we now clarified the terminology, drawing the parallel between the different fields of study at line 79:

“These waves resulted from the interplay between the diffusion current (also known in biology/biophysics as the gap junction mediated current) and the bi-stable state that was induced in the illuminated region.”

(23) Lines 357-58: 'Such ectopic sources are typically initiated by high frequency pacing' - While this might be true during clinical testing, how would you explain this when not externally imposed? What could be biological high-frequency triggers?

Biological high-frequency triggers could include sudden increases in heart rates, such as those induced by physical activity or emotional stress. Another possibility is the occurrence of paroxysmal atrial or ventricular fibrillation, which could then give rise to an ectopic source.

(24) Lines 419-420: 'large ionic cell currents and small repolarising coupling currents'. Are coupling currents actually small in comparison to cellular currents? Can you provide relative numbers (~ratio)?

Coupling currents are indeed small compared to cellular currents. This can be inferred from the I-V curve shown in Figure 8C1, which dips below 0 and creates bi-stability only because of the small coupling current. If the coupling current were larger, the system would revert to a monostable regime. To make this more concrete, we have now provided the exact value of the coupling current used in Figure 8C1.

“Otherwise, if the hills and dips of the N-shaped steady-state IV curve were large (Figure 8C-1), they would have similar magnitudes as the large currents of fast ion channels, preventing the subtle interaction between these strong ionic cell currents and the small repolarising coupling currents (-0.103649 ≈ 0.1 pA).”

(25) Line 426: Please explain how ‘voltage shocks’ were modelled.

We would like to refer the reviewer to our response to comment (20) regarding how we model voltage shocks. In the context of line 426, a typical voltage shock corresponds to a tissue-wide stimulus of 50 mV. Independent of our computational model, line 426 also cites other publications showing that, in clinical settings, high-voltage shocks are unable to terminate ectopic sustained activity, consistent with our findings.

(26) Lines 429 ff: 0.2pA/pF would correspond to 20 pA for a small cardiomyocyte of 100 pF, this current should be measurable using patch-clamp recordings.

In trying to be succinct, we may have caused some confusion. The difference between the dips (≈-0.07 pA/pF) and hills (_≈_0.11 pA/pF) is approximately 0.18 pA/pF. For a small cardiomyocyte, this corresponds to deviations from zero of roughly ±10 pA. Considering that typical RMS noise levels in whole-cell patch-clamp recordings range from 2-10 pA , it is understandable that detecting these peaks and dips in an I-V curve (average current after holding a voltage for an extended period)  is difficult. Achieving statistical significance would therefore require patching a large number of cells.

Given the already extensive scope of our manuscript in terms of techniques and concepts, we decided not to pursue these additional patch-clamp experiments.

Reviewer #2 (Recommendations for the authors):

Given the deluge of conditions to consider, there are several areas of improvement possible in communicating the authors' findings. I have the following suggestions to improve the manuscript.

(1) Please change "pulse train" straight pink bar OR add stimulation marks (such as "*", or individual pulse icons) to provide better visual clarity that the applied stimuli are "short ON, long OFF" electrical pulses. I had significant initial difficulty understanding what the pulse bars represented in Figures 2, 3, 4A-B, etc. This may be partially because stimuli here could be either light (either continuous or pulsed) or electrical (likely pulsed only). To me, a solid & unbroken line intuitively denotes a continuous stimulation. I understand now that the pink bar represents the entire pulse-train duration, but I think readers would be better served with an improvement to this indicator in some fashion. For instance, the "phases" were much clearer in Figures 7C and 8D because of how colour was used on the Vm(t) traces. (How you implement this is up to you, though!)

We have addressed the reviewer’s concern and updated the figures by marking each external pulse with a small vertical line (see below).

(2) Please label the electrical stimulation location (akin to the labelled stimulation marker in circle 2 state in Figure 1A) in at least Figures 2 and 4A, and at most throughout the manuscript. It is unclear which "edge" or "pixel" the pulse-train is originating from, although I've assumed it's the left edge of the 2D tissue (both in vitro and silico). This would help readers compare the relative timing of dark blue vs. orange optical signal tracings and to understand how the activation wavefront transverses the tissue.

We indicated the pacing electrode in the optical voltage recordings with a grey asterisk. For the in silico simulations, the electrode was assumed to be far away, and the excitation was modelled as a parallel wave originating from the top boundary, indicated with a grey zone.

(3) Given the prevalence of computational experiments in this study, I suggest considering making a straightforward video demonstrating basic examples of STA, OSC, and TR.OSC states. I believe that a video visualizing these states would be visually clarifying to and greatly appreciated by readers. Appendix 2 Figure 3 would be the no-motion visualization of the examples I'm thinking of (i.e., a corresponding stitched video could be generated for this). However, this video-generation comment is a suggestion and not a request.

We have included a video showing all relevant states, which is now part of the Supplementary Material.

(4) Please fix several typos that I found in the manuscript:

(4A) Line 279: a comma is needed after i.e. when used in: "peculiar, i.e. a standard". However, this is possibly stylistic (discard suggestion if you are consistent in the manuscript).

(4B) Line 382: extra period before "(Figure 3C)".

(4C) Line 501: two periods at end of sentence "scientific purposes.." .

We would like to thank the reviewer for pointing out these typos. We have corrected them and conducted an additional check throughout the manuscript for minor errors.

self-direction 自らによる方向決定、自己主導性

universalism 普遍主義、《神学》万人救済説◆通例Universalism

Reviewer #2 (Public review):

Summary:

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation.

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis.

Strengths:

The 15N,1H and 13C,methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin with both kD and description of the interfaces.

Weaknesses:

This reviewer has identified only minor weaknesses with the study.

(1) I don't observe two overlapping spectra of Arrestin2 (1-393) +/- CLTC NTD in Supp Figure 1

(2) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition and is this what is shown in Supp Fig 2D

(3) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems?

(4) At one point the authors added excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface?

(5) Once phosphopeptide activates arrestin-2 and AP2 binds can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

(6) I'd be tempted to move the discussion of class A and class B GPCRs and their presumed differences to the intro and then motivate the paper with specific questions.

(7) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors.

Reviewer #3 (Public review):

Summary:

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field.

Strengths:

Strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL 376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Fig. 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2.

SEC and NMR data suggest that full-length arr2 (1-418) binding with 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Fig. 3). The pp6 peptide shows the highest degree of arr2 activation, and 2-adaptin binding, compared to less phosphorylated peptide or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the AP2 interaction is necessary for CCR5 endocytosis.

To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes onto endosomes (Fig. 4). The data suggest that complex internalization is dependent on AP2 binding not clathrin (Fig. 5).

The addition of the antagonist experiment/data adds rigor to the study.

Overall, this is a solid study that will be of interest to the field.

Author response:

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

Reviewer #1 (Public review): 

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data are particularly compelling, clearly demonstrating chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants. 

To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization. 

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays. 

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fitsall model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure. 

We thank the referee for this positive assessment of our manuscript. Indeed, by stepping away from the common receptor models for understanding internalization (b2AR and V2R), we revealed the phosphorylation level of the receptor as a key factor in driving the sequestration of the receptor from the plasma membrane. We hope that the proposed mechanistic model will aid further studies to obtain an even more detailed understanding of forces driving receptor internalization.

Reviewer #2 (Public review): 

Summary: 

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin-mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time, a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation. 

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis. 

Strengths: 

The 15N,1H, and 13C, methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of the activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin, with both kD and description of the interfaces. 

We sincerely thank the referee for this encouraging evaluation of our work and appreciate the recognition of the NMR efforts and insights into the arrestin–clathrin–AP2 interactions.

Weaknesses: 

This reviewer has identified only minor weaknesses with the study.

(1) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition, and is this what is shown in Supplementary Figure 2D? 

We believe the reviewer is referring to Figure 3 - figure supplement 1. In this figure, the panels E and F show resonances of arrestin2^1-418 (apo state shown with black outline) disappear upon the addition of CCR5pp6 (arrestin2^1-418•CCR5pp6 complex spectrum in red). The panels C and D show resonances of arrestin2^1-418 (apo state shown with black outline), which remain unchanged upon addition of AP2b2^701-937 (orange), indicating no complex formation. We also recorded a spectrum of the arrestin2^1-418 •CCR5pp6 complex under addition of AP2b2 ^701-937(not shown), but the arrestin2 resonances in the arrestin2¹⁴¹⁸ •CCR5pp6 complex were already too broad for further analysis. This had been already explained in the text.

“In agreement with the AP2b2 NMR observations, no interaction was observed in the arrestin2 methyl and backbone NMR spectra upon addition of AP2b2 in the absence of phosphopeptide (Figure 3-figure supplement 1C, D). However, the significant line broadening of the arrestin2 resonances upon phosphopeptide addition (Figure 3-figure supplement 1E, F) precluded a meaningful assessment of the effect of the AP2b2 addition on arrestin2 in the presence of phosphopeptide””.

(2) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems. 

We thank the referee for this comment. We would like to clarify that in general a broadened spectrum beyond what is expected from the rotational correlation time does not necessarily correlate with sample stability problems. It is rather evidence of conformational intermediate exchange on the micro- to millisecond time scale.

The displayed ¹H-¹⁵ N spectra of apo arrestin2 already suffer from line broadening due to such intrinsic mobility of the protein. These spectra were recorded with acquisition times of 50 ms (¹⁵N) and 55 ms (¹H) and resolution-enhanced by a 60˚-shifted sine-bell filter for ¹⁵N and a 60˚-shifted squared sine-bell filter for ¹H, respectively, which leads to the observed resolution with still reasonable sensitivity. The ¹H-¹⁵ resonances in Fig. 1b (arrestin2^1-393) look particularly narrow. However, this region contains a large number of flexible residues. The full spectrum, e.g. Figure 1-figure supplement 2, shows the entire situation with a clear variation of linewidths and intensities. The linewidth variation becomes stronger when omitting the resolution enhancement filters.

The addition of the CCR5pp6 phosphopeptide does not change protein stability, which we assessed by measuring the melting temperature of arrestin2^1-418 and arrestin2^1-418 •CCR5pp6 complex (Tm = 57°C in both cases). We believe that the explanation for the increased broadening of the arrestin2 resonances is that addition of the CCR5pp6, possibly due to the release of the arrestin2 strand b20, amplifies the mentioned intermediate timescale protein dynamics. This results in the disappearance of arrestin2 resonances. 

We have now included the assessment of arrestin2^1-418 and arrestin2^1-418 •CCR5pp6 stability in the manuscript:

“The observed line broadening of arrestin2 in the presence of phosphopeptide must be a result of increased protein motions and is not caused by a decrease in protein stability, since the melting temperature of arrestin2 in the absence and presence of phosphopeptide are identical (56.9 ± 0.1 °C)”.

(3) At one point, the authors added an excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface? 

Unfortunately, when we titrate arrestin2 with CCR5pp6 (please see Isaikina & Petrovic et. al, Mol. Cell, 2023 for more details), the arrestin2 resonances undergo fast-to-intermediate exchange upon binding. In the presence of phosphopeptide excess, very few resonances remain, the majority of which are in the disordered region, including resonances from the clathrin-binding loop. Due to the peak overlap, we could not unambiguously assign arrestin2 resonances in the bound state, which precluded our assessment of the arrestin2-clathrin interaction in the presence of phosphopeptide. We have made this now clearer in the paragraph ‘The arrestin2-clathrin interaction is independent of arrestin2 activation’

“Due to significant line broadening and peak overlap of the arrestin2 resonances upon phosphopeptide addition, the influence of arrestin activation on the clathrin interaction could not be detected on either backbone or methyl resonances”.

(4) Once phosphopeptide activates arrestin-2 and AP2 binds, can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

This would be an interesting mechanism. In principle, this should be possible as long as the other (phosphorylated) receptor outcompetes the initial phosphopeptide with higher affinity towards the binding site. However, we do not have experiments to assess this process directly. Therefore, we rather wish not to further speculate.

(5) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors. 

We thank the referee for this question. Indeed, we tested whether PIP2 can stabilize the arrestin2•CCR5pp6•AP2 complex by SEC experiments. Unfortunately, the addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. The resolution of SEC experiments was not sufficient to distinguish arrestin2 in oligomeric form or in arrestin2•CCR5pp6•AP2 complex. We now mention this in the text: 

“We also attempted to stabilize the arrestin2-AP2b2-phosphopetide complex through the addition of PIP2, which can stabilize arrestin complexes with the receptor (Janetzko et al., 2022). The addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. Unfortunately, the resolution of the SEC experiments was not sufficient to separate the arrestin2 oligomers from complexes with AP2b2”.

Reviewer #3 (Public review): 

Summary: 

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field. 

Strengths: 

(1) The strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL 376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Figure 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2.

(2) SEC and NMR data suggest that full-length arr2 (1-418) binding with the 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Figure 3). The pp6 peptide shows the highest degree of arr2 activation and 2-adaptin binding, compared to less phosphorylated peptides or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the AP2 interaction is necessary for CCR5 endocytosis. 

(3) To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes into endosomes (Figure 4). The data suggest that complex internalization is dependent on AP2 binding, not clathrin (Figure 5). 

We thank the referee for the careful and encouraging evaluation of our work. We appreciate the recognition of the solidity of our data and the support for our conclusions regarding the distinct roles of AP2 and clathrin in arrestin-mediated receptor internalization.

Weaknesses:

The interaction of truncated arr2 (1-393) was not impacted by CCR5 phospho-peptide pp6, suggesting the interaction with clathrin is not dependent on arrestin activation (Figure 2). This raises some questions.

We thank the referee for raising this concern, as we were also surprised by the discovery that the interaction does not depend on arrestin activation. However, the NMR data clearly show at atomic resolution that arrestin activation does not influence the interaction with clathrin in vitro. Evolutionary, the arrestin-clathrin interaction appears not to be conserved as the visual arrestin completely lacks a clathrin-binding motif. For that reason, we believe that the weak arrestin-clathrin interaction provides more of a supportive role during the internalization rather than the regulatory interaction with AP2, which requires and quantitatively depends on the arrestin2 activation. We have reflected on this in the Discussion:

“Although the generalization of this mechanism from CCR5 to other arr-class B receptors has to be explored further, it is indirectly corroborated in the visual rhodopsin-arrestin1 system. The arr-class B receptor rhodopsin (Isaikina et al., 2023) also undergoes CME (Moaven et al., 2013) with arrestin1 harboring the conserved AP2 binding motif, but missing the clathrinbinding motif (Figure 1-figure supplement 1A)”.

Overall, the data are solid, but for added rigor, can these experiments be repeated without tagged receptor and/or arr2? My concern stems from the fact that the stability of the interaction between arr2 and the receptor may be related to the position of the tags.

We thank the referee for this suggestion, which refers to the cellular experiments; the biophysical experiments were carried out without tags. To eliminate the possibility of tags contributing to receptor-arrestin2 binding in the cellular experiments, we also performed the experiments in the presence of CCR5 antagonist [5P12]CCL5 (Figure 4). These data show that in the case of inactive CCR5, arrestin2 is not recruited to CCR5, nor does it form internalization complexes, which would be the case if the tags were increasing the receptorarrestin interaction. In contrast, if the tags were decreasing the interaction, we would not expect such a strong internalization. As indicated below, we have also attempted to perform our cellular experiments using an N-terminally SNAP-tagged CCR5. Unfortunately, this construct did not express in HeLa cells indicating that SNAP-CCR5 was either toxic or degraded.

Reviewing Editor Comments: 

Overall, the reviewers did not suggest much by way of additional experiments. They do suggest several aspects of the manuscript that would benefit from further clarification. 

Reviewer #1 (Recommendations for the authors): 

(1) The distinction between arrestin 2S and arrestin 2L as relates to the canonical and non-canonical clathrin binding sites would benefit from clarification, particularly because the second binding site depends on the splice variant. This is something that some readers may not be familiar with (particularly young ones that are hopefully part of the intended readership).

We thank the referee for this suggestion. We would like to emphasize that in our work, only the long arrestin2 splice variant was used, which contains both binding sites. We have now introduced the splice variants and their relation to the clathrin binding sites in the text. 

In section ‘Localizing and quantifying the arrestin2-clathrin interaction by NMR spectroscopy’:

“Clathrin and arrestin interact in their basal state (Goodman et al., 1996), and a structure of a complex between arrestin2 and the clathrin heavy chain N-terminal domain (residues 1-363, named clathrin-N in the following) has been solved by X-ray crystallography (PDB:3GD1) in the absence of an arrestin2-activating phosphopeptide (Kang et al., 2009). This structure (Figure 1-figure supplement 1B) suggests a 2:1 binding model between arrestin2 and clathrinN. The first interaction (site I) is observed between the ³⁷⁶LIELD³⁸⁰ clathrin-binding motif of the arrestin2 CBL and the edge of the first two β-sheet blades of clathrin-N, whereas the second interaction (site II) occurs between arrestin2 residues ³³⁴LLGDLA³³⁹ and the 4th and 5th blade of clathrin-N. The latter arrestin interaction site is not present in the arrestin2 splice variant arrestin2S (for short) where an 8-amino acid insert (residues 334-341) between β-strands 18 and 19 is removed (Kang et al., 2009)”.

Section ‘The arrestin2-clathrin interaction is independent of arrestin2 activation’

“Figure 2A (left) shows the intensity changes (full spectra in Figure 2-figure supplement 1A) of the clathrin-N ¹H-¹⁵N TROSY resonances [assignments transferred from BMRB, ID:25403 (Zhuo et al., 2015)] upon addition of a one-molar equivalent of arrestin2^1-393. A significant intensity reduction due to line broadening is detected for clathrin-N residues 39-40, 48-50, 62-72, 83-90, 101-106, and 108. These residues form a clearly defined binding region at the edges of blade 1 and blade 2 of clathrin-N (Figure 2A, right), which corresponds to interaction site I in the 3GD1 crystal structure, involving the conserved arrestin2 ³⁷⁶LIELD³⁸⁰ motif. However, no significant signal attenuation was observed for clathrin-N residues in blade 4 and blade 5, which would correspond to the crystal interaction site II with arrestin2 residues ³³⁴LLGDLA³³⁹ that are absent in the arrestin2S splice variant. Thus only one arrestin2 binding site in clathrin-N is detected in solution, and site II of the crystal structure may be a result of crystal packing”.

(2) Acronym density is high throughout. While many are standard in the clathrin literature, this could hinder accessibility for readers with a GPCR or arrestin focus.

We agree with the referee. The acronyms were hard to avoid. The most non-obvious acronym seems ‘CLTC-NTD’ for the N-terminal domain of the clathrin heavy chain, which uses the non-obvious, but common gene name CLTC for the clathrin heavy chain. We have now replaced ‘CLTC-NTD’ by ‘clathrin-N’ and hope that this makes the text easier to follow.

(3) The NMR section, while impressive in scope, had writing that was more difficult to follow than the rest. I am curious what percentage of resonance could be assigned. 

We apologize if the NMR sections of this manuscript were unclear. We attempted to provide a very detailed description of the experimental setup and the spectral results. Being experienced NMR spectroscopists, we have tried very hard to obtain good 3D triple resonance spectra for assignments, but their sensitivity is very low. We believe that this is due to the microsecond dynamics present in the system, which makes the heteronuclear transfers inefficient. So far, we have been able to assign ~30% of the visible arrestin2 resonances. We are still validating the assignments and are working on the analysis and an explanation for this arrestin2 behavior. Therefore, at this point, we want to refrain from stronger statements besides that considerable intrinsic microsecond dynamics is impeding the assignment process.

(4) It may be worth noting in the main text that truncated arrestins have slightly higher basal activation. I was curious why the truncated arrestin was not chosen for the AP2 NMR titrations. Presumably, an effect would be more likely to be seen.

While some truncated arrestin2 variants (comprising residues 1-382 or 1-360) indeed show higher basal activity than the full-length arrestin2, they typically completely lack the b20 strand (residues 386-390), which is crucial for the formation of a parallel b-sheet with strand b1, and whose release governs arrestin activation. Our truncated arrestin2 construct comprises residues 1-393 and contains strand b20. In our experience, no significant difference in basal activity, as assessed by Fab30 binding, was detected for arrestin2^1-393 and arrestin2^1-418 (Author response image 1).

Author response image 1.

SEC profiles showing arrestin2^1–393 (left) and arrestin2^1-418 (right) activation by the CCR5pp6 phosphopeptide as assayed by Fab30 binding. The active ternary arrestin2-phosphopeptide-Fab30 complex elutes at a lower volume than the inactive apo arrestin2 or the binary arrestin2-phosphopeptide complex. Both arrestin2 constructs are activated by the phosphopeptide to a similar level as assessed by the integrated SEC volumes.

We want to emphasize that we used full-length arrestin2^1-418 in order to assess the AP2 interaction, as the crystal structure of arrestin2 peptide-AP2 (PDB:2IV8) shows residues past the residue 393 involved in binding.

PDB codes are currently not accompanied by corresponding literature citations throughout. Please add these. 

Thank you for this suggestion. In the manuscript, we were careful to provide the full literature citation the first time each PDB code is mentioned. To avoid redundancy and maintain clarity, we rather do not want to repeat the citations with every subsequent mentioning of the PDB code.

(5) The AlphaFold model could benefit from a more transparent discussion of prediction confidence and caveats. The younger crowd (part of the presumed intended readership) tends to be more certain that computational output is 'true'. Figure 1A shows long loops that are likely regions of low confidence in the prediction. Displaying expected disordered regions as transparent or color-coded would help highlight these as flexible rather than stable, especially for that same younger readership. 

We need to explain that the AlphaFold model of arrestin2 was only used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available apo bovine (PDB:1G4M) and apo human (PDB:8AS4) arrestin2 crystal structures. However, the AlphaFold model of arrestin2 is basically identical to the crystal structures in the regions that are visible in the crystal structures. We have clarified this now in the caption to Figure 1.

“The model was used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available crystal structures of apo arrestin2 [bovine: PDB 1G4M (Han et al., 2001), human: PDB 8AS4 (Isaikina et al., 2023)]. In the other structured regions, the model is virtually identical to the crystal structures”.

(6) Several figure panels were difficult to interpret due to their small size. Especially microscopy insets, where I needed to simply trust that the authors were accurately describing the data. Enlarging panels is essential, and this may require separating them into different figures.

We appreciate the referee’s concern regarding figure readability. However, we want to indicate that all our figures are provided as either high-resolution pixel or scalable vector graphics, which allow for zooming in to very fine detail, either electronically or in print. This ensures that microscopy insets and other small panels can be examined clearly when viewed appropriately. We believe the current layout of the figures is necessary to be able to efficiently compare the data between different conditions.

Many figure panels had text size that was too small. Font inconsistencies across figures also stand out. 

We apologize for this. We have now enlarged the font size in the figures and made the styles more consistent.

For Fig. 1F, consider adding individual data points and error bars.

Thank you for this suggestion. However, Figure 1F already contains the individual data points, with colored circles corresponding to the titration condition. As we did not have replicates of the titration, no error bars are shown. However, the close agreement of the theoretical fit with the individual measured data points stemming from different experiments shows that the statistical errors are indeed very small. We have estimated an overall error for the Kd (as indicated in panel F, right) by error propagation based on an estimate of the chemical shift error as obtained in the NMR software POKY (based on spectral noise). 

Reviewer #2 (Recommendations for the authors):

(1) I don't observe two overlapping spectra of Arrestin2 (1393) +/- CLTC NTD in Supplementary Figure 1.

As explained above all the spectra are shown as scalable vector graphics. The overlapping spectra are visible when zoomed in.

(2) I'd be tempted to move the discussion of class A and class B GPCRs and their presumed differences to the intro and then motivate the paper with specific questions.

We appreciate the referee’s suggestion and had a similar idea previously. However, as we do not have data on other class-A or class-B receptors, we rather don’t want to motivate the entire manuscript by this question.

Reviewer #3 (Recommendations for the authors): 

(1) What happens with full-length arr2 (1-418) when the phospho-peptide pp6 is added to the reaction? It's unclear to me that 1-418 would behave the same as 1-393 because the arr2 tail of 1-393 is likely sufficiently mobile to accommodate binding to CLTC NTD. I suggest attempting this experiment for added rigor.

We believe that there is a misunderstanding. The 1-393 and 1-418 constructs differ by the disordered C-terminal tail, which is not involved in the clathrin interaction with the arrestin2 376-380 (LIELD) residues. Accordingly, both 1-393 and 1-418 constructs show almost identical interactions with clathrin (Figure 2A and 2C). Moreover, the phospho-activated arrestin2^1-393 (Figure 2B) interacts identically with clathrin as inactive arrestin2^1-393 and inactive arrestin2^1-418. We believe that this comparison is sufficient for the conclusion that arrestin activation does not play a role in arrestin-clathrin binding.

(2) If the tags were moved to the N-terminus of the receptor and/or arr2, I wonder if the complex is as stable (Figure 4)? 

We thank the referee for their suggestion. We have indeed attempted to perform our experiments using an N-terminally SNAP-tagged CCR5. Unfortunately, this construct did not express in the HeLa cells indicating that SNAP-CCR5 was either toxic or degraded. Unfortunately, as the lab is closing due to the retirement of the PI, we are not able to repeat these experiments with further differently positioned tags. We refer also to our answer above that the experiments with the antagonist [5P12]CCL5 present a certain control.

(3) A biochemical assay to measure receptor internalization, in addition to the cell biological approach (Figure 5), would add additional rigor to the study and conclusions.

We tried to measure internalization using a biochemical approach. We tried to pull-down CCR5 from HeLa cells and assess arrestin binding. Unfortunately, even using different buffer conditions, we found that CCR5 was aggregating once solubilized from membranes, preventing us from doing this analysis. We had a similar problem when we exogenously expressed CCR5 in insect cells for purification purposes. We have long experience with CCR5, and this receptor is very aggregation-prone due to extended charged surfaces, which interact with the chemokines.

As an alternative, and in support of the cellular immunofluorescence assays, we also attempted to obtain internalization data via FACS using a CCR5 surface antibody (CD195 Monoclonal Antibody eBioT21/8). CD195 recognizes the N-terminus of the receptor. Unfortunately, the presence of the chemokine ligand (~ 8 kDa) interferes with antibody binding, precluding the quantitative biochemical assessment of the arrestin2 mutants on the CCR5 internalization.

For these reasons, we were particularly careful to quantify CCR5 internalization from the immunofluorescence microscopy data using colocalization coefficients as well as puncta counting (Figure 4+5).

Reviewer #2 (Public review):

Summary:

In this paper, the authors defined the "channelome," consisting of 419 predicted human ion channels as well as 48,000 ion channel orthologs from other organisms. Using this information, the ion channels were clustered into groups, which can potentially be used to make predictions about understudied ion channels in the groups. The authors then focused on the CALHM ion channel family, mutating conserved residues and assessing channel function.

Strengths:

The curation of the channelome provides an excellent resource for researchers studying ion channels. Supplemental Table 1 is well organized with an abundance of useful information.

Comments on revisions:

The authors have thoroughly addressed my concerns and the manuscript is substantially improved. I have just a few suggestions regarding wording/clarification.

In Supplemental Figure 4, the Western blots (n=3) were quantitated, but the surface biotinylation was not. While I suppose that it is fine to just show one representative experiment for the biotinylation assay, the authors should indicate in the legend how many times this was done. It is essential to know whether these data in Supplemental Figure 4E, F are reproducible as they are absolutely critical for interpretation of all of the data in Figure 5.

Author response:

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

Reviewing Editor Comments:

(A) Revisions related to the first part, regarding data mining and curation:

(1) One question that arises with the part of the manuscript that discusses the identification and classification of ion channels is whether these will be made available to the wider public. For the 419 human sequences, making a small database to share this result so that these sequences can be easily searched and downloaded would be desirable. There are a variety of acceptable formats for this: GitHub/figshare/zenodo/university website that allows a wider community to access their hard work. Providing such a resource would greatly expand the impact of this paper. The same question can be asked of the 48,000+ ion channels from diverse organisms.

We thank the reviewer for providing this important feedback. While the long term plan is to provide access to these sequences and annotations through a knowledge base resource like Pharos, we agree with the comments that it would be beneficial to have these sequences made available with the manuscript as well. We have compiled 3 fasta files containing the following: 1) Full length sequences for the curated 419 ion channel sequences. 2) Pore containing domain sequences for the 343 pore domain containing human ion channel sequences. 3) All the identified orthologs for the human ion channels.

For each sequence in these files, we have extended the ID line to include the most pertinent annotation information to make it readily available. For example, the id>sp|P48995|TRPC1_HUMAN|TRP:VGIC--TRP-TRPC|pore-forming|dom:387-637 provides the classification, unit and domain bounds for the human TRPC1 in the fasta file itself.

These files have been uploaded to Zenodo and are available for download with doi 10.5281/zenodo.16232527. We have included this in the Data Availability statement of the manuscript as well.

(2) Regarding the 48,000+ sequences, what checks have been done to confirm that they all represent bona fide, full-length ion channel sequences? Uniprot contains a good deal of unreviewed sequences, especially from single-celled organisms. The process by which true orthologues were identified and extraneous hits discarded should be discussed in more detail, and all inclusion criteria should be described and justified, clearly illustrating that the risk of gene duplicates and fragments in this final set of ion channel orthologues has been avoided. Related to this, does this analysis include or exclude isoforms?

We thank the reviewer for raising this important point. Our selection of curated proteomes and the KinOrtho pipeline for orthology detection returns, up to an extent, reliable orthologous sequence sets. In brief, our database sequences are retrieved from full proteomes that only include proteins that are part of an official proteome release. Thus, they are mapped from a reference genome to ensure species-specific relevance and avoid redundancy. The >1500 proteomes in this analysis were selected based on their wider use in other orthology detection pipelines like OMA and InParanoid. Our orthology detection pipeline, KinOrtho, performs a fulllength and a domain-based orthology detection which ensures that the orthologous relationships are being defined based on the pore-domain sequence similarity. 

But we agree with the reviewer that this might leave room for extraneous, fragments or misannotated sequences to be included in our results. Taking this into careful consideration, we have expanded our sequence validation pipeline to include additional checks such as checking the uniport entry type, protein existence evidence and sequence level checks such as evaluating the compositional bias, non-standard codons and sequence lengths. These validation steps are now described in detail in the Methods section under orthology analysis (lines 768-808). All the originally listed orthologous sequences passed this validation pipeline and thus provide additional confidence that they are bona fide full length ion channel sequences.

We have also expanded this section (lines 758 – 766) to provide more details of the KinOrtho pipeline for orthology detection, which is a previously published method used for orthology detection in kinases by our lab.

Finally, our orthology analysis excludes isoforms and only spans the primary canonical sequences that are part of the UniProt Proteomes annotated sequence set. The isoforms that are generally available in UniProt Proteomes in a separate file named *_additional.fasta were not included in this analysis.

(3) The decision to show the families of ion channels in Figure 1 as pie charts within a UMAP embedding is intriguing but somewhat non-intuitive and difficult to understand. Illustrating these results with a standard tree-like visualization of the relationship of these channels to each other would be preferred.

We appreciate the feedback provided by the reviewer, and understand that a standard tree-like visualization would be much easier to interpret and familiar than a bubble chart based on UMAP embeddings. However, we opted to use the bubble chart for the following reasons:

Low sequence similarity: the 419 human ICs share very minimal sequence similarity, falling in the twilight zone or lower ( Dolittle, 1992; PMID:1339026). Thus, traditional multiple sequence alignment and phylogenetic reconstruction methods perform very poorly and generate unreliable or even misleading results. To explore the practicality of this option, we pursued performing a multiple sequence alignment of just 3 of the possibly related IC families as suggested by reviewer 2 (CALHM, Pannexins, and Connexins) using the state of the art structure based sequence alignment method Foldmason (doi: https://doi.org/10.1101/2024.08.01.606130). Even then, the sequence alignment and the resulting tree for just these 3 families were poor and unreliable, as illustrated in the attached Author response Image 2.

Protein embeddings based clustering: Novel LLM based approaches such as the protein language model embeddings offer ways to overcome these limitations by capturing sequence, structure, function and evolutionary properties in a high-dimensional space. Thus, we employed this model using DEDAL followed by UMAP for dimensionality reduction, which preserves biologically meaningful local and global relationships.

Abstraction at family level: In Figure 1, we aggregate individual channels into family bubbles with their positions representing the average UMAP coordinates of their members. This offers a balance between an intuitive view of how IC families are distributed in the embedding space and reflects potential functional and evolutionary proximities, while not being impeded by individual IC relationships across families.

We have revised the figure legend (lines 1221 – 1234) with additional description of the visualization and the process used to generate it, and the manuscript text (lines 248-270) provides the rationale behind the selection of this method.

(4) A strength of this paper is the visualization of 'dark' ion channels. However, throughout the paper, this could be emphasized more as the key advantage of this approach and how this or similar approaches could be used for other families of proteins. Specifically, in the initial statement describing 'light' vs 'dark channels', the importance of this distinction and the historical preference in science to study that which has already been studied can be discussed more, even including references to other studies that take this kind of approach. An example of a relevant reference here is to the Structural Genomics Consortium and its goals to achieve structures of proteins for which functions may not be well-characterized. Clarifying these motivations throughout the entire paper would strengthen it considerably.

We thank the reviewer for this constructive comment and agree that highlighting the strength of visualizing “dark” channels and prioritizing them for future studies would strengthen the paper. As suggested, we have revised the text throughout the paper (lines 84-89, 176-180) to contextualize and emphasize this distinction. We have also added a reference for the Structural Genomics Consortium, which, along with resources like IDG, has provided significant resources for prioritizing understudied proteins.

(5) Since the authors have generated the UMAP visualization of the channome, it would be interesting to understand how the human vs orthologue gene sets compare in this space.

We appreciate the reviewer’s input. It is an interesting idea to explore the UMAP embedding space for the human ICs along with their orthologs. The large number of orthologous sequences (>37,000) would certainly impose a computational challenge to generate embeddings-based pairwise alignments across all of them. Downstream dimensionality reduction from such a large set and the subsequent visualization would also suffer from accuracy and interpretability concerns. However, to follow up on the reviewer’s comments, we selected orthologous sequences from a subset of 12 model organisms spanning all taxa (such as mouse, zebrafish, fruit fly, C. elegans, A. thaliana, S. cerevisiae, E. coli, etc.).This increased the number of sequences for analysis to 1094 from 343, which is still manageable for UMAP. Using the exact same method, we generated the UMAP embeddings plot for this set as shown below. 

Author response image 1.

UMAP embeddings of the human ICs alongside orthologs from 12 model organisms

As shown above, we observed that each orthologous set forms tight, well-defined clusters, preserving local relationships among closely related sequences. For example, a large number of VGICs cluster more closely together compared to Supplementary Figure 1 (with only the human ICs). However, families that were previously distant from others now appear to be even more scattered or pushed further away, indicating a loss of global structure. This pattern suggests that while local distances are well preserved, the global topology of the embedding space could be compromised. Moreover, we find that the placement of ICs with respect to other families is highly sensitive to the parameter choices (e.g., n_neighbors and min_dist), an issue which we did not encounter when using only the human IC sequences. The inclusion of a large number of orthologous sequences that are highly similar to a single human IC but dissimilar to others skews the embedding space, emphasizing local structure at the expense of global relationships.

Since UMAP and similar dimensionality reduction methods prioritize local over global structure, the resulting embeddings accurately reflect strong ortholog clustering but obscure broader interfamily relationships. Consequently, interpreting the spatial arrangement of human IC families with respect to one another becomes unreliable. We have made this plot available as part of this response, and anyone interested can access this in the response document.   

(6) Figure 1 should say more clearly that this is an analysis of the human gene set and include more of the information in the text: 419 human ion channel sequences, 75 sequences previously unidentified, 4 major groups and 55 families, 62 outliers, etc. Clearer visualizations of these categories and numbers within the UMAP (and newly included tree) visualization would help guide the reader to better understand these results. Specifically, which are the 75 previously unidentified sequences?

We thank the reviewer for the comments. To address this, we have revised Figure 1 and added more information, including a clear header that states that these are only human IC sets, numbers showing the total number of ICs, and the number of ICs in each group. We have further included new Supplementary Figure 2 and Supplementary Table 2, which show the overlap of IC sequences across the different resources. Supplementary Figure 2 is an upset plot that provides a snapshot of the overlap between curated human ICs in this study compared to KEGG, GtoP, and Pharos. Supplementary Table 2 provides more details on this overlap by listing, for each human IC, whether they are curated as an IC in the 3 IC annotation resources. We believe these additions should provide all the information, including the unidentified sequences we are adding to this resource.

(7) Overall, the manuscript needs to provide a clearer description of the need for a better-curated sequence database of ion channels, as well as how existing resources fall short.

We thank the reviewer for pointing out this important gap in the description. As suggested, we have revised the text thoroughly in the Introduction section to address this comment. Specifically, we have added sections to describe existing resources at sequence and structure levels that currently provide details and/or classification of human ion channels. Then, we highlight the facts that these resources are missing some characterized pore-containing ICs, do not include any information on auxiliary channels, and lack a holistic evolutionary perspective, which raises the need for a better-curated database of ion channels. Please refer to lines 57-63, 73-79, and 95 – 119 for these changes and additions.

(8) Some of the analysis pipeline is unclear. Specifically, the RAG analysis seems critical, but it is unclear how this works - is it on top of the GPT framework and recursively inquires about the answer to prompts? Some example prompts would be useful to understand this.

We thank the reviewer for highlighting this gap in explanation. We understand that the details provided in the Methods and Supplementary Figure 1 may not have sufficiently explained the pipeline, and are missing some important details. The RAG pipeline leverages vector-based retrieval integrated with OpenAI’s GPT-4o model to systematically search literature and generate evidence-based answers. The process is as follows:

Literature sources (PubMed articles) relevant to the annotated ion channels were converted into vector representations stored in a Qdrant database.

Queries constructed from the annotated IC dataset were submitted to the vector database, retrieving contextually relevant literature segments.

Retrieved contexts served as inputs to the GPT-4o model, which produced structured JSON-formatted responses containing direct evidence regarding ion selectivity and gating mechanisms, along with associated confidence scores.

To clarify this further, we have rewritten the relevant subsection in lines 649 - 718. Now, this section provides a detailed description of the RAG pipeline. Also, we have improved Supplementary Figure 1 to provide a clearer description of the pipeline. We have also provided an example prompt template to illustrate the query. These additions clarify how the pipeline functions and demonstrate its practical utility for IC annotation.

(9) The existence of 76 auxiliary non-pore containing 'ion channel' genes in this analysis is a little confusing, as it seems a part of the pipeline is looking for pore-lining residues. Furthermore, how many of these are picked up in the larger orthologues search? Are these harder to perform checks on to ensure that they are indeed ion channel genes? A further discussion of the choice to include these auxiliary sequences would be relevant. This could just be further discussion of the literature that has decided to do this in the past.

We thank the reviewer for this comment, and agree that further clarification of our selection and definition of auxiliary IC sequences would be helpful. As the reviewer has pointed out, one of the annotation pipeline steps is indeed looking for the pore-lining residues. Any sequences that do not have a pore-containing domain are then considered to be auxiliary, and we search for additional evidence of their binding with one of the annotated pore-containing ICs. If such evidence is not found in the literature, we remove them from our curated IC list. 

In response to the above comment, we have revised the manuscript text to provide these details. In the Introduction section, we have added references to previous literature that have described auxiliary ICs and also pointed out that the existing ion channel resources do not account for such auxiliary channels (lines 73-79, 107-108,148-149). We have also expanded the Methods section to describe the selection and definition of auxiliary channels (lines 640-646).

With regards to the orthology analysis, since auxiliary channels do not have a pore domain, and our orthology pipeline requires a pore domain similarity search and hit, we did not include them in this part of the analysis. We have clarified the text in the Results section to ensure this is communicated properly throughout the manuscript (lines 212-215, 260-263). 

(10) Why are only evolutionary relationships between rat, mouse, and human shown in Figure 3A? These species are all close on the evolutionary timeline.

We thank the reviewer for this comment. Figure 3A currently provides a high-level evolutionary relationship across the 6 human CALHM members as a pretext for the pattern based Bayesian analysis. However, since this analysis is based on a wider set of orthologs that span taxa, we agree that a larger tree that includes more orthologs is warranted.

We have now revised Figure 3A to include an expanded tree that includes 83 orthologs from all 6 human CALHM members spanning 14 organisms from different taxa, ranging from mammals, fishes, birds, nematodes, and cnidarians. The overall structure of the tree is still consistent with 2 major clades as before, with CALHM 1 and 3 in the first clade and CALHM 2,4,5, and 6 in the second clade, with good branch support.

(B) Revisions related to the second part, regarding the analysis of CAHLM channel mutations:

(1) It would strengthen the manuscript if it included additional discussion and references to show that previous methods to analyze conserved residues in CALHM were significantly lacking. What results would previous methods give, and why was this not enough? Were there just not enough identified CALHM orthologues to give strong signals in conservation analysis? Also, the amino acid conservation between CLHM-1 and CALHM1 is extremely low. Thus, there are other CALHM orthologs that give strong signals in conservation analysis. There are ~6 papers that perform in-depth analysis of the role of conserved residues in the gating of CALHM channels (human and C. elegans) that were not cited (Ma et al, Am J Physiol Cell Physiol, 2025; Syrjanen et al, Nat Commun, 2023; Danielli et al, EMBO J, 2023; Kwon et al, Mol Cells, 2021; Tanis et al, Am J Physiol Cell Physiol, 2017; Tanis et al, J Neurosci, 2013; Ma et al, PNAS, 2013) - these data needs to be discussed in the context of the present work.

We thank the reviewer for the comment and agree that these are excellent studies that have advanced understanding of conserved residues in CALHM gating. While their analyses compared a limited set of sequences, focusing on residues conserved in specific CALHM homologs or species like C. elegans, our analysis encompasses thousands of sequences across the entire CALHM family, allowing us to identify residues conserved across all family members over evolution. We also coupled this sequence analysis with hypotheses derived from our published structural studies (Choi et al., Nature, 2019), which highlighted the NTH/S1 region as a critical element in channel gating. Based on this, we focused on evolutionarily conserved residues in the S1–S2 linker and at the interface of S1 with the rest of the TMD, reasoning that if S1 movement is essential for gating, these two structural elements (acting as a hinge and stabilizing interface, respectively) would be key determinants of the conformational dynamics of S1. These regions have been largely overlooked in previous studies. As a result, the residues highlighted in our study do not overlap with those previously reported but instead provide complementary insights into gating mechanisms in this unique channel family. Together, our study and the published literature suggest that many regions and residues in CALHM proteins are critical for gating: while some are conserved across the entire family evolutionarily, others appear conserved only within certain species or subfamilies.

To address the reviewer’s comment, and to highlight the points mentioned above, we have added a brief discussion of these studies and the relevant citations in the revised manuscript (lines 378– 385, 563–576).

(2) Whereas the current-voltage relations for WT channels are clearly displayed, the data that is shown for the mutants does not allow for determining if their gating properties are indeed different than WT.

First, the current amplitudes for the mutants were quantified at just one voltage, which makes it impossible to determine if their voltage-dependence was different than WT, which would be a strong indicator for an effect in gating. Current-voltage relations as done for the WT channels should be included for at least some key mutations, which should include additional relevant controls like the use of Gd3+ as an inhibitor to rule out the contribution of some endogenous currents.

We thank the reviewer for this comment. To address this, we performed additional experiments using a multi-step pulse protocol to obtain current-voltage relations for WT CALHM1, CALHM1(I109W), WT CALHM6, and CALHM6(W113A). Our initial two-step protocol (−80 mV and +120 mV) covers both the physiological voltage range and the extended range commonly used in biophysical characterization of ion channels. Most mutants did not exhibit channel activation even within this broad range. We therefore focused on the three mutants that did show substantial activation to perform full I–V analysis as suggested. In all groups, currents activated at 37 °C were significantly inhibited by Gd³⁺, consistent with published reports (Ma et al., AJP 2025; Danielli et al., EMBO J 2023; Syrjänen et al., Nat Commun 2023). Notably, for CALHM6(Y51A), while this mutation did not significantly alter current amplitudes at positive membrane potentials, it markedly reduced currents at negative potentials, rendering the channel outwardly rectifying and altering its voltage dependence. These new data are incorporated into Figure 5 (panels A–O) and discussed in the manuscript. Figure 5 now also shows current amplitudes at both +120 mV and −80 mV in 0 mM Ca²⁺ at 37 °C to facilitate direct comparison between WT and mutants. The previous data at 5 mM Ca²⁺ and 0 mM Ca²⁺ at 22 °C have been moved to Supplementary Figure 5 as requested.

Second, it is unclear whether the three experimental conditions (5 mM Ca²⁺, and 0 Ca²⁺, at 22 and 37C) were measured in the same cell in each experiment, or if they represent different experiments. This should be clarified. If measurements at each condition were done in the same experiment, direct comparison between the three conditions within each individual experiment could further help identify mutations with altered gating.

We thank the reviewer for pointing this out and apologize for the confusion. All three conditions (5 mM Ca²⁺ at 22 °C, 0 mM Ca²⁺ at 22 °C, and 0 mM Ca²⁺ at 37 °C) were sequentially measured in the same cell within each experiment. The currents were then averaged across cells and plotted for each group.

Third, in line 334, the authors state that "expression levels of wild-type proteins and mutants are comparable." However, Western blots showing CALHM protein abundance (Supplementary Fig. 3) are not of acceptable quality; in the top blot, WT CALHM1 appears too dim, representative blots were not shown for all mutants, and individual data points should be included on the group data quantitation of the blots, together with a statistical test comparing mutants with the WT control.

We thank the reviewer for the comment and agree that representative blots were not shown for all mutants. Supplementary Figure 4 (previously Supplementary Figure 3) has been updated to include representative blots for all mutants, individual data points in the quantification, and statistical tests comparing each mutant to the WT control.

A more serious concern is that the total protein quantitation is not very informative about the functional impact of mutations in ion channels, because mutations can severely impact channel localization in the plasma membrane without reducing the total protein that is translated. In mammalian cells, CALHM6 is localized to intracellular compartments and only translocates to the plasma membrane in response to an activating stimulus (Danielli et al, EMBO J, 2023). Thus, if CALHM6 is only intracellular, the protein amount would not change, but the measured current would. Abundant intracellular CALHM1 has also been observed in mammalian cells transfected with this protein (Dreses-Werringloer et al., Cell, 2008). Quantitation of surface-biotinylated channels would provide information on whether there are differences between the constructs in relation to surface expression rather than gating. An alternative approach to biotinylation would be to express GFP-tagged constructs in Xenopus oocytes and look for surface expression. This is what has been done in previous CALHM channel studies.

Without evidence for the absence of defects in localization or clear alterations in gating properties, it is not possible to conclude whether mutant channels have altered activity. Does the analysis of sequences provide any testable hypotheses about substitutions with different side chains at the same position in the sequence?

We thank the reviewer for this very important comment. We agree that total protein levels alone do not distinguish between intracellular retention and proper trafficking to the plasma membrane. To address this, we performed surface biotinylation assays for all WT and mutant CALHM1 and CALHM6 constructs to assess their plasma membrane localization. The results show that mutants have either comparable or substantially higher surface expression levels than WT, consistent with the Western blot data. Together, these findings support our original interpretation that the observed differences in electrophysiological currents are not due to trafficking defects but reflect functional effects. These new data are presented in Supplementary Figure 5.

(3) Line 303 - 13 aligned amino acids were conserved across all CALHM homologs - are these also aligned in related connexin and pannexin families? It is likely that cysteines and proline in TM2 are since CALHM channels overall share a lot of similarities with connexins and pannexins (Siebert et al, JBC, 2013). As in line 207, it would be expected that pannexins, connexins, and CALHM channel families would group together. Related to this, see Line 406 - in connexins, there is also a proline kink in TM2 that may play a role in mediating conformational changes between channel states (Ri et al, Biophysical Journal, 1999). This should be discussed.

We thank the reviewer for the suggestion. We attempted a structure based sequence alignment of representative structures from all 3 families (CALHM, connexins and pannexins), but the resulting alignments are very poor and have a lot of gapped regions, making it very difficult to comment on the similarities mentioned in this comment. This is actually expected, as although CALHM, connexins, and pannexins are all considered “large-pore” channels, the TMD arrangement and conformation of CALHM are distinct from those of connexins and pannexins. Below, we have included a snapshot of the alignment at the conserved cysteine regions of the CALHM homologs, along with the resulting tree, which has very low support values and has difficulty placing the connexins properly, making it difficult to interpret.

Author response image 2.

Structure based sequence alignment and phylogenetic analysis of available crystal structures of members from the CALHM, Pannexin and Connexin families. Top: The resulting sequence alignment is very sparse and does not show conservation of residues in the TM regions. The CPC motif with conserved cysteines in CALHM family is shown. Bottom: Phylogenetic tree based on the alignment has low support values making it difficult to interpret.

(4) Line 36 - This work does not have experimental evidence to show that the selected evolutionarily conserved residues alter gating functions.

Our electrophysiology data demonstrate that the selected evolutionarily conserved residues have a major impact on CALHM1 and CALHM6 gating. As shown in Figure 5, mutations at these residues produce two distinct phenotypes: (1) nonconductive channels, and (2) altered voltage dependence, resulting in outward rectification. Importantly, these functional changes occur despite normal total expression and surface trafficking, as confirmed by Western blotting and surface biotinylation (Supplementary Figure 4). These findings indicate that the affected residues are critical for the conformational dynamics underlying channel gating rather than for protein expression or localization.

(5) Line 296-297 - This could also be put in the context of what we already know about CALHM gating. While all cryo EM structures of CALHM channels are in the open state, we still do understand some things about gating mechanism (Tanis et al Am J Physiol Cell Physiol, Cell Physiol 2017; Ma et al Am J Physiol Cell Physiol, Cell Physiol 2025) with the NT modulating voltage dependence and stabilizing closed channel states and the voltage dependent gate being formed by proximal regions of TM1.

Thank you for providing this suggestion. As suggested, we have revised the text to place our findings in the context of current knowledge about CALHM gating and have added the relevant citations (lines 370-373).

(6) Lines 314-315 - Just because residues are conserved does not mean that they play a role in channel gating. These residues could also be important for structure, ion selectivity, etc.

We agree that evolutionary conservation alone does not imply a role in gating. However, our hypothesis derives from the positioning of these conserved residues, and previous studies that have indicated the importance of the NTH/S1 region for channel gating function. More importantly, our electrophysiology data indicate that these conserved residues specifically impact channel gating in CALHM1 and CALHM6. We have revised the text in lines 404-406 to clarify this further.

(7) Line 333 - while CALHM6 is less studied than CALHM1, there is knowledge of its function and gating properties. Should CALHM6 be considered a "dark" channel? The IDG development level in Pharos is Tbio. There have been multiple papers published on this channel (ex: Ebihara et al, J Exp Med, 2010; Kasamatsu et al, J Immunol 2014; Danielli et al, EMBO J, 2023).

We thank the reviewer for noting this important discrepancy. We have updated the text and labels related to CALHM6 to reflect its status as Tbio in the manuscript.

(8) Please cite Jeon et al., (Biochem Biophys Res Commun, 2021), who have already shown temperature-dependence of CALHM1.

Thank you for the comment. We have added the citation.  

(9) It would be helpful to have a schematic showing amino acid residues, TM domains, highlighted residues mutated, etc.

Thank you for the suggestion. We have revised the figure and added labels for the TM domains, and highlighted the mutated residues.

Reviewer #1 (Recommendations for the authors):

(1) Why in the title is 'ion-channels' hyphenated but in the text it is not?

This has been changed.

(2) Line 78: 'Cryo-EM' is not defined before the acronym is used.

This has been fixed.

(3) Typo in line 519: KinOrthto.

This has been fixed.

(4) Capitalizing 'Tree of Life' is a bit strange in section 2 of the results and the Discussion.

We have removed the capitalization as suggested.

(5) In Figure 3 and Supplementary Figure 4A, the gene names in the tree are CAHM and not CALHM - I assume this is an error.

This has been made consistent to CALHM.

(6) Font sizes throughout all figures, with the exception of Figure 1, need to be more legible. The X-axis labels in Figure 2A are hard to read, for example (though I can see that there is also the CAHM/CALHM typo here...). A good rule of thumb is that they should be the same size as the manuscript text. Furthermore, the grey backgrounds of Figure 4 and Figure 5 are off-putting; just having a white background here should be sufficient.

This has been addressed. We have increased the font size in all figures with these revisions. The styling for Figure 4 and 5 has also been made consistent with other figures.

Reviewer #2 (Recommendations for the authors):

(1) Line 36 - This work does not have experimental evidence to show that the selected evolutionarily conserved residues alter gating functions.

Addressed in comment #4 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(2) Line 168 - should also be Supplemental Table 1.

This has been addressed.

(3) Line 170 - 419 human ion channel sequences were identified and this was an increase of 75 sequences over previous number. Which 75 proteins are these?

This is now shown in Supplementary Figure 2 and Supplementary Table 2. Supplementary Figure 2 shows an Upset plot with the number of sequences that overlap across databases and the novel sequences that we have added as part of this study. The 75 specifically refers to the sequences that were not included in Pharos, which was chosen to refer to this number since it has the highest number of ICs listed out of all the other resources. Further, Supplementary Table 2 now provides a list of individual ICs and whether they were present in each of the 3 databases compared.

(4) Line 289 - Ca2+ (not Ca); other similar mistakes throughout the manuscript

These have been fixed.

(5) Line 291-292 - Please include more about functions for CALHM channels; ex. CALHM1 regulates cortical neuron excitability (Ma et al, PNAS 2012), CLHM-1 regulates locomotion and induces neurodegeneration in C. elegans (Tanis et al. Journal of Neuroscience 2013); see above for references on CALHM6 function.

We have added the functions as suggested.

(6) Line 296-297 - This could also be put in the context of what we already know about CALHM gating. While all cryo EM structures of CALHM channels are in the open state, we still do understand some things about gating mechanism (Tanis et al Am J Physiol Cell Physiol, Cell Physiol 2017; Ma et al Am J Physiol Cell Physiol, Cell Physiol 2025) with the NT modulating voltage dependence and stabilizing closed channel states and the voltage dependent gate being formed by proximal regions of TM1.

Addressed in comment #5 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(7) Lines 314-315 - Just because residues are conserved does not mean that they play a role in channel gating. These residues could also be important for structure, ion selectivity, etc.

Addressed in comment #6 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(8) Line 333 - While CALHM6 is less studied than CALHM1, there is knowledge of its function and gating properties. Should CALHM6 be considered a "dark" channel? The IDG development level in Pharos is Tbio. There have been multiple papers published on this channel (ex: Ebihara et al, J Exp Med, 2010; Kasamatsu et al, J Immunol 2014; Danielli et al, EMBO J, 2023).

Addressed in comment #7 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(9) Line 627 - Do you mean that 5 mM CaCl2 was replaced with 5 mM EGTA in 0 Ca2+ solution?

This is correct.  

(10) Why are only evolutionary relationships between rat, mouse, and human shown in Figure 3A? These species are all close on the evolutionary timeline.

Addressed in comment #10 for Part A Revisions related to the first part, regarding data mining and curation above.

(11) Figure 5 - no need to show the currents at room temperature in the main text since there are robust currents at 37 degrees; this could go into the supplement. Also, please cite Jeon et al. (Biochem Biophys Res Commun, 2021), who have already shown temperature-dependence of CALHM1.

Addressed in comment #8 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(12) It would be helpful to have a schematic showing amino acid residues, TM domains, highlighted residues mutated etc.

Addressed in comment #9 for Part B Revisions related to the second part, regarding the analysis of CAHLM channel mutations above.

(13) Use of S1-S4 to refer to the transmembrane "segments" is not standard; rather, TM1-TM4 would generally be used to refer to transmembrane domains.

We have used the S1–S4 helix notation to maintain consistency with the nomenclature employed in our previous study (Choi et al., Nature, 2019).

Author Response:

Reviewer #1 (Public Review):

[...] The major limitation of the manuscript lies in the framing and interpretation of the results, and therefore the evaluation of novelty. Authors claim for an important and unique role of beliefs-of-other-pain in altruistic behavior and empathy for pain. The problem is that these experiments mainly show that behaviors sometimes associated with empathy-for-pain can be cognitively modulated by changing prior beliefs. To support the notion that effects are indeed relating to pain processing generally or empathy for pain specifically, a similar manipulation, done for instance on beliefs about the happiness of others, before recording behavioural estimation of other people's happiness, should have been performed. If such a belief-about-something-else-than-pain would have led to similar results, in terms of behavioural outcome and in terms of TPJ and MFG recapitulating the pattern of behavioral responses, we would know that the results reflect changes of beliefs more generally. Only if the results are specific to a pain-empathy task, would there be evidence to associate the results to pain specifically. But even then, it would remain unclear whether the effects truly relate to empathy for pain, or whether they may reflect other routes of processing pain.

We thank Reviewer #1's for these comments/suggestions regarding the specificity of belief effects on brain activity involved in empathy for pain. Our paper reported 6 behavioral/EEG/fMRI experiments that tested effects of beliefs of others’ pain on empathy and monetary donation (an empathy-related altruistic behavior). We showed not only behavioral but also neuroimaging results that consistently support the hypothesis of the functional role of beliefs of others' pain in modulations of empathy (based on both subjective and objective measures as clarified in the revision) and altruistic behavior. We agree with Reviewer 1# that it is important to address whether the belief effect is specific to neural underpinnings of empathy for pain or is general for neural responses to various facial expressions such as happy, as suggested by Reviewer #1. To address this issue, we conducted an additional EEG experiment (which can be done in a limited time in the current situation), as suggested by Reviewer #1. This new EEG experiment tested (1) whether beliefs of authenticity of others’ happiness influence brain responses to perceived happy expressions; (2) whether beliefs of happiness modulate neural responses to happy expressions in the P2 time window as that characterized effects of beliefs of pain on ERPs.

Our behavioral results in this experiment (as Supplementary Experiment 1 reported in the revision) showed that the participants reported less feelings of happiness when viewing actors who simulate others' smiling compared to when viewing awardees who smile due to winning awards (see the figure below). Our ERP results in Supplementary Experiment 1 further showed that lack of beliefs of authenticity of others’ happiness (e.g., actors simulate others' happy expressions vs. awardees smile and show happy expressions due to winning an award) reduced the amplitudes of a long-latency positive component (i.e., P570) over the frontal region in response to happy expressions. These findings suggest that (1) there are possibly general belief effects on subjective feelings and brain activities in response to facial expressions; (2) beliefs of others' pain or happiness affect neural responses to facial expressions in different time windows after face onset; (3) modulations of the P2 amplitude by beliefs of pain may not be generalized to belief effects on neural responses to any emotional states of others. We reported the results of this new ERP experiment in the revision as Supplementary Experiment 1 and also discussed the issue of specificity of modulations of empathic neural responses by beliefs of others' pain in the revised Discussion (page 49-50).

Supplementary Experiment Figure 1. EEG results of Supplementary Experiment 1. (a) Mean rating scores of happy intensity related to happy and neutral expressions of faces with awardee or actor/actress identities. (b) ERPs to faces with awardee or actor/actress identities at the frontal electrodes. The voltage topography shows the scalp distribution of the P570 amplitude with the maximum over the central/parietal region. (c) Mean differential P570 amplitudes to happy versus neutral expressions of faces with awardee or actor/actress identities. The voltage topographies illustrate the scalp distribution of the P570 difference waves to happy (vs. neutral) expressions of faces with awardee or actor/actress identities, respectively. Shown are group means (large dots), standard deviation (bars), measures of each individual participant (small dots), and distribution (violin shape) in (a) and (c).

In the revised Introduction we cited additional literatures to explain the concept of empathy, behavioral and neuroimaging measures of empathy, and how, similar to previous research, we studied empathy for others' pain using subjective (self reports) and objective (brain responses) estimation of empathy (page 6-7). In particular, we mentioned that subjective estimation of empathy for pain depends on collection of self-reports of others' pain and ones' own painful feelings when viewing others' suffering. Objective estimation of empathy for pain relies on recording of brain activities (using fMRI, EEG, etc.) that differentially respond to painful or non-painful stimuli applied to others. fMRI studies revealed greater activations in the ACC, AI, and sensorimotor cortices in response to painful or non-painful stimuli applied to others. EEG studies showed that event-related potentials (ERPs) in response to perceived painful stimulations applied to others' body parts elicited neural responses that differentiated between painful and neutral stimuli over the frontal region as early as 140 ms after stimulus onset (Fan and Han, 2008; see Coll, 2018 for review). Moreover, the mean ERP amplitudes at 140–180 ms predicted subjective reports of others' pain and ones' own unpleasantness. Particularly related to the current study, previous research showed that pain compared to neutral expressions increased the amplitude of the frontal P2 component at 128–188 ms after stimulus onset (Sheng and Han, 2012; Sheng et al., 2013; 2016; Han et al., 2016; Li and Han, 2019) and the P2 amplitudes in response to others' pain expressions positively predicted subjective feelings of own unpleasantness induced by others' pain and self-report of one's own empathy traits (e.g., Sheng and Han, 2012). These brain imaging findings indicate that brain responses to others' pain can (1) differentiate others' painful or non-painful emotional states to support understanding of others' pain and (2) predict subjective feelings of others' pain and one's own unpleasantness induced by others' pain to support sharing of others' painful feelings. These findings provide effective subjective and objective measures of empathy that were used in the current study to investigate neural mechanisms underlying modulation of empathy and altruism by beliefs of others’ pain.

In addition, we took Reviewer #1’s suggestion for VPS analyses which examined specifically how neural activities in the empathy-related regions identified in the previous research (Krishnan et al., 2016, eLife) were modulated by beliefs of others’ pain. The results (page 40) provide further evidence for our hypothesis. We also reported new results of RSA analyses(page 39) that activities in the brain regions supporting affective sharing (e.g., insula), sensorimotor resonance (e.g., post-central gyrus), and emotion regulation (e.g., lateral frontal cortex) provide intermediate mechanisms underlying modulations of subjective feelings of others' pain intensity due to lack of BOP. We believe that, putting all these results together, our paper provides consistent evidence that empathy and altruistic behavior are modulated by BOP.

Reviewer #2 (Public Review):

[...] 1. In laying out their hypotheses, the authors write, "The current work tested the hypothesis that BOP provides a fundamental cognitive basis of empathy and altruistic behavior by modulating brain activity in response to others' pain. Specifically, we tested predictions that weakening BOP inhibits altruistic behavior by decreasing empathy and its underlying brain activity whereas enhancing BOP may produce opposite effects on empathy and altruistic behavior." While I'm a little dubious regarding the enhancement effects (see below), a supporting assumption here seems to be that at baseline, we expect that painful expressions reflect real pain experience. To that end, it might be helpful to ground some of the introduction in what we know about the perception of painful expressions (e.g., how rapidly/automatically is pain detected, do we preferentially attend to pain vs. other emotions, etc.).

Thanks for this suggestion! We included additional details about previous findings related to processes of painful expressions in the revised Introduction (page 7-8). Specifically, we introduced fMRI and ERP studies of pain expressions that revealed structures and temporal procedure of neural responses to others' pain (vs. neutral) expressions. Moreover, neural responses to others' pain (vs. neutral) expressions were associated with self-report of others' feelings, indicating functional roles of pain-expression induced brain activities in empathy for pain.

1. For me, the key takeaway from this manuscript was that our assessment of and response to painful expressions is contextually-sensitive - specifically, to information reflecting whether or not targets are actually in pain. As the authors state it, "Our behavioral and neuroimaging results revealed critical functional roles of BOP in modulations of the perception-emotion-behavior reactivity by showing how BOP predicted and affected empathy/empathic brain activity and monetary donations. Our findings provide evidence that BOP constitutes a fundamental cognitive basis for empathy and altruistic behavior in humans." In other words, pain might be an incredibly socially salient signal, but it's still easily overridden from the top down provided relevant contextual information - you won't empathize with something that isn't there. While I think this hypothesis is well-supported by the data, it's also backed by a pretty healthy literature on contextual influences on pain judgments (including in clinical contexts) that I think the authors might want to consider referencing (here are just a few that come to mind: Craig et al., 2010; Twigg et al., 2015; Nicolardi et al., 2020; Martel et al., 2008; Riva et al., 2015; Hampton et al., 2018; Prkachin & Rocha, 2010; Cui et al., 2016).

Thanks for this great suggestion! Accordingly, we included an additional paragraph in the revised Discussion regarding how social contexts influence empathy and cited the studies mentioned here (page 46-47).

1. I had a few questions regarding the stimuli the authors used across these experiments. First, just to confirm, these targets were posing (e.g., not experiencing) pain, correct? Second, the authors refer to counterbalancing assignment of these stimuli to condition within the various experiments. Was target gender balanced across groups in this counterbalancing scheme? (e.g., in Experiment 1, if 8 targets were revealed to be actors/actresses in Round 2, were 4 female and 4 male?) Third, were these stimuli selected at random from a larger set, or based on specific criteria (e.g., normed ratings of intensity, believability, specificity of expression, etc.?) If so, it would be helpful to provide these details for each experiment.

We'd be happy to clarify these questions. First, photos of faces with pain or neutral expressions were adopted from the previous work (Sheng and Han, 2012). Photos were taken from models who were posing but not experience pain. These photos were taken and selected based on explicit criteria of painful expressions (i.e., brow lowering, orbit tightening, and raising of the upper lip; Prkachin, 1992). In addition, the models' facial expressions were validated in independent samples of participants (see Sheng and Han, 2012). Second, target gender was also balanced across groups in this counterbalancing scheme. We also analyzed empathy rating score and monetary donations related to male and female target faces and did not find any significant gender effect (see our response to Point 5 below). Third, because the face stimuli were adopted from the previous work and the models' facial expressions were validated in independent samples of participants regarding specificity of expression, pain intensity, etc (Sheng and Han, 2012), we did not repeat these validation in our participants. Most importantly, we counterbalanced the stimuli in different conditions so that the stimuli in different conditions (e.g., patient vs. actor/actress conditions) were the same across the participants in each experiment. The design like this excluded any potential confound arising from the stimuli themselves.

1. The nature of the charitable donation (particularly in Experiment 1) could be clarified. I couldn't tell if the same charity was being referenced in Rounds 1 and 2, and if there were multiple charities in Round 2 (one for the patients and one for the actors).

Thanks for this comment! Yes, indeed, in both Rounds 1 and 2, the participants were informed that the amount of one of their decisions would be selected randomly and donated to one of the patients through the same charity organization (we clarified these in the revised Method section, page 55-56). We made clear in the revision that after we finished all the experiments of this study, the total amount of the participants' donations were subject to a charity organization to help patients who suffer from the same disease after the study.

1. I'm also having a hard time understanding the authors' prediction that targets revealed to truly be patients in the 2nd round will be associated with enhanced BOP/altruism/etc. (as they state it: "By contrast, reconfirming patient identities enhanced the coupling between perceived pain expressions of faces and the painful emotional states of face owners and thus increased BOP.") They aren't in any additional pain than they were before, and at the outset of the task, there was no reason to believe that they weren't suffering from this painful condition - therefore I don't see why a second mention of their pain status should increase empathy/giving/etc. It seems likely that this is a contrast effect driven by the actor/actress targets. See the Recommendations for the Authors for specific suggestions regarding potential control experiments. (I'll note that the enhancement effect in Experiment 2 seems more sensible - here, the participant learns that treatment was ineffective, which may be painful in and of itself.)

Thanks for comments on this important point! Indeed, our results showed that reassuring patient identities in Experiment 1 or by noting the failure of medical treatment related to target faces in Experiment 2 increased rating scores of others' pain and own unpleasantness and prompted more monetary donations to target faces. The increased empathy rating scores and monetary donations might be due to that repeatedly confirming patient identity or knowing the failure of medical treatment increased the belief of authenticity of targets' pain and thus enhanced empathy. However, repeatedly confirming patient identity or knowing the failure of medical treatment might activate other emotional responses to target faces such as pity or helplessness, which might also influence altruistic decisions. We agree with Reviewer #2 that, although our subjective estimation of empathy in Exp. 1 and 2 suggested enhanced empathy in the 2nd_round test, there are alternative interpretations of the results and these should be clarified in future work. We clarified these points in the revised Discussion (page 41-42).

1. I noted that in the Methods for Experiment 3, the authors stated "We recruited only male participants to exclude potential effects of gender difference in empathic neural responses." This approach continues through the rest of the studies. This raises a few questions. Are there gender differences in the first two studies (which recruited both male and female participants)? Moreover, are the authors not concerned about target gender effects? (Since, as far as I can tell, all studies use both male and female targets, which would mean that in Experiments 3 and on, half the targets are same-gender as the participants and the other half are other-gender.) Other work suggests that there are indeed effects of target gender on the recognition of painful expressions (Riva et al., 2011).

Thanks for raising this interesting question! Therefore, we reanalyzed data in Exp. 1 by including participants' gender or face gender as an independent variable. The three-way ANOVAs of pain intensity scores and amounts of monetary donations with Face Gender (female vs. male targets) × Test Phase (1st vs. 2nd_round) × Belief Change (patient-identity change vs. patient-identity repetition) did not show any significant three-way interaction (F(1,59) = 0.432 and 0.436, p = 0.514 and 0.512, ηp2 = 0.007 and 0.007, 90% CI = (0, 0.079) and (0, 0.079), indicating that face gender do not influence the results (see the figure below). Similarly, the three-way ANOVAs with Participant Gender (female vs. male participants) × Test Phase × Belief Change did not show any significant three-way interaction (F(1,58) = 0.121 and 1.586, p = 0.729 and 0.213, ηp2 = 0.002 and 0.027, 90% CI = (0, 0.055) and (0, 0.124), indicating no reliable difference in empathy and donation between men and women. It seems that the measures of empathy and altruistic behavior in our study were not sensitive to gender of empathy targets and participants' sexes.

Figure legend: (a) Scores of pain intensity and amount of monetary donations are reported separately for male and female target faces. (b) Scores of pain intensity and amount of monetary donations are reported separately for male and female participants.

1. I was a little unclear on the motivation for Experiment 4. The authors state "If BOP rather than other processes was necessary for the modulation of empathic neural responses in Experiment 3, the same manipulation procedure to assign different face identities that do not change BOP should change the P2 amplitudes in response to pain expressions." What "other processes" are they referring to? As far as I could tell, the upshot of this study was just to demonstrate that differences in empathy for pain were not a mere consequence of assignment to social groups (e.g., the groups must have some relevance for pain experience). While the data are clear and as predicted, I'm not sure this was an alternate hypothesis that I would have suggested or that needs disconfirming.

Thanks for this comment! We feel sorry for not being able to make clear the research question in Exp. 4. In the revised Results section (page 27-28) we clarified that the learning and EEG recording procedures in Experiment 3 consisted of multiple processes, including learning, memory, identity recognition, assignment to social groups, etc. The results of Experiment 3 left an open question of whether these processes, even without BOP changes induced through these processes, would be sufficient to result in modulation of the P2 amplitude in response to pain (vs. neutral) expressions of faces with different identities. In Experiment 4 we addressed this issue using the same learning and identity recognition procedures as those in Experiment 3 except that the participants in Experiment 4 had to learn and recognize identities of faces of two baseball teams and that there is no prior difference in BOP associated with faces of beliefs of the two baseball teams. If the processes involved in the learn and reorganization procedures rather than the difference in BOP were sufficient for modulation of the P2 amplitude in response to pain (vs. neutral) expressions of faces, we would expect similar P2 modulations in Experiments 4 and 3. Otherwise, the difference in BOP produced during the learning procedure was necessary for the modulation of empathic neural responses, we would not expect modulations of the P2 amplitude in response to pain (vs. neutral) expressions in Experiment 4. We believe that the goal and rationale of Exp. 4 are clear now.

Author Response:

We thank the editors and the reviewers for their careful reading and rigorous evaluation of our manuscript. We thank them for their positive comments and constructive feedback, which led us to add further lines of evidence in support of our central hypothesis that intrinsic neuronal resonance could stabilize heterogeneous grid-cell networks through targeted suppression of low-frequency perturbations. In the revised manuscript, we have added a physiologically rooted mechanistic model for intrinsic neuronal resonance, introduced through a slow negative feedback loop. We show that stabilization of patterned neural activity in a heterogeneous continuous attractor network (CAN) model could be achieved with this resonating neuronal model. These new results establish the generality of the stabilizing role of neuronal resonance in a manner independent of how resonance was introduced. More importantly, by specifically manipulating the feedback time constant in the neural dynamics, we establish the critical role of the slow kinetics of the negative feedback loop in stabilizing network function. These results provide additional direct lines of evidence for our hypothesis on the stabilizing role of resonance in the CAN model employed here. Intuitively, we envisage intrinsic neuronal resonance as a specific cellular-scale instance of a negative feedback loop. The negative feedback loop is a well-established network motif that acts as a stabilizing agent and suppresses the impact of internal and external perturbations in engineering applications and biological networks.

Reviewer #1 (Public Review):

The authors succeed in conveying a clear and concise description of how intrinsic heterogeneity affects continuous attractor models. The main claim, namely that resonant neurons could stabilize grid-cell patterns in medial entorhinal cortex, is striking.

We thank the reviewer for their time and effort in evaluating our manuscript, and for their rigorous evaluation and positive comments on our study.

I am intrigued by the use of a nonlinear filter composed of the product of s with its temporal derivative raised to an exponent. Why this particular choice? Or, to be more specific, would a linear bandpass filter not have served the same purpose?

Please note that the exponent was merely a mechanism to effectively tune the resonance frequency of the resonating neuron. In the revised manuscript, we have introduced a new physiologically rooted means to introduce intrinsic neuronal resonance, thereby confirming that network stabilization achieved was independent of the formulation employed to achieve resonance.

The magnitude spectra are subtracted and then normalized by a sum. I have slight misgivings about the normalization, but I am more worried that, as no specific formula is given, some MATLAB function has been used. What bothers me a bit is that, depending on how the spectrogram/periodogram is computed (in particular, averaged over windows), one would naturally expect lower frequency components to be more variable. But this excess variability at low frequencies is a major point in the paper.

We have now provided the specific formula employed for normalization as equation (16) of the revised manuscript. We have also noted that this was performed to account for potential differences in the maximum value of the homogeneous vs. heterogeneous spectra. The details are provided in the Methods subsection “Quantitative analysis of grid cell temporal activity in the spectral domain” of the revised manuscript. Please note that what is computed is the spectra of the entire activity pattern, and not a periodogram or a scalogram. There was no tiling of the time-frequency plane involved, thus eliminating potential roles of variables there on the computation here.

In addition to using variances of normalized differences to quantify spectral distributions, we have also independently employed octave-based analyses (which doesn’t involve normalized differences) to strengthen our claims about the impact of heterogeneities and resonance on different bands of frequency. These octave-based analyses also confirm our conclusions on the impact of heterogeneities and neuronal resonance on low-frequency components.

Finally, we would like to emphasize that spectral computations are the same for different networks, with networks designed in such a way that there was only one component that was different. For instance, in introducing heterogeneities, all other parameters of the network (the specific trajectory, the seed values, the neural and network parameters, the connectivity, etc.) remained exactly the same with the only difference introduced being confined to the heterogeneities. Computation of the spectral properties followed identical procedures with activity from individual neurons in the two networks, and comparison was with reference to identically placed neurons in the two networks. Together, based on the several routes to quantifying spectral signatures, based on the experimental design involved, and based on the absence of any signal-specific tiling of the time-frequency plane, we argue that the impact of heterogeneities or the resonators on low-frequency components is not an artifact of the analysis procedures.

We thank the reviewer for raising this issue, as it helped us to elaborate on the analysis procedures employed in our study.

Which brings me to the main thesis of the manuscript: given the observation of how heterogeneities increase the variability in the low temporal frequency components, the way resonant neurons stabilize grid patterns is by suppressing these same low frequency components.

I am not entirely convinced that the observed correlation implies causality. The low temporal frequeny spectra are an indirect reflection of the regularity or irregularity of the pattern formation on the network, induced by the fact that there is velocity coupling to the input and hence dynamics on the network. Heterogeneities will distort the pattern on the network, that is true, but it isn't clear how introducing a bandpass property in temporal frequency space affects spatial stability causally.

Put it this way: imagine all neurons were true oscillators, only capable of oscillating at 8 Hz. If they were to synchronize within a bump, one will have the field blinking on and off. Nothing wrong with that, and it might be that such oscillatory pattern formation on the network might be more stable than non-oscillatory pattern formation (perhaps one could even demonstrate this mathematically, for equivalent parameter settings), but this kind of causality is not what is shown in the manuscript.

The central hypothesis of our study was that intrinsic neuronal resonance could stabilize heterogeneous grid-cell networksthrough targeted suppression of low-frequency perturbations.

In the revised manuscript, we present the following lines of evidence in support of this hypothesis (mentioned now in the first paragraph of the discussion section of the revised manuscript):

1. Neural-circuit heterogeneities destabilized grid-patterned activity generation in a 2D CAN model (Figures 2–3).

2. Neural-circuit heterogeneities predominantly introduced perturbations in the lowfrequency components of neural activity (Figure 4).

3. Targeted suppression of low-frequency components through phenomenological (Figure 5C) or through mechanistic (new Figure 9D) resonators resulted in stabilization of the heterogeneous CAN models (Figure 8 and new Figure 11). We note that the stabilization was achieved irrespective of the means employed to suppress low-frequency components: an activity-independent suppression of low-frequencies (Figure 5) or an activity-dependent slow negative feedback loop (new Figure 9).

4. Changing the feedback time constant τm in mechanistic resonators, without changes to neural gain or the feedback strength allowed us to control the specific range of frequencies that would be suppressed. Our analyses showed that a slow negative feedback loop, which results in targeted suppression of low-frequency components, was essential in stabilizing grid-patterned activity (new Figure 12). As the slow negative feedback loop and the resultant suppression of low frequencies mediates intrinsic resonance, these analyses provide important lines of evidence for the role of targeted suppression of low frequencies in stabilizing grid patterned activity.

5. We demonstrate that the incorporation of phenomenological (Figure 13A–C) or mechanistic (new Figure panels 13D–F) resonators specifically suppressed lower frequencies of activity in the 2D CAN model.

6. Finally, the incorporation of resonance through a negative feedback loop allowed us to link our analyses to the well-established role of network motifs involving negative feedback loops in inducing stability and suppressing external/internal noise in engineering and biological systems. We envisage intrinsic neuronal resonance as a cellular-scale activitydependent negative feedback mechanism, a specific instance of a well-established network motif that effectuates stability and suppresses perturbations across different networks (Savageau, 1974; Becskei and Serrano, 2000; Thattai and van Oudenaarden, 2001; Austin et al., 2006; Dublanche et al., 2006; Raj and van Oudenaarden, 2008; Lestas et al., 2010; Cheong et al., 2011; Voliotis et al., 2014). A detailed discussion on this important link to the stabilizing role of this network motif, with appropriate references to the literature is included in the new discussion subsection “Slow negative feedback: Stability, noise suppression, and robustness”.

We thank the reviewer for their detailed comments. These comments helped us to introducing a more physiologically rooted mechanistic form of resonance, where we were able to assess the impact of slow kinetics of negative feedback on network stability, thereby providing more direct lines of evidence for our hypothesis. This also allowed us to link resonance to the wellestablished stability motif: the negative feedback loop. We also note that our analyses don’t employ resonance as a route to introducing oscillations in the network, but as a means for targeted suppression of low-frequency perturbations through a negative feedback loop. Given the strong quantitative links of negative feedback loops to introducing stability and suppressing the impact of perturbations in engineering applications and biological networks, we envisage intrinsic neuronal resonance as a stability-inducing cellular-scale activity-dependent negative feedback mechanism.

Reviewer #2 (Public Review):

[...] The pars construens demonstrates that similar networks, but comprised of units with different dynamical behavior, essentially amputated of their slowest components, do not suffer from the heterogeneities - they still produce grids. This part proceeds through 3 main steps: a) defining "resonator" units as model neurons with amputated low frequencies (Fig. 5); b) showing that inserted into the same homogeneous CAN network, "resonator" units produce the same grids as "integrator" units (Figs. 6,7); c) demonstrating that however the network with "resonator" units is resistant to heterogeneities (Fig. 8). Figs. 9 and 10 help understand what has produced the desired grid stabilization effect. This second part is on the whole also well structured, and its step c) is particularly convincing.

We thank the reviewer for their time and effort in evaluating our manuscript, and for their rigorous evaluation and positive comments on our study.

Step b) intends to show that nothing important changes, in grid pattern terms, if one replaces the standard firing rate units with the ad hoc defined units without low frequency behavior. The exact outcome of the manipulation is somewhat complex, as shown in Figs. 6 and 7, but it could be conceivably summed up by stating that grids remain stable, when low frequencies are removed. What is missing, however, is an exploration of whether the newly defined units, the "resonators", could produce grid patterns on their own, without the CAN arising from the interactions between units, just as a single-unit effect. I bet they could, because that is what happens in the adaptation model for the emergence of the grid pattern, which we have studied extensively over the years. Maybe with some changes here and there, but I believe the CAN can be disposed of entirely, except to produce a common alignment between units, as we have shown.

Step a), finally, is the part of the study that I find certainly not wrong, but somewhat misleading. Not wrong, because what units to use in a model, and what to call them, is a legitimate arbitrary choice of the modelers. Somewhat misleading, because the term "resonator" evokes a more specific dynamical behavior that than obtained by inserting Eqs. (8)-(9) into Eq. (6), which amounts to a brute force amputation of the low frequencies, without any real resonance to speak of. Unsurprisingly, Fig. 5, which is very clear and useful, does not show any resonance, but just a smooth, broad band-pass behavior, which is, I stress legitimately, put there by hand. A very similar broad band-pass would result from incorporating into individual units a model of firing rate adaptation, which is why I believe the "resonator" units in this study would generate grid patterns, in principle, without any CAN.

We thank the reviewer for these constructive comments and questions, as they were extremely helpful in (i) formulating a new model for rate-based resonating neurons that is more physiologically rooted; (ii) demonstrating the stabilizing role of resonance irrespective of model choices that implemented resonance; and (iii) mechanistically exploring the impact of targeted suppression of low frequency components in neural activity. We answer these comments of the reviewer in two parts, the first addressing other models for grid-patterned activity generation and the second addressing the reviewer’s comment on “brute force amputation of the low frequencies” in the resonator neuron presented in the previous version of our manuscript.

I. Other models for grid-patterned activity generation.

In the adaptation model (Kropff and Treves, 2008; Urdapilleta et al., 2017; Stella et al., 2020), adaptation in conjunction with place-cell inputs, Hebbian synaptic plasticity, and intrinsic plasticity (in gain and threshold) to implement competition are together sufficient for the emergence of the grid-patterned neural activity. However, the CAN model that we chose as the substrate for assessing the impact of neural circuit heterogeneities on functional stability is not equipped with the additional components (place-cell inputs, synaptic/intrinsic plasticity). Therefore, we note that decoupling the single unit (resonator or integrator) from the network does not yield grid-patterned activity.

However, we do agree that a resonator neuron endowed with additional components from the adaptation model would be sufficient to elicit grid-patterned neural activity. This is especially clear with the newly introduced mechanistic model for resonance through a slow feedback loop (Figure 9). Specifically, resonating conductances such as HCN and M-type potassium channels can effectuate spike-frequency adaptation. One of the prominent channels that is implicated in introducing adaptation, the calcium-activated potassium channels implement a slow activitydependent negative feedback loop through the slow calcium kinetics. Neural activity drives calcium influx, and the slow kinetics of the calcium along with the channel-activation kinetics drive a potassium current that completes a negative feedback loop that inhibits neural activity. Consistently, one of the earliest-reported forms of electrical resonance in cochlear hair cells was shown to be mediated by calcium-activated potassium channels (Crawford and Fettiplace, 1978, 1981; Fettiplace and Fuchs, 1999). Thus, adaptation realized as a slow negative-feedback loop, in conjunction with place-cell inputs and intrinsic/synaptic plasticity would elicit gridpatterned neural activity as demonstrated earlier (Kropff and Treves, 2008; Urdapilleta et al., 2017; Stella et al., 2020).

There are several models for the emergence of grid-patterned activity, and resonance plays distinct roles (compared to the role proposed through our analyses) in some of these models (Giocomo et al., 2007; Kropff and Treves, 2008; Burak and Fiete, 2009; Burgess and O'Keefe, 2011; Giocomo et al., 2011b; Giocomo et al., 2011a; Navratilova et al., 2012; Pastoll et al., 2012; Couey et al., 2013; Domnisoru et al., 2013; Schmidt-Hieber and Hausser, 2013; Yoon et al., 2013; Schmidt-Hieber et al., 2017; Urdapilleta et al., 2017; Stella et al., 2020; Tukker et al., 2021). However, a common caveat that spans many of these models is that they assume homogeneous networks that do not account for the ubiquitous heterogeneities that span neural circuits. Our goal in this study was to take a step towards rectifying this caveat, towards understanding the impact of neural circuit heterogeneities on network stability. We chose the 2D CAN model for grid-patterned activity generation as the substrate for addressing this important yet under-explored question on the role of biological heterogeneities on network function. As we have mentioned in the discussion section, this choice implies that our conclusions are limited to the 2D CAN model for grid patterned generation; these conclusions cannot be extrapolated to other networks or other models for grid-patterned activity generation without detailed analyses of the impact of neural circuit heterogeneities in those models. As our focus here was on the stabilizing role of resonance in heterogeneous neural networks, with 2D CAN model as the substrate, we have not implemented the other models for grid-patterned generation. The impact of biological heterogeneities and resonance on each of these models should be independently addressed with systematic analyses similar to our analyses for the 2D CAN model. As different models for grid-patterned activity generation are endowed with disparate dynamics, and have different roles for resonance, it is conceivable that the impact of biological heterogeneities and intrinsic neuronal resonance have differential impact on these different models. We have mentioned this as a clear limitation of our analyses in the discussion section, also presenting future directions for associated analyses(subsection: “Future directions and considerations in model interpretation”).

II. Brute force amputation of the low frequencies in the resonator model.

We completely agree with the reviewer on the observation that the resonator model employed in the previous version of our manuscript was rather artificial, with the realization involving brute force amputation of the lower frequencies. To address this concern, in the revised manuscript, we constructed a new mechanistic model for single-neuron resonance that matches the dynamical behavior of physiological resonators. Specifically, we noted that physiological resonance is elicited by a slow activity-dependent negative feedback (Hutcheon and Yarom, 2000). To incorporate resonance into our rate-based model neurons, we mimicked this by introducing a slow negative feedback loop into our single-neuron dynamics (the motivations are elaborated in the new results subsection “Mechanistic model of neuronal intrinsic resonance: Incorporating a slow activity-dependent negative feedback loop”). The singleneuron dynamics of mechanistic resonators were defined as follows:

Here, S governed neuronal activity, τ defined the feedback state variable, g represented the integration time constant, Ie was the external current, and g represented feedback strength. The slow kinetics of the negative feedback was controlled by the feedback time constant (τm). In order to manifest resonance, τm > τ (Hutcheon and Yarom, 2000). The steady-state feedback kernel (m∞) of the negative feedback is sigmoidally dependent on the output of the neuron (S), defined by two parameters: half-maximal activity (S1/2) and slope (k). The single-neuron dynamics are elaborated in detail in the methods section (new subsection: Mechanistic model for introducing intrinsic resonance in rate-based neurons).

We first demonstrate that the introduction of a slow-negative feedback loop introduce resonance into single-neuron dynamics (new Figure 9D–E). We performed systematic sensitivity analyses associated with the parameters of the feedback loop and characterized the dependencies of intrinsic neuronal resonance on model parameters (new Figure 9F–I). We demonstrate that the incorporation of resonance through a negative feedback loop was able to generate grid-patterned activity in the 2D CAN model employed here, with clear dependencies on model parameters (new Figure 10; new Figure 10-Supplements1–2). Next, we incorporated heterogeneities into the network and demonstrated that the introduction of resonance through a negative feedback loop stabilized grid-patterned generation in the heterogeneous 2D CAN model (new Figure 11).

The mechanistic route to introducing resonance allowed us to probe the basis for the stabilization of grid-patterned activity more thoroughly. Specifically, with physiological resonators, resonance manifests only when the feedback loop is slow (new Figure 9I; Hutcheon and Yarom, 2000). This allowed us an additional mechanistic handle to directly probe the role of resonance in stabilizing the grid patterned activity. We assessed the emergence of grid-patterned activity in heterogeneous CAN models constructed with networks constructors with neurons with different τm values (new Figure 12). Strikingly, we found that when τm value was small (resulting in fast feedback loops), there was no stabilization of gridpatterned activity in the CAN model, especially with the highest degree of heterogeneities (new Figure 12). With progressive increase in τm, the patterns stabilized with grid score increasing with τm=25 ms (new Figure 12) and beyond (new Figure 11B; τm=75 ms). Finally, our spectral analyses comparing frequency components of homogeneous vs. heterogeneous resonator networks (new Figure panels 13D–F) showed the suppression of low-frequency perturbations in heterogeneous CAN networks.

We gratefully thank the reviewer for raising the issue with the phenomenological resonator model. This allowed us to design the new resonator model and provide several new lines of evidence in support of our central hypothesis. The incorporation of resonance through a negative feedback loop also allowed us to link our analyses to the well-established role of network motifs involving negative feedback loops in inducing stability and suppressing external/internal noise in engineering and biological systems. We envisage intrinsic neuronal resonance as a cellular-scale activity-dependent negative feedback mechanism, a specific instance of a well-established network motif that effectuates stability and suppresses perturbations across different networks (Savageau, 1974; Becskei and Serrano, 2000; Thattai and van Oudenaarden, 2001; Austin et al., 2006; Dublanche et al., 2006; Raj and van Oudenaarden, 2008; Lestas et al., 2010; Cheong et al., 2011; Voliotis et al., 2014). A detailed discussion on this important link to the stabilizing role of this network motif, with appropriate references to the literature is included in the new discussion subsection “Slow negative feedback: Stability, noise suppression, and robustness”.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review): 

The manuscript by Ivan et al aimed to identify epitopes on the Abeta peptide for a large set of anti-Abeta antibodies, including clinically relevant antibodies. The experimental work was well done and required a major experimental effort, including peptide mutational scanning, affinity determinations, molecular dynamics simulations, IP-MS, WB, and IHC. Therefore, it is of clear interest to the field. The first part of the work is mainly based on an assay in which peptides (15-18-mers) based on the human Abeta sequence, including some containing known PTMs, are immobilized, thus preventing aggregation. Although some results are in agreement with previous experimental structural data (e.g. for 3D6), and some responses to diseaseassociated mutations were different when compared to wild-type sequences (e.g. in the case of Aducanumab) - which may have implications for personalized treatment - I have concerns about the lack of consideration of the contribution of conformation (as in small oligomers and large aggregates) in antibody recognition patterns. The second part of the study used fulllength Abeta in monomeric or aggregated forms to further investigate the differential epitope interaction between Aducanumab, Donanemab, and Lecanemab (Figures 5-7). Interestingly, these results confirmed the expected preference of these antibodies for aggregated Abeta, thus reinforcing my concerns about the conclusions drawn from the results obtained using shorter and immobilized forms of Abeta. Overall, I understand that the work is of interest to the field and should be published without the need for additional experimental data. However, I recommend a thorough revision of the structure of the manuscript in order to make it more focused on the results with the highest impact (second part).

We thank the reviewer for highlighting this critical aspect. Our rationale for beginning with the high-resolution, aggregation-independent peptide microarray was to systematically dissect sequence requirements, including PTMs, truncations, and elongations, at single–amino acid resolution. This platform defines linear epitope preferences without the confounding influence of aggregation and enabled analyses that would not have been technically feasible with fulllength Aβ. This rationale is now clarified in the Introduction (lines 72–77).

At the same time, the physiological relevance of antibody binding can only be assessed in the context of aggregation. Prompted by the reviewer’s comments, we restructured the manuscript to foreground the full-length, aggregation-dependent data (Figures 5–7). These assays demonstrate that Aducanumab preferentially recognizes aggregated peptide over monomers and that pre-adsorption with fibrils, but not monomers, blocks tissue reactivity (lines 585–599; Fig. 5B). They also show that Lecanemab can capture soluble Aβ in CSF by IP-MS (lines 544–547; Fig. 4B, Fig. 6–Supplement 1), and that Donanemab strongly binds low-molecular-weight pyroGlu-Aβ while also recognizing highly aggregated Aβ1-42 (lines 668–684; Fig. 7).

The revised Conclusion now explicitly states the complementarity of the two approaches: microarrays for precise sequence and modification mapping, and full-length aggregation assays for context and physiological relevance (lines 705–714).

Finally, prompted by the reviewer’s feedback, we refined the discussion of therapeutic antibodies to move beyond a descriptive dataset and provide mechanistic clarity. Specifically, the dimerization-supported, valency-dependent binding mode of Aducanumab and the additional structural contributions required for Lecanemab binding to aggregated Aβ are now integrated into the reworked Conclusion (lines 725–741).

Reviewer #2 (Public review):  

This paper investigates binding epitopes of different anti-Abeta antibodies. Background information on the clinical outcome of some of the antibodies in the paper, which might be important for readers to know, is lacking. There are no references to clinical outcomes from antibodies that have been in clinical trials. This paper would be much more complete if the status of the antibodies were included. The binding characteristics of aducanumab, donanemab, and Lecanemab should be compared with data from clinical phase 3 studies. 

Aducanumab was identified at Neurimmune in Switzerland and licensed to Biogen and Eisai. Aducanumab was retracted from the market due to a very high frequency of the side-effect amyloid-related imaging abnormalities-edema (ARIA-E). Gantenerumab was developed by Roche and had two failed phase 3 studies, mainly due to a high frequency of ARIA-E and low efficacy of Abeta clearance. Lecanemab was identified at Uppsala University, humanized by BioArctic, and licensed to Eisai, who performed the clinical studies. Eisai and Biogen are now marketing Lecanemab as Leqembi on the world market. Donanemab was developed by Ely Lilly and is sold in the US as Kisunla. 

We thank the reviewer for this valuable suggestion. In the revised manuscript, we have included a concise overview of the clinical status and outcomes of the therapeutic antibodies in the Introduction. This new section (lines 81–99) summarizes the origins, phase 3 trial outcomes, and current regulatory status of Aducanumab, Lecanemab, and Donanemab, as well as mentioning Gantenerumab as a comparator. Key aspects such as ARIA-E incidence, amyloid clearance efficacy, and regulatory decisions are now referenced to provide the necessary clinical context.

These additions directly link our epitope mapping data with the clinical performance and safety profiles of the antibodies, thereby making the translational implications of our results clearer for both research and therapeutic applications.

Limitations: 

(1) Conclusions are based on Abeta antigens that may not be the primary targets for some conformational antibodies like aducanumab and Lecanemab. There is an absence of binding data for soluble aggregated species.

We thank the reviewer for raising this important point. To address the absence of data on soluble aggregated species, we added IP-MS experiments using pooled human CSF as a physiologically relevant source of endogenous Aβ. Lecanemab enriched several endogenous soluble Aβ variants (Aβ1–40, Aβ1–38, Aβ1–37, Aβ1–39, and Aβ1–42), whereas Aducanumab did not yield detectable signals (Figure 4B; lines 544–547). These results directly distinguish between synthetic and patient-derived Aβ and highlight Lecanemab’s capacity to capture soluble Aβ species under biologically relevant conditions.

(2) Quality controls and characterization of different Abeta species are missing. The authors need to verify if monomers remain monomeric in the blocking studies for Figures 5 and 6. 

We thank the reviewer for this comment. In Figure 5 we show that pre-adsorption with monomeric Aβ1–42 does not prevent Aducanumab binding, whereas fibrillar Aβ1–42 completely abolishes staining, consistent with Aducanumab’s avidity-driven preference for higher-order aggregates.

For Lecanemab (Figure 6), we observed a partial preference for aggregated Aβ1–42 over HFIP-treated monomeric and low-n oligomeric forms. We note, as now stated in the revised manuscript (lines 622–623), that monomeric preparations may partially re-aggregate under blocking conditions, which represents an inherent limitation of such experiments.

To further address this, we performed additional blocking experiments using shorter Aβ peptides, which are less prone to aggregation. These peptides did not block immunohistochemical staining (Figure 6 – Supplement 1), underscoring that both epitope length and conformational state contribute to Lecanemab binding. This conclusion is also consistent with recent data presented at AAIC 2023.

(3) The authors should discuss the limitations of studying synthetic Abeta species and how aggregation might hide or reveal different epitopes. 

We thank the reviewer for this important comment. We now explicitly discuss the limitations of using synthetic Aβ peptides, including that aggregation state can mask or expose epitopes in ways that differ from endogenous species. This discussion has been added in the revised manuscript (lines 737–742).

As noted in our replies to Points (2) and (4) here, and to Reviewer #1, we addressed this experimentally by complementing the high-resolution, aggregation-independent mapping with blocking studies using aggregated and monomeric Aβ preparations, and by validating key findings with IP-MS of human CSF as a physiologically relevant source of soluble Aβ. Together, these complementary approaches mitigate the limitations of synthetic peptides and provide a more comprehensive picture of antibody–Aβ interactions

(4) The authors should elaborate on the differences between synthetic Abeta and patientderived Abeta. There is a potential for different epitopes to be available. 

We thank the reviewer for this comment. In the revised manuscript we now discuss how comparisons between synthetic and patient-derived Aβ species reveal additional, likely conformational epitopes that are not accessible in short or monomeric synthetic forms. To address this directly, we performed IP-MS with pooled human CSF. Lecanemab enriched a diverse set of endogenous soluble Aβ1–X species (Aβ1–40, Aβ1–38, Aβ1–37, Aβ1–39, and Aβ1–42), whereas Aducanumab did not yield measurable pull-down (Figure 4B; lines 544– 547). These results emphasize that patient-derived Aβ displays distinct aggregation dynamics and epitope accessibility.

We have expanded on this point in the Conclusion (lines 737–742), underscoring the

importance of integrating both synthetic and native Aβ sources to capture the full range of antibody targets. 

Reviewer #1 (Recommendations for the authors): 

This revision should prioritize the presentation of results obtained using the full-length Abeta peptide, given its more direct relevance to expected antibody recognition patterns in physiological contexts, and discuss the evidence for using synthetic Abeta. 

We thank the reviewer for this recommendation. The revised manuscript now places stronger emphasis on results obtained with full-length Aβ peptides, particularly in Figures 5–7, which analyze binding preferences across monomeric, oligomeric, and fibrillar states (lines 585–599, 609–623, 668–684). We also expanded the Discussion to outline both the rationale and the limitations of using synthetic Aβ. The microarray approach provides high-resolution, aggregation-independent sequence and modification mapping, but must be complemented by experiments with full-length Aβ1–42 under physiologically relevant conditions, such as IP-MS from CSF (lines 544–547) and blocking in IHC (lines 585–599, 622–623, 684), to capture conformational epitopes and validate functional relevance.

Figure 6. = Please review/better explain the following statement "Lecanemab recognized Aβ140, Aβ1-42, Aβ3-40, Aβ-3-40 and phosphorylated pSer8-Aβ1-40 on CIEF-immunoassay and Bicine-Tris SDS-PAGE/ Western blot, indicating that the Lecanemabbs epitope is located in the N-terminal region of the Aβ sequence". Is it possible that N-truncated peptides do not form aggregates as efficiently as (or conformationally distinct from) full-length ones? 

In the revised text we now clarify that Lecanemab recognized Aβ1-40, Aβ1-42, Aβ3-40, Aβ-340, and phosphorylated pSer8-Aβ1-40 on CIEF-immunoassay (Figure 6A; lines 612–619) and Bicine-Tris SDS-PAGE/Western blot (Figure 6C; lines 639–640). In contrast, shorter Ntruncated variants such as Aβ4-40 and Aβ5-40 did not generate detectable signals under the tested conditions. This is consistent with our initial microarray data (Figure 1), which indicated that Lecanemab binding depends on residues 3–7 of the N-terminus.

On gradient Bistris SDS-PAGE/Western blot, Lecanemab showed a partial but not exclusive preference for aggregated Aβ1-42 over monomeric or low-n oligomeric forms in the HFIPtreated preparation (Figure 6B; lines 632–633). Immunohistochemical detection of Aβ deposits in AD brain sections was efficiently blocked by pre-adsorption with monomerized, oligomeric, or fibrillar Aβ1-42 (Figure 6E; lines 643–645), but not by shorter synthetic peptides such as Aβ1-16, Aβ1-34, or Aβ1-38 (Figure 6 – Supplement 1; lines 654–663).

We also note, as now stated in the Results, that re-aggregation of HFIP-treated Aβ1-42 monomers during incubation cannot be entirely excluded (lines 622–623). Taken together, these experiments indicate that both N-terminal sequence length and conformational context are critical for Lecanemab binding, and that truncated peptides may indeed fail to reproduce the aggregate-associated conformations required for full recognition.

Reviewer #2 (Recommendations for the authors): 

Introduction: 

(1) Include examples of Lecanemab, donanemab, and gantenerumab, along with relevant references. 

We expanded the clinical-context paragraph that already covers Aducanumab, Lecanemab, and Donanemab (lines 81–96) and added Gantenerumab. 

(2) Address why gantenerumab was not included in the study. 

Due to the focus of our current study on antibodies with recently approved or late-stage clinical use (Aducanumab, Donanemab, Lecanemab), Gantenerumab was not included. 

(3) Table 1: Correct the reference for Lecanemab, should be reference 44. 

Table 1 has been updated to correct the Lecanemab reference.

(4) Line 84: Add Uppsala University and Eisai alongside Biogen for Lecanemab. 

Line 84 has been revised to acknowledge Uppsala University and Eisai alongside Biogen for the development of Lecanemab (lines 90–96).

(5) Line 539: Include the reference: "Lecanemab, Aducanumab, and Gantenerumab - Binding Profiles to Different Forms of Amyloid-Beta Might Explain Efficacy and Side Effects in Clinical Trials for Alzheimer's Disease. doi: 10.1007/s13311-022-01308-6. 

We thank the reviewer for drawing attention to this important reference (now cited as Ref. 83) provides a state-of-the-art comparison of binding profiles of Lecanemab, Aducanumab, and Gantenerumab, and we have now properly incorporated it into our manuscript. 

(6) Line 657-659: State that the findings are also applicable to Lecanemab. 

Discrepancies between analysis of the short synthetic fragments and the full-length Abeta are now resolved for Aducanumab and Lecanemab and put into context in the results section and the conclusion lines 725-740. 

(7) Figures 5 and 6: Discuss how to ensure that monomers remain monomers under the study conditions, considering the aggregation-prone nature of Abeta1-42. This aggregation could impact Lecanemab's binding to "monomers." To our knowledge, Lecanemab does not bind to monomers. The binding properties observed diverge from previously described properties for Lecanemab. Explore reasons for these discrepancies and suggest conducting complementary experiments using a solution-based assay, as per Söderberg et al, 2023. In Figure 6, note that Lecanemab is strongly avidity-driven, potentially causing densely packed monomers to expose Abeta as aggregated, affecting binding interpretation on SDS-PAGE. 

We thank the reviewer for this important point. In the revised Results and Discussion we explicitly note that HFIP-treated Aβ1–42 monomers may partially re-aggregate during incubation, which cannot be fully excluded (lines 622–623).

To complement these data, we show that Lecanemab successfully enriched soluble endogenous Aβ species (Aβ1–40, Aβ1–38, Aβ1–37, Aβ1–39, and Aβ1–42) in IP-MS from pooled CSF (lines 544–547; Fig. 4B), demonstrating its ability to bind soluble Aβ under physiologically relevant conditions.

We also now cite the Söderberg et al. (2023, PMID: 36253511) study, which reported weak but detectable binding of Lecanemab to monomeric Aβ (their Fig. 1 and Table 6). This supports our interpretation that Lecanemab is aggregation-sensitive rather than strictly aggregationdependent, in contrast to Aducanumab.

To further address sequence and conformational contributions, we performed blocking experiments with shorter, non-HFIP-treated Aβ peptides (Aβ1–16, Aβ1–34, Aβ1–38). These peptides did not block Lecanemab staining in IHC (lines 654–657; Fig. 6 – Supplement 1), indicating that both extended sequence and conformational context are necessary for recognition.

Finally, our findings are in line with preliminary data by Yamauchi et al. (AAIC 2023, DOI: 10.1002/alz.065104), who proposed that Lecanemab recognizes either a conformational epitope spanning the N-terminus and mid-region, or a structural change in the mid-region induced by the N-terminus.

Reviewer #3 (Public review):

Shimogawa et al. describe the generation of acetylated aSyn variants by genetic code expansion to elucidate effects on vesicle binding, aggregation, and seeding effects. The authors compared a semi-synthetic approach to obtain acetylated aSyn variants with genetic code expansion and concluded that the latter was more efficient in generating all 12 variants studied here, despite the low yields for some of them. Selected acetylated variants were used in advanced NMR, FCS, and cryo-EM experiments to elucidate structural and functional changes caused by acetylation of aSyn. Finally, site-specific differences in deacetylation by HDAC 8 were identified.

The study is of high scientific quality, andthe results are convincingly supported by the experimental data provided. The challenges the authors report regarding semi-synthetic access to aSyn are somewhat surprising, as this protein has been made by a variety of different semi-synthesis strategies in satisfactory yields and without similar problems being reported.

The role of PTMs such as acetylation in neurodegenerative diseases is of high relevance for the field, and a particular strength of this study is the use of authentic acetylated aSyn instead of acetylation-mimicking mutations. The finding that certain lysine acetylations can slow down aggregation even when present only at 10-25% of total aSyn is exciting and bears some potential for diagnostics and therapeutic intervention.

Author response:

We thank you for your efforts in reviewing our manuscript.  We sincerely appreciate that the reviewers were all enthusiastic about our comparison of native chemical ligation (NCL) and non-canonical amino acid (ncAA) mutagenesis methods for installing acetyl lysine (AcK) in alpha-synuclein, as well as the wide variety of biochemical experiments enabled by our ncAA approach.  We respond to the critiques specific to each reviewer here.

Reviewer #1:

Expressed concern that in vitro studies of effects on membrane binding were not followed up with neurotransmitter trafficking experiments.  While we certainly think that such studies would be interesting, they would presumably require the use of acetylation mimic mutants (Lys-to-Gln mutations), which we would want to validate by comparison to our semi-synthetic proteins with authentic AcK.  Such experiments are planned for a follow-up manuscript, and we will investigate the reviewer’s suggested experiment at that time.

Reviewer #1 Noted that the method of in vitro seeding really reports on the impact of acetylation on the elongation phase of aggregation.  We will clarify this in our revisions.  They also expressed concern that this was different than the role that acetylation would play in seeding cellular aggregation with pre-acetylated fibrils.  We will also acknowledge and clarify this in our revisions.  Having the monomer population acetylated in cells presents technical challenges that might also be addressed with Gln mutant mimics, and we plan to pursue such experiments in the follow-up manuscript noted above.

Reviewer #1 Criticized the fact that the pre-formed fibrils used in seeding would not have the same polymorph as PD or MSA fibrils derived from patient material.  They were also critical of how our cryo-EM structure of AcK80 fibrils related to the PD and MSA polymorphs.  Finally, while the reviewer liked the MS experiments used to quantify acetylation levels from patient samples, they felt that our findings then threw the physiological relevance of our structural and biochemical experiments into question.  We believe that all of these critiques can be addressed by clarifying our purpose.  We are not necessarily trying to claim that our AcK80 fold is populated in health or disease, but that by driving Lys80 acetylation, one could push fibrils to adopt this conformation, which is less aggregation-prone.  A similar argument has been made in investigations of alpha-synuclein glycosylation and phosphorylation.  Our results in Figure 9 imply that this could be done with HDAC8 inhibition.  We will revise the manuscript to make these ideas clearer, while being sure to acknowledge the limitations noted by Reviewer #1.

Reviewer #2:

Expressed concern over our use of SDS micelles for initial investigation of the 12 AcK variants, rather than the phospholipid vesicles used in later FCS and NMR experiments.  We will note this shortcoming in revisions of our manuscript, but we do not believe that using vesicles instead would change the conclusions of these experiments (that only AcK43 produces an effect, and a modest one at that).

We will add additional detail to the figure captions, as requested by Reviewer #2.

Reviewer #2 shared some of the concerns of Reviewer #1 regarding the distinctions of which phase of aggregation we were investigating in our in vitro experiments.  As noted above, we will clarify this language.

Finally, Reviewer #2 stated that “It is not clear from the EM data that the structures of the different lysine acetylated variants are different.”  We feel that it is quite clear from structures in Figure 8 and the EM density maps in Figure S38 that the AcK80 fold is indeed different.  Although the overall polymorphs are somewhat similar to WT, the position of K80 clearly changes upon acetylation, altering the local fold significantly and the global fold more moderately.

Reviewer #3:

Found the results convincing, including the potential therapeutic implications.  The only concern noted was that they found the difficulties in semi-synthesis of AcK-modified alpha-synuclein surprising given that it has been made many times before through NCL.  Indeed, our own laboratory has made alpha-synuclein through NCL, and the yields reported here are in keeping with our own previous results.  However, since NCL did not give higher yields than ncAA methods, and it is significantly easier to scan AcK positions using ncAAs, we felt that ncAAs are the method of choice in this case.  We will clarify this position in the revised manuscript.

In conclusion, on behalf of all authors, I again thank the reviewers for both their positive and negative observations in helping us to improve our manuscript.  We will revise it to strive for greater clarity as we have noted in this letter.

Reviewer #1 (Public review):

Summary:

The study by Akita B. Jaykumar et al. explored an interesting and relevant hypothesis whether serine/threonine With-No-lysine (K) kinases (WNK)-1, -2, -3, and -4 engage in insulin-dependent glucose transporter-4 (GLUT4) signaling in the murine central nervous system. The authors especially focused on the hippocampus as this brain region exhibits high expression of insulin and GLUT4. Additionally, disrupted glucose metabolism in the hippocampus has been associated with anxiety disorders, while impaired WNK signaling has been linked to hypertension, learning disabilities, psychiatric disorders or Alzheimer's disease. The study took advantage of selective pan-WNK inhibitor WNK 643 as the main tool to manipulate WNK 1-4 activity both in vivo by daily, per-oral drug administration to wild-type mice, and in vitro by treating either adult murine brain synaptosomes, hippocampal slices, primary cortical cultures, and human cell lines (HEK293, SH-SY5Y). Using a battery of standard behavior paradigms such as open field test, elevated plus maze test, and fear conditioning, the authors convincingly demonstrate that the inhibition of WNK1-4 results in behavior changes, especially in enhanced learning and memory of WNK643-treated mice. To shed light on the underlying molecular mechanism, the authors implemented multiple biochemical approaches including immunoprecipitation, glucose-uptake assay, surface biotylination assay, immunoblotting, and immunofluorescence. The data suggest that simultaneous insulin stimulation and WNK1-4 inhibition results in increased glucose uptake and the activity of insulin's downstream effectors, phosphorylated Akt and phosphorylated AS160. Moreover, the authors demonstrate that insulin treatment enhances the physical interaction of the WNK effector OSR1/SPAK with Akt substrate AS160. As a result, combined treatment with insulin and the WNK643 inhibitor synergistically increases the targeting of GLUT4 to the plasma membrane. Collectively, these data strongly support the initial hypothesis that neuronal insulin- and WNK-dependent pathways do interact and engage in cognitive functions.

In response to our initial comments, the authors mildly revised the manuscript, which did not improve the weaknesses to a sufficient level. Our follow-up comments are labeled under "Revisions 1".

Strengths:

The insulin-dependent signaling in the central nervous system is relatively understudied. This explorative study delves into several interesting and clinically relevant possibilities, examining how insulin-dependent signaling and its crosstalk with WNK kinases might affect brain circuits involved in memory formation and/or anxiety. Therefore, these findings might inspire follow-up studies performed in disease models for disorders that exhibit impaired glucose metabolism, deficient memory, or anxiety, such as Diabetes mellitus, Alzheimer's disease, or most of psychiatric disorders.

The graphical presentation of the figures is of high quality, which helps the reader to obtain a good overview and to easily understand the experimental design, results, and conclusions.

The behavioral studies are well conducted and provide valuable insights into the role of WNK kinases in glucose metabolism and their effect on learning and memory. Additionally, the authors evaluate the levels of basal and induced anxiety in Figures 1 and 2, enhancing our understanding of how WNK signaling might engage in cognitive function and anxiety-like behavior, particularly in the context of altered glucose metabolism.

The data presented in Figures 3 and 4 are notably valuable and robust. The authors effectively utilize a variety of in vivo and in vitro models, combining different treatments in a clear manner. The experimental design is well-controlled, efficiently communicated, and well-executed, providing the reader with clear objectives and conclusions. Overall, these data represent particularly solid and reproducible evidence on the enhanced glucose uptake, GLUT4 targeting, and downstream effectors' activation upon insulin and WNK/OSR1 signaling crosstalk.

Weaknesses:

(1) The study used a WNK643 inhibitor as the only tool to manipulate WNK1-4 activity. This inhibitor seems selective; however, it has been reported that it exhibits different efficiency in inhibiting the individual WNK kinases among each other (e.g. PMID: 31017050, PMID: 36712947). Additionally, the authors do not analyze nor report the expression profiles or activity levels of WNK1, WNK2, WNK3, and WNK4 within the relevant brain regions (i.e. hippocampus, cortex, amygdala). Combined, these weaknesses raise concerns about the direct involvement of WNK kinases within the selected brain regions and behavior circuits. It would be beneficial if the authors provided gene profiling for WNK1, 2, 3, and -4 (e.g. using Allen brain atlas). To confirm the observations, the authors should either add results from using other WNK inhibitors or, preferentially, analyze knock-down or knock-out animals/tissue targeting the single kinases.

Revisions 1: The authors added Fig. S1A during the revisions to show expression of Wnt1-4. While the expression data from humans is interesting, the experimental part of the study is performed in mice. It would be more informative for the authors to add expression profiles from mice or overview the expression pattern with suitable references in the introduction to address this point. The authors did not add data from knock down or knockout tissue targeting the single kinases.

(2) The authors do not report any data on whether the global inhibition of WNKs affects insulin levels as such. Since the authors demonstrate the synergistic effect of simultaneous insulin treatment and WNK1-4 inhibition, such data are missing.

Revisions 1: The authors added Fig. S5A to address this point. It is appreciated that authors performed the needed experiment. Unfortunately, no significant change was found, therefore, the authors still cannot conclude that they demonstrate a synergistic effect of simultaneous insulin treatment and WNT1-4 inhibition. It is a missed opportunity that the authors did not measure insulin in the CSF or tissue lysate to support the data.

(3) The study discovered that the Sortilin receptor binds to OSR1, leading the authors to speculate that Sortilin may be involved in the insulin-dependent GLUT4 surface trafficking. The authors conclude in the result section that "WNK/OSR1/SPAK influences insulin-sensitive GLUT4 trafficking by balancing GLUT4 sequestration in the TGN via regulation of Sortilin with GLUT4 release from these vesicles upon insulin stimulation via regulation of AS160." However, the authors do not provide any evidence supporting Sortilin's involvement in such regulation, thus, this conclusion should be removed from the section. Accordingly, the first paragraph of the discussion should be also rephrased or removed.

Revisions 1: The authors added Fig. 5M-N to address this point. The new experiment is appreciated. However, the authors still do not show that sortilin is involved in insulin or WNK-dependent GLUT4 trafficking in their set up since the authors do not demonstrate any changes in GLUT4 sorting or binding. The conclusions should therefore be rephrased or included purely in the discussion. Moreover, the discussion was not adjusted either, leading to over interpretation based on the available data.

(4) The background relevant to Figure 5, as well as the results and conclusions presented in Figure 5 are quite challenging to follow due to the lack of a clear introduction to the signaling pathways. Consequently, understanding the conclusions drawn from the data is also difficult. It would be beneficial if the authors addressed this issue with either reformulations or additional sections in the introduction. Furthermore, the pulldown experiments in this figure lack some of the necessary controls.

Revisions 1: The Authors insufficiently addressed this point during the revisions and did not rewrite the introduction as suggested.

(5) The authors lack proper independent loading controls (e.g. GAPDH levels) in their immunoblots throughout the paper, and thus their quantifications lack this important normalization step. The authors also did not add knock-out or knock-down controls in their co-IPs. This is disappointing since these improvements were central and suggested during the revision process.

(6) The schemes that represent only hypotheses (Fig. 1K, 4A) are unnecessary and confusing and thus should be omitted or placed at the end of each figure if the conclusions align.

(7) Low-quality images, such as Fig. 5H should be replaced with high-resolution photos, moved to the supplementary, or omitted.

Author response:

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

Joint Public Review:

Summary:

The major issues are the need for more information concerning WNK expression in brain regions and additional confirmation of the role of sortilin on WNT signaling. There is a lack of sufficient evidence supporting sortilin's involvement in insulin- and WNK-dependent GLUT4 regulation. The recommendation is to examine what WNK kinase is selectively expressed in the region of interest and then explore its engagement with the sortilin and GLUT4 pathways. Further identification of components of the WNK/OSr1/SPAK-sortilin pathway that regulate GLUT4 in brain slices or primary neurons will be helpful in confirming the results. The use of knock-down or knock-out models would be helpful to explore the direct interaction of the pathways. Immortalized and primary cells also represent useful models.

Together our results indicate that one or more WNK family members regulate insulin sensitivity.  As all WNK family members are expressed in relevant brain regions, whether the results are due to actions of a single WNK family member or more likely due to their combined impact will be an important question to ask in the future.  

There are multiple publications describing how sortilin is involved in insulin-dependent Glut4 trafficking; thus, we did not further address that issue.  We have added data on an additional action of WNK463 which indicates that it can block association of OSR1 with sortilin.  While these results do not delve further into how sortilin works, they support the conclusion that WNK/OSR1/SPAK can influence insulin-dependent glucose transport via distinct cellular events (AS160, sortilin, Akt) which are WNK463 sensitive.  

Altogether we added 12 new panels of data from new and previously performed experiments and we modified 3 existing subfigures in response to comments.

Weaknesses:

(1) The study used a WNK643 inhibitor as the only tool to manipulate WNK1-4 activity. This inhibitor seems selective; however, it has been reported that it exhibits different efficiency in inhibiting the individual WNK kinases among each other (e.g. PMID: 31017050, PMID: 36712947). Additionally, the authors do not analyze nor report the expression profiles or activity levels of WNK1, WNK2, WNK3, and WNK4 within the relevant brain regions (i.e. hippocampus, cortex, amygdala). Combined, these weaknesses raise concerns about the direct involvement of WNK kinases within the selected brain regions and behavior circuits. It would be beneficial if the authors provided gene profiling for WNK1, 2, 3, and -4 (e.g. using Allen brain atlas). To confirm the observations, the authors should either add results from using other WNK inhibitors or, preferentially, analyze knock-down or knock-out animals/tissue targeting the single kinases.

Thank you for the excellent suggestion to include mRNA data for the four WNKs. We have included a supplementary figure showing expression of WNK1-4 mRNAs in prefrontal cortex and the hippocampus curated from the Allen Brain Atlas. As per the Allen Brain Atlas, all four WNKs are detected in these regions with WNK4 mRNA the most highly expressed followed by WNK2, WNK3 and then WNK1 (Figure S1A).   

With regard to the use of WNK463, we continue to use WNK463 because we have examined its actions in cell lines that only express WNK1, e.g. A549 (Haman Center lung cancer RNA-seq data), and in A549 with WNK1 deleted using CRISPR in which we saw no effects of WNK463 on several assays we use for WNK1 including suppression of autophagy.  WNK463 was reported in the literature to inhibit only the four WNKs out of more than 400 kinases tested, indicating more selectivity than many small molecules used to target other enzymes.  In other cell lines, we also use WNK1 knockdown which replicates the effect of WNK463 (Figure S7A-D). However, in SHSY5Y cells, WNK1 knockdown did not replicate the effect of WNK463 on pAKT levels (Figure S7E-F), suggesting a cooperativity among other WNK family members in neuronal cells. This makes WNK463 an ideal tool to test our hypotheses in this study as it targets all 4 WNKs (WNK1-4).  

(2) The authors do not report any data on whether the global inhibition of WNKs affects insulin levels. Since the authors wish to demonstrate the synergistic effect of simultaneous insulin treatment and WNK1-4 inhibition, such data are missing.

Thank you for this comment. To obtain this information, we treated C57BL/6J mice with WNK463 for 3 days once daily at a dose of 6 mg/kg and then fasted overnight. Plasma insulin levels were measured. Results showed that the plasma insulin levels trended upwards in the WNK463 treated animals compared to the vehicle treated groups but failed to reach any statistical significance. We have now included these data in supplementary figure S5A.

The study discovered that the Sortilin receptor binds to OSR1, leading the authors to speculate that Sortilin may be involved in the insulin-dependent GLUT4 surface trafficking. However, the authors do not provide any evidence supporting Sortilin's involvement in insulin- or WNKdependent GLUT4 trafficking. Thus, this conclusion should be qualified, rephrased, or additional data included.

Work from several groups have shown that sortilin is involved in insulin-dependent GLUT4 trafficking, for example [9-11,135-139] as we noted in the manuscript. We now show that WNK463 blocks co-immunoprecipitation of Flag-tagged sortilin with endogenous OSR1 in HEK293T cells. This result supports our model for WNK/OSR1/SPAK- insulin mediated regulation of sortilin.  We included these data in figures 5M, 5N.

Minor issues:

(1) The method and result sections lack information regarding the gender and age of mice used in the behavioral experiments. This information should be added.

Thank you for pointing this out. We apologize for the omission. The requested information has now been added in the methods section.

(2) The authors present an analysis of relative protein levels in Figure 1B and Figure 4B, however, the original immunoblots (?) are not included in the study. These data should be added to provide complete and transparent evidence for the analysis.

Thank you for this request. The blots have now been included in the supplementary figure S2A and Figure 4B, respectively.  

(3) The basis for Figure 3A needs to be explained and supported with suitable references either in the background or in the result section.

Thank you for pointing this out. Figure 3A has been moved to Figure 3H as it represents the model summary of the data presented in Figure 3. Other figure numbers have been changed accordingly.  This figure 3A (now 3H) and the model diagram of Figure 5 (now Figure 5O) are now cited in the Discussion, where the results are considered in detail.      

(4) Figure 4E should be labeled as 'Primary cortical neurons' for clarity, as the major focus is on the hippocampus. To increase consistency, the authors should consider performing the same experiment on hippocampal cultures or explaining using cortical neurons.

Thank you for the suggestion. Figure 4E (now 4F) has been labelled as Primary cortical neurons for clarity. The major focus of this study is to understand the regulation of WNKmediated regulation of insulin signaling in the areas of the brain that are insulin sensitive such as the hippocampus and the prefrontal cortex. Therefore, we included cortical neurons to test this hypothesis.  

(5) Figure 5B: The use of whole brain extracts is inconsistent with the rest of the study, especially considering the indication of differing insulin activity in selected brain regions. The authors should explain why they could not use only hippocampal tissue.

In this manuscript, we are trying to test our hypothesis in insulin-sensitive neuronal cells which includes, but not limited to, the hippocampus. Figure 5B used whole brain extracts, which contain brain regions that are insulin-sensitive as well as insulin-insensitive regions, to show the association between OSR1 and AS160. However, this observation was replicated in the insulin-sensitive SH-SY5Y cell model suggesting that association of OSR1 and AS160 is modulated in the presence of insulin as shown in Figure 5B, 5C. We added data from SH-SY5Y cells showing effects of WNK463. These data support the concept that this is an interaction that is modulated by WNKs and will occur as long as both OSR1/SPAK and AS160 are expressed.

(6) Figure 5B-C - Knock-out or knock-down condition should be included in the co-IP experiment. This is especially straightforward to generate in the SH-SY5Y cells. Moreover, these figures lack loading controls.

If we understand correctly, the issue with regard to including knockdown conditions stems from the issues raised regarding specificity of the antibody which we have addressed in point 10 below. We have now included input blots for both AS160 and OSR1 which serve as the loading control for the IP experiment in figure 5B and 5C.

(7) Figure 5C-D - A condition with WNK463 inhibition alone is missing. This condition is necessary for evaluating the effects of WNK643 inhibition with and without insulin stimulation.

Thank you for this observation. We have now added the data for that condition.  The aim of this experiment in Figure 5C (now 5B and 5C) is to show that insulin is important to facilitate interaction between OSR1 and AS160 in differentiated SHSY5Y cells and the effect of WNK463 to diminish this insulin-dependent interaction. With only WNK463, there was minimal interaction between AS160 and OSR1 as now shown in Figure 5B, 5C.

(8) Figure 5G - This figure shows the overexpression of plasmids in HEK cells, however, it lacks samples that overexpress the plasmid individually (single expression). Such data should be added, especially when the addition of the blocking peptide does not fully disable the interaction between AS160 and SPAK. Additionally, this figure also lacks a loading control, which is essential for validating the results.

Thank you for this comment. Figure 5G (now Figure 5F, 5G) is an in vitro IP in which we have mixed a purified Flag-SPAK fragment residues 50-545 with a lysate from cells expressing Myc-AS160 (residues 193-446). This is essentially an in vitro IP; because it is not an IP experiment from cell lysates where we overexpressed these plasmids which would require a loading control. The lysates were divided in half and one half did not receive the blocking peptide while the other half did, creating a control. From our experience, this blocking peptide does not completely block interactions between SPAK/OSR1 and NKCC2 fragments which are well-characterized interacting partners [a]. The reason for the partial block in interactions could also be attributed to the multivalent nature of interaction between these proteins. This confusion in our methodology used has been noted and we have tried to explain it with more clarity in the methods, results and the figure legend section. Our Commun. Biol. paper [134] that describes this assay and uses it extensively is now available online.

(a) Piechotta K, Lu J, Delpire E. Cation chloride cotransporters interact with the stressrelated kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) J Biol Chem. 2002;277:50812–50819. doi: 10.1074/jbc.M208108200.

(9) Figure 5J, L - These figures are missing negative controls. The authors should add Sortilin knock-down or knock-out conditions for the immunoprecipitation experiments. Also, the figures lack loading controls. Moreover, the labeling "Control" should be specified, as it is unclear what this condition represents.

Thank you for noting the lack of clarity in the controls provided. Controls in Figure 5J and 5L refer to IgG Control which serves as the negative control in this case. This has now been specified in the figures (and added Figures 5M and 5N, as well). The issue with OSR1 and sortilin antibody specificity and cross-reaction has been addressed in point 10.

(10) Figure 5I - The fluorescent signals for the individual channels of OSR1 and Sortilin appear identical (even within the background signal). This raises concerns about potential antibody cross-reaction. One potential solution would be to include additional stainings with different antibodies and perform staining of each protein alone to ensure the specificity of the colocalization.

Thank you for pointing this out and giving us an opportunity to provide better images that will address the issues raised regarding antibody cross-reaction and antibody specificity. We realize that the images that we originally provided appeared to show all the puncta colocalize which could give rise to the concern about potential antibody cross-reaction. We have replaced them with more appropriate representative images that clearly show some selected regions of common staining as well as regions where there is no overlap.  

(11) Figures 5D, 5F, 5H, 5L, 5M: These analyses should be first normalized to the loading control such as GAPDH.

In Figure 5F (now 5E), the analysis has been normalized to the total AS160 protein levels. Because we are reporting changes in pAS160 protein, normalizing it to the total AS160 gives a better idea about the changes in the phosphorylated AS160 form compared to the whole protein and this is more appropriate compared to other loading controls such as GAPDH.  

In Figure 5H (now Figure 5G), the analysis is an in vitro IP assay using purified protein fragments. Therefore, using GAPDH as a control is not applicable in this case. Please refer to our response to comment 8 for details.

In Figures 5L, 5M and 5D (now 5K, 5L, 5C) shown, the IP proteins have been normalized to the input protein levels serving as a loading control for the IP experiment. 

(12) Figure 5K: The significance/meaning of the red star is unclear. It should be explained in the figure legend.

Thank you for the opportunity to enhance the readability of our manuscript. The meaning of red star denotes the condition in the yeast two-hybrid assay which shows the binding of CCT of OSR1 with C-terminus of sortilin. This has now been clarified in the figure legend.

(13) Differences in WNK643 dosage and administration periods can affect the results. There is a lack of explanation with regard to the divergent WNK643 treatments of mice across different behavior conditions of fear conditioning, the novel object test, and the elevated plus maze test. This should be considered.

Thank you for pointing out that the explanation regarding the WNK463 dosage and times are unclear. WNK463 was dosed 3 days before the start of the behavior experiment daily at a dose of 6 mg/kg and continued throughout the test protocol. This is the same protocol used for all experiments.  The text describing the protocol has been reworded with more clarity on dosage and times in methods and result section.

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Reviewer #1 (Public review):

Summary:

This study identifies HSD17B7 as a cholesterol biosynthesis gene enriched in sensory hair cells, with demonstrated importance for auditory behavior and potential involvement in mechanotransduction. Using zebrafish knockdown and rescue experiments, the authors show that loss of hsd17b7 reduces cholesterol levels and impairs hearing behavior. They also report a heterozygous nonsense variant in a patient with hearing loss. The gene mutation has a complex and somewhat inconsistent phenotype, appearing to mislocalize, reduce mRNA and protein levels, and alter cholesterol distribution, supporting HSD17B7 as a potential deafness gene.

While the study presents an interesting candidate and highlights an underexplored role for cholesterol in hair cell function, several important claims are insufficiently supported, and the mechanistic interpretations remain somewhat murky.

Strengths:

(1) HSD17B7 is a new candidate deafness gene with plausible biological relevance.

(2) Cross-species RNAseq convincingly shows hair-cell enrichment.

(3) Lipid metabolism, particularly cholesterol homeostasis, is an emerging area of interest in auditory function.

(4) The connection between cholesterol levels and MET is potentially impactful and, if substantiated, would represent a significant advance.

Weaknesses:

(1) The pathogenic mechanism of the E182STOP variant is unclear: The mutant protein presumably does not affect WT protein localization, arguing against a dominant-negative effect. Yet, overexpression of HSD17B7-E182* alone causes toxicity in zebrafish, and it binds and mislocalizes cholesterol in HEI-OC1 cells, suggesting some gain-of-function or toxic effect. In addition, the mRNA of the variant has a low expression level, suggesting nonsense-mediated decay. This complexity and inconsistency need clearer explanation.

(2) The link to human deafness is based on a single heterozygous patient with no syndromic features. Given that nearly all known cholesterol metabolism disorders are syndromic, this raises concerns about causality or specificity. The term "novel deafness gene" is premature without additional cases or segregation data.

(3) The localization of HSD17B7 should be clarified better: In HEI-OC1 cells, HSD17B7 localizes to the ER, as expected. In mouse hair cells, the staining pattern is cytosolic and almost perfectly overlaps with the hair cell marker used, Myo7a. This needs to be discussed. Without KO tissue, HSD17B7 antibody specificity remains uncertain.

Reviewer #2 (Public review):

A summary of what the authors were trying to achieve.

The authors aim to determine whether the gene Hsb17b7 is essential for hair cell function and, if so, to elucidate the underlying mechanism, specifically the HSB17B7 metabolic role in cholesterol biogenesis. They use animal, tissue, or data from zebrafish, mouse, and human patients.

Strengths:

(1) This is the first study of Hsb17b7 in the zebrafish (a previous report identified this gene as a hair cell marker in the mouse utricle).

(2) The authors demonstrate that Hsb17b7 is expressed in hair cells of zebrafish and the mouse cochlea.

(3) In zebrafish larvae, a likely KO of the Hsb17b7 gene causes a mild phenotype in an acoustic/vibrational assay, which also involves a motor response.

(4) In zebrafish larvae, a likely KO of the Hsb17b7 gene causes a mild reduction in lateral line neuromast hair cell number and a mild decrease in the overall mechanotransduction activity of hair cells, assayed with a fluorescent dye entering the mechanotransduction channels.

(5) When HSB17B7 is overexpressed in a cell line, it goes to the ER, and an increase in Cholesterol cytoplasmic puncta is detected. Instead, when a truncated version of HSB17B7 is overexpressed, HSB17B7 forms aggregates that co-localize with cholesterol.

(6) It seems that the level of cholesterol in crista and neuromast hair cells decreases when Hsb17b7 is defective (but see comment below).

Weakness:

(1) The statement that HSD17B7 is "highly" expressed in sensory hair cells in mice and zebrafish seems incorrect for zebrafish:

(a) The data do not support the notion that HSB17B7 is "highly expressed" in zebrafish. Compared to other genes (TMC1, TMIE, and others), the HSB17B7 level of expression in neuromast hair cells is low (Figure 1F), and by extension (Figure 1C), also in all hair cells. This interpretation is in line with the weak detection of an mRNA signal by ISH (Figure 1G I"). On this note, the staining reported in I" does not seem to label the cytoplasm of neuromast hair cells. An antisense probe control, along with a positive control (such as TMC1 or another), is necessary to interpret the ISH signal in the neuromast.

(b) However, this is correct for mouse cochlear hair cells, based on single-cell RNA-seq published databases and immunostaining performed in the study. However, the specificity of the anti-HSD17B7 antibody used in the study (in immunostaining and western blot) is not demonstrated. Additionally, it stains some supporting cells or nerve terminals. Was that expression expected?

(2) A previous report showed that HSD17B7 is expressed in mouse vestibular hair cells by single-cell RNAseq and immunostaining in mice, but it is not cited:

Spatiotemporal dynamics of inner ear sensory and non-sensory cells revealed by single-cell transcriptomics.

Jan TA, Eltawil Y, Ling AH, Chen L, Ellwanger DC, Heller S, Cheng AG.

Cell Rep. 2021 Jul 13;36(2):109358. doi: 10.1016/j.celrep.2021.109358.

(3) Overexpressed HSD17B7-EGFP C-terminal fusion in zebrafish hair cells shows a punctiform signal in the soma but apparently does not stain the hair bundles. One limitation is the consequence of the C-terminal EGFP fusion to HSD17B7 on its function, which is not discussed.

(4) A mutant Zebrafish CRISPR was generated, leading to a truncation after the first 96 aa out of the 340 aa total. It is unclear why the gene editing was not done closer to the ATG. This allele may conserve some function, which is not discussed.

(5) The hsd17b7 mutant allele has a slightly reduced number of genetically labeled hair cells (quantified as a 16% reduction, estimated at 1-2 HC of the 9 HC present per neuromast). On a note, it is unclear what criteria were used to select HC in the picture. Some Brn3C:mGFP positive cells are apparently not included in the quantifications (Figure 2F, Figure 5A).

(6) The authors used FM4-64 staining to evaluate the hair cell mechanotransduction activity indirectly. They found a 40% reduction in labeling intensity in the HCs of the lateral line neuromast. Because the reduction of hair cell number (16%) is inferior to the reduction of FM4-64 staining, the authors argue that it indicates that the defect is primarily affecting the mechanotransduction function rather than the number of HCs. This argument is insufficient. Indeed, a scenario could be that some HC cells died and have been eliminated, while others are also engaged in this path and no longer perform the MET function. The numbers would then match. If single-cell staining can be resolved, one could determine the FM4-64 intensity per cell. It would also be informative to evaluate the potential occurrence of cell death in this mutant. On another note, the current quantification of the FM4-64 fluorescence intensity and its normalization are not described in the methods. More importantly, an independent and more direct experimental assay is needed to confirm this point. For example, using a GCaMP6-T2A-RFP allele for Ca2+ imaging and signal normalization.

(7) The authors used an acoustic startle response to elicit a behavioral response from the larvae and evaluate the "auditory response". They found a significative decrease in the response (movement trajectory, swimming velocity, distance) in the hsd17b7 mutant. The authors conclude that this gene is crucial for the "auditory function in zebrafish".

This is an overstatement:

(a) First, this test is adequate as a screening tool to identify animals that have lost completely the behavioral response to this acoustic and vibrational stimulation, which also involves a motor response. However, additional tests are required to confirm an auditory origin of the defect, such as Auditory Evoked Potential recordings, or for the vestibular function, the Vestibulo-Ocular Reflex.

(b) Secondly, the behavioral defects observed in the mutant compared to the control are significantly different, but the differences are slight, contained within the Standard Deviation (20% for velocity, 25% for distance). To this point, the Figure 2 B and C plots are misleading because their y-axis do not start at 0.

(8) Overexpression of HSD17B7 in cell line HEI-OC1 apparently "significantly increases" the intensity of cholesterol-related signal using a genetically encoded fluorescent sensor (D4H-mCherry). However, the description of this quantification (per cell or per surface area) and the normalization of the fluorescent signal are not provided.

(9) When this experiment is conducted in vivo in zebrafish, a reduction in the "DH4 relative intensity" is detected (same issue with the absence of a detailed method description). However, as the difference is smaller than the standard deviation, this raises questions about the biological relevance of this result.

(10) The authors identified a deaf child as a carrier of a nonsense mutation in HSB17B7, which is predicted to terminate the HSB17B7 protein before the transmembrane domain. However, as no genetic linkage is possible, the causality is not demonstrated.

(11) Previous results obtained from mouse HSD17B7-KO (citation below) are not described in sufficient detail. This is critical because, in this paper, the mouse loss-of-function of HSD17B7 is embryonically lethal, whereas no apparent phenotype was reported in heterozygotes, which are viable and fertile. Therefore, it seems unlikely that heterozygous mice exhibit hearing loss or vestibular defects; however, it would be essential to verify this to support the notion that the truncated allele found in one patient is causal.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Jokela H, Rantakari P, Lamminen T, Strauss L, Ola R, Mutka AL, Gylling H, Miettinen T, Pakarinen P, Sainio K, Poutanen M.<br /> Endocrinology. 2010 Apr;151(4):1884-92. doi: 10.1210/en.2009-0928. Epub 2010 Feb 25.

(12) The authors used this truncated protein in their startle response and FM4-64 assays. First, they show that contrary to the WT version, this truncated form cannot rescue their phenotypes when overexpressed. Secondly, they tested whether this truncated protein could recapitulate the startle reflex and FM4-64 phenotypes of the mutant allele. At the homozygous level (not mentioned by the way), it can apparently do so to a lesser degree than the previous mutant. Again, the differences are within the Standard Deviation of the averages. The authors conclude that this mutation found in humans has a "negative effect" on hearing, which is again not supported by the data.

(13) The authors looked at the distribution of the HSB17B7 in a cell line. The WT version goes to the ER, while the truncated one forms aggregates. An interesting experiment consisted of co-expressing both constructs (Figure S6) to see whether the truncated version would mislocalize the WT version, which could be a mechanism for a dominant phenotype. However, this is not the case.

(14) Through mass spectrometry of HSB17B7 proteins in the cell line, they identified a protein involved in ER retention, RER1. By biochemistry and in a cell line, they show that truncated HSB17B7 prevents the interaction with RER1, which would explain the subcellular localization.

Hydroxysteroid (17beta) dehydrogenase 7 activity is essential for fetal de novo cholesterol synthesis and for neuroectodermal survival and cardiovascular differentiation in early mouse embryos.

Jokela H, Rantakari P, Lamminen T, Strauss L, Ola R, Mutka AL, Gylling H, Miettinen T, Pakarinen P, Sainio K, Poutanen M.<br /> Endocrinology. 2010 Apr;151(4):1884-92. doi: 10.1210/en.2009-0928. Epub 2010 Feb 25.

(15) Information and specificity validation of the HSB17B7 antibody are not presented. It seems that it is the same used on mice by IF and on zebrafish by Western. If so, the antibody could be used on zebrafish by IF to localize the endogenous protein (not overexpression as done here). Secondly, the specificity of the antibody should be verified on the mutant allele. That would bring confidence that the staining on the mouse is likely specific.

I found this source fascinating because it changes how I think about disinformation. Starbird and her co-authors argue that misinformation online isn’t always the work of one bad actor—it’s often collaborative, created and spread by everyday users who unintentionally participate in shaping false narratives. This makes me think about how easily people can get caught up in sharing misleading content without realizing they’re part of a larger system. It connects to the chapter’s idea of ad hoc crowdsourcing—just like people online come together to solve problems, they can also come together to spread rumors or false information. It’s a reminder that online collaboration can be powerful, but it also requires awareness and responsibility.

1.excel 文件解析 - 读取时不区分 tab，直接读取所有 tab 的统一社会信用代码： - 如果系统中有对应的集团，则匹配对应的集团（一级公司） - 如果系统中没有对应的集团，则匹配为“其他” - 注意：比对的时候要取并集（Excel+系统） - 所有的比对结果不用存，都是单次 - 注：股权比对最后做，可以先按照如下格式解析；如果处理不了则按照 % 核对股东数量 “宜昌产投控股集团有限公司持股99%； 湖北同富创业投资管理有限公司持股1%”

2.比对结果的统计数据 - 一致/差异 是指有差异（只要有打❌这家就算差异），全部为✅则为一致。一致率=一致企业数/总企业数

3.企业列表： - 默认仅显示差异企业，点击后显示所有企业。 - 根据系统和 Excel 数据，一致显示✅，不一致显示❌。 - 分页默认可以多点，一次按照 50.

4.导出： 点击导出后一次性按照所有集团导出，每个集团一个Excel，内容即为列表显示内容即可（其他也导出）。

1.excel 文件解析 - 读取时不区分 tab，直接读取所有 tab 的统一社会信用代码： - 如果系统中有对应的集团，则匹配对应的集团（一级公司） - 如果系统中没有对应的集团，则匹配为“其他” - 注意：比对的时候要取并集（Excel+系统） - 所有的比对结果不用存，都是单次 - 注：股权比对最后做，可以先按照如下格式解析；如果处理不了则按照 % 核对股东数量 “宜昌产投控股集团有限公司持股99%； 湖北同富创业投资管理有限公司持股1%”

2.比对结果的统计数据 - 一致/差异 是指有差异（只要有打❌这家就算差异），全部为✅则为一致。一致率=一致企业数/总企业数

3.企业列表： - 默认仅显示差异企业，点击后显示所有企业。 - 根据系统和 Excel 数据，一致显示✅，不一致显示❌。 - 分页默认可以多点，一次按照 50.

4.导出： 点击导出后一次性按照所有集团导出，每个集团一个Excel，内容即为列表显示内容即可（其他也导出）。

Raising a family while pursuing education offers pros like improved family relationships and setting a positive example for your children, but also has cons such as significant time and financial strain

that he should raise a family later when he's more financially stable also that he should spend more time on getting money instead of using all of his time on learning new skills

breaking the problem into smaller manageable pieces are that he wants to spend 4 years for an education also that he wants to raise a family

that he wants to start a family but he also wants to higher his education. that he's not able to do all of that in the amount of time that he's given. the related issue is that he doesn't have the time to do all of the raising a kid and hiring his education. the requirements for a solution is that he should put the raising a family later on instead of doing it now.

different types of of academic books

Textbooks help you understand the basics, research books give you detailed info you can use as evidence, and edited collections let you see different experts’ ideas.

Analyse des Interventions contre la Pauvreté : L'Émergence des Dons Directs en Espèces

Résumé

L'analyse des stratégies de lutte contre la pauvreté révèle un changement de paradigme significatif, s'éloignant des modèles philanthropiques traditionnels pour se tourner vers les dons directs en espèces.

Des décennies d'interventions conventionnelles, incluant l'éducation, la formation professionnelle et la microfinance, ont montré des résultats décevants et un impact minimal sur l'augmentation des revenus, comme l'ont démontré des essais contrôlés randomisés à la fin des années 1990.

En contraste, les dons directs en espèces, initialement considérés comme une approche contre-productive, ont produit des résultats remarquables. Une étude de cas emblématique menée en 2018 dans le village d'Ahenyo, au Kenya, a montré qu'un versement unique de 500 dollars par adulte a entraîné une augmentation de 65 % des revenus des entreprises, une amélioration des conditions de vie et une réduction des problèmes sociaux en seulement deux ans. Ces résultats positifs sont corroborés par de nombreuses autres études, indiquant que l'impact des dons en espèces dépasse souvent celui des programmes d'aide traditionnels et peut même stimuler l'économie locale de manière significative.

Le principe fondamental de cette approche est de reconnaître que les personnes en situation de pauvreté sont les mieux placées pour identifier et répondre à leurs propres besoins.

Cependant, cette méthode n'est pas une solution miracle.

La pauvreté est un problème générationnel et les effets à très long terme de ces dons ne sont pas encore entièrement compris, comme l'illustre une étude ougandaise aux résultats fluctuants. Néanmoins, les ressources financières pour éliminer l'extrême pauvreté existent déjà, avec 200 milliards de dollars d'aide internationale annuelle et 1,5 billion de dollars dans des fondations privées.

Le véritable défi consiste pour ces institutions à adopter une nouvelle philosophie : faire confiance à l'expertise des personnes qu'elles cherchent à aider.

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1. L'Échec des Modèles Philanthropiques Traditionnels

Depuis les années 1960, les organisations caritatives ont investi des milliards de dollars dans des programmes visant à sortir les pays de la pauvreté.

Cependant, des évaluations rigoureuses ont remis en question l'efficacité de ces approches conventionnelles.

L'Impact Limité de l'Aide au Développement

Les efforts philanthropiques se sont historiquement concentrés sur des interventions structurées dans l'espoir de créer un environnement propice à l'indépendance financière.

• Domaines d'intervention : Éducation, formation professionnelle, développement agricole, projets d'infrastructure et programmes de santé.

• Objectif théorique : Créer un "terreau de connaissances et de capitaux" pour soutenir les économies en difficulté.

• Constat des chercheurs (fin des années 1990 - début 2000) : Des essais contrôlés randomisés ont révélé que ce type d'aide avait souvent un impact minimal.

◦ Les fournitures scolaires n'ont pas amélioré la qualité de l'enseignement.   

◦ La formation professionnelle n'a pas systématiquement conduit à une augmentation des revenus.  

◦ Les bénéfices de l'éducation nutritionnelle variaient considérablement d'un groupe à l'autre.

Les Limites de la Microfinance

Un modèle plus récent, la microfinance, a également fait l'objet d'un examen critique.

Conçue pour offrir de petits prêts aux entrepreneurs dans les économies pauvres, cette approche n'a pas non plus tenu toutes ses promesses.

Bien que les bénéficiaires aient régulièrement remboursé leurs prêts avec intérêts, cela n'a pas contribué à une augmentation significative de leurs revenus.

2. Les Dons Directs en Espèces : Une Stratégie Efficace

Face aux résultats décevants des modèles traditionnels, les chercheurs ont commencé à explorer une stratégie radicalement différente : les transferts monétaires directs et inconditionnels.

Une Approche Initialement Discréditée

La plupart des philanthropes rejetaient cette idée, la qualifiant de "ridicule" et de "pire forme de philanthropie à courte vue".

La crainte dominante était que les bénéficiaires dépenseraient rapidement l'argent reçu pour ensuite retourner à leur situation initiale sans aucune amélioration durable.

L'Expérience Révélatrice d'Ahenyo

En 2018, une organisation à but non lucratif a mené une expérience dans le village d'Ahenyo, au Kenya, où la plupart des familles vivaient dans l'extrême pauvreté.

Chaque adulte a reçu 500 dollars, soit l'équivalent du salaire annuel de la plupart des habitants, sans aucune condition.

Les résultats, observés deux ans plus tard, ont été "étonnants" :

Indicateur

Impact Observé

Économique

Augmentation de 65 % des revenus des entreprises.

Financier

Augmentation de l'épargne des familles.

Social

Amélioration des résultats scolaires des enfants.

Réduction de l'alcoolisme, de la dépression et de la violence domestique.

Diminution des inégalités entre les familles.

Nutrition

Augmentation de la consommation alimentaire.

Confirmation à plus Grande Échelle

Les résultats d'Ahenyo ne sont pas un cas isolé.

Depuis cette étude, les dons directs en espèces sont devenus l'une des interventions les plus étudiées.

Les données démontrent de manière constante que leurs impacts dépassent souvent ceux des programmes d'aide traditionnels.

Une étude ultérieure menée dans des centaines de villages kényans a même révélé que l'économie locale avait connu une croissance équivalente au double du montant total distribué, un an seulement après les dons.

3. Limites et Incertitudes

Malgré leurs succès avérés, les dons directs en espèces ne constituent pas une "solution miracle" et des questions subsistent quant à leur durabilité.

• Le défi de la durabilité : La pauvreté est un problème générationnel qui requiert des changements à long terme. L'intervention étant relativement récente, ses effets sur la durée ne sont pas encore totalement compris.

• L'exemple de l'Ouganda : Une étude initiée en 2008 a montré des résultats complexes.

Un transfert d'argent a amélioré les revenus de certaines familles durant les quatre premières années, mais cet effet positif a disparu au cours des cinq années suivantes.

Il est cependant réapparu sous la pression de la pandémie de COVID-19, illustrant la complexité de l'évolution de ces impacts dans le temps.

4. Le Changement de Paradigme Fondamental

Au-delà des résultats économiques, la théorie derrière le succès des dons directs en espèces propose une refonte complète de la manière d'envisager la lutte contre la pauvreté.

• Programmes traditionnels : Ils partent du principe que les philanthropes et les experts extérieurs sont les mieux placés pour connaître les besoins d'une communauté.

• Dons directs en espèces : Ils reposent sur l'idée que les personnes en situation de pauvreté sont les véritables experts de leur propre situation et comprennent le mieux ce dont elles ont besoin pour s'en sortir.

Cette approche permet une flexibilité totale, reconnaissant que les priorités varient d'un individu à l'autre.

Pour une personne, la réparation de sa maison peut être un investissement plus crucial pour sa réussite à long terme que le lancement d'une entreprise.

Pour une autre, assurer l'éducation de son enfant peut représenter la voie la plus sûre vers des revenus futurs plus élevés.

Conclusion : Les Moyens Existent, la Confiance est la Clé

Les ressources financières nécessaires pour mettre fin à l'extrême pauvreté sont déjà disponibles.

Les pays riches dépensent 200 milliards de dollars par an en aide internationale, et les fondations philanthropiques privées disposent de 1,5 billion de dollars supplémentaires.

Le principal obstacle n'est pas financier, mais philosophique.

Pour réussir, ces institutions devront opérer un changement fondamental : faire confiance à l'expertise, au jugement et à la capacité d'action des personnes qui vivent réellement dans la pauvreté.

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

Authors’ reply (____Ono et al)

Review Commons Refereed Preprint #RC-2025-03137

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

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

We greatly appreciate the reviewer’s supportive comments. The reviewer has accurately recognized our new findings concerning the collaborative roles of condensin II and cohesin in establishing and maintaining interphase chromosome territories.

Major point:

They propose a functional 'handover' from condensin II to cohesin, for the organization of CTs at the M-to-G1 transition. However, the 'handover', i.e. difference in timing of executing their functions, was not experimentally substantiated. Ideally, they can deplete condensin II and cohesin at different times to prove the 'handover'. However, this would require the use of two different degron tags and go beyond the revision of this manuscript. At least, based on the literature, the authors should discuss why they think condensin II and cohesin should work at different timings in the CT organization.

We take this comment seriously, especially because Reviewer #2 also expressed the same concern.　

First of all, we must admit that the basic information underlying the “handover” idea was insufficiently explained in the original manuscript. Let us make it clear below:

• Condensin II bound to chromosomes and is enriched along their axes from anaphase through telophase (Ono et al., 2004; Hirota et al., 2004; Walther et al., 2018).
• In early G1, condensin II is diffusely distributed within the nucleus and does not bind tightly to chromatin, as shown by detergent extraction experiments (Ono et al., 2013).
• Cohesin starts binding to chromatin when the cell nucleus reassembles (i.e., during the cytokinesis stage shown in Fig. 1B), apparently replacing condensins I and II (Brunner et al., 2025).
• Condensin II progressively rebinds to chromatin from S through G2 phase (Ono et al., 2013). The cell cycle-dependent changes in chromosome-bound condensin II and cohesin summarized above are illustrated in Fig. 1A. We now realize that Fig. 1B in the original manuscript was inconsistent with Fig. 1A, creating unnecessary confusion, and we sincerely apologize for this. The fluorescence images shown in the original Fig. 1B were captured without detergent extraction prior to fixation, giving the misleading impression that condensin II remained bound to chromatin from cytokinesis through early G1. This was not our intention. To clarify this, we have repeated the experiment in the presence of detergent extraction and replaced the original Fig. 1B with a revised panel. Figs. 1A and 1B are now more consistent with each other. Accordingly, we have modified the correspsonding sentences as follows:

Although condensin II remains nuclear throughout interphase, its chromatin binding is weak in G1 and becomes robust from S phase through G2 (Ono et al., 2013). Cohesin, in contrast, replaces condensin II in early G1 (Fig. 1 B)(Abramo et al., 2019; Brunner et al., 2025), and establishes topologically associating domains (TADs) in the G1 nucleus (Schwarzer et al., 2017; Wutz et al., 2017)*. *

While there is a loose consensus in the field that condensin II is replaced by cohesin during the M-to-G1 transition, it remains controversial whether there is a short window during which neither condensin II nor cohesin binds to chromatin (Abramo et al., 2019), or whether there is a stage in which the two SMC protein complexes “co-occupy” chromatin (Brunner et al., 2025). Our images shown in the revised Fig. 1B cannot clearly distinguish between these two possibilities.

From a functional point of view, the results of our depletion experiments are more readily explained by the latter possibility. If this is the case, the “interplay” or “cooperation” rather than the “handover” may be a more appropriate term to describe the functional collaboration between condensin II and cohesin during the M-to-G1 transition. For this reason, we have avoided the use of the word “handover” in the revised manuscript. It should be emphasized, however, that given their distinct chromosome-binding kinetics, the cooperation of the two SMC complexes during the M-to-G1 transition is qualitatively different from that observed in G2. Therefore, the central conclusion of the present study remains unchanged.

For example, a sentence in Abstract has been changed as follows:

a functional interplay between condensin II and cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus.

While the reviewer suggested one experiment, it is clearly beyond the scope of the current study. It should also be noted that even if such a cell line were available, the proposed application of sequential depletion to cells progressing from mitosis to G1 phase would be technically challenging and unlikely to produce results that could be interpreted with confidence.

Other points:

Figure 2E: It seems that the chromosome length without IAA is shorter in Rad21-aid cells than H2-aid cells or H2-aid Rad21-aid cells. How can this be interpreted? This comment is well taken. A related comment was made by Reviewer #3 (Major comment #2). Given the substantial genetic manipulations applied to establish multiple cell lines used in the present study, it is, strictly speaking, not straightforward to compare the -IAA controls between different cell lines. Such variations are most prominently observed in Fig. 2E, although they can also be observed to lesser extent in other experiments (e.g., Fig. 3E). This issue is inherently associated with all studies using genetically manipulated cell lines and therefore cannot be completely avoided. For this reason, we focus on the differences between -IAA and +IAA within each cell line, rather than comparing the -IAA conditions across different cell lines. In this sense, a sentence in the original manuscript (lines 178-180) was misleading. In the revised manuscript, we have modified the corresponding and subsequent sentence as follows:

Although cohesin depletion had a marginal effect on the distance between the two site-specific probes (Fig.2, C and E), double depletion did not result in a significant change (Fig.2, D and E), consistent with the partial restoration of centromere dispersion (Fig. 1G).

• *

In addition, we have added a section entitled “Limitations of the study” at the end of the Discussion to address technical issues that are inevitably associated with the current approach.

Figure 3: Regarding the CT morphology, could they explain further the difference between 'elongated' and 'cloud-like (expanded)'? Is it possible to quantify the frequency of these morphologies? In the original manuscript, we provided data that quantitatively distinguished between the “elongated” and “cloud-like” phenotypes. Specifically, Fig. 2E shows that the distance between two specific loci (Cen 12 and 12q15) is increased in the elongated phenotype but not in the cloud-like phenotype. In addition, the cloud-like morphology was clearly deviated from circularity, as indicated by the circularity index (Fig. 3F). However, because circularity can also decrease in rod-shaped chromosomes, these datasets alone may not be sufficiently convincing, as the reviewer pointed out. We have now included an additional parameter, the aspect ratio, defined as the ratio of an object’s major axis to its minor axis (new Fig. 3F). While this intuitive parameter was altered upon condensin II depletion and double depletion, again, we acknowledge that it is not sufficient to convincingly distinguish between the elongated and cloud-like phenotypes proposed in the original manuscript. For these reasons, in the revised manuscript, we have toned down our statements regarding the differences in CT morphology between the two conditions. Nonetheless, together with the data from Figs. 1 and 2, it is that the Rabl configuration observed upon condensin II depletion is further exacerbated in the absence of cohesin. Accordingly, we have modified the main text and the cartoon (Fig 3H) to more accurately depict the observations summarized above.

Figure 5: How did they assign C, P and D3 for two chromosomes? The assignment seems obvious in some cases, but not in other cases (e.g. in the image of H2-AID#2 +IAA, two D3s can be connected to two Ps in the other way). They may have avoided line crossing between two C-P-D3 assignments, but can this be justified when the CT might be disorganized e.g. by condensin II depletion? This comment is well taken. As the reviewer suspected, we avoided line crossing between two sets of assignments. Whenever there was ambiguity, such images were excluded from the analysis. Because most chromosome territories derived from two homologous chromosomes are well separated even under the depleted conditions as shown in Fig. 6C, we did not encounter major difficulties in making assignments based on the criteria described above. We therefore remain confident that our conclusion is valid.

That said, we acknowledge that our assignments of the FISH images may not be entirely objective. We have added this point to the “Limitations of the study” section at the end of the Discussion.

Figure 6F: The mean is not indicated on the right-hand side graph, in contrast to other similar graphs. Is this an error? We apologize for having caused this confusion. First, we would like to clarify that the right panel of Fig. 6F should be interpreted together with the left panel, unlike the seemingly similar plots shown in Figs. 6G and 6H. In the left panel of Fig. 6F, the percentages of CTs that contact the nucleolus are shown in grey, whereas those that do not are shown in white. All CTs classified in the “non-contact” population (white) have a value of zero in the right panel, represented by the bars at 0 (i.e., each bar corresponds to a collection of dots having a zero value). In contrast, each CT in the “contact” population (grey) has a unique contact ratio value in the right panel. Because the right panel consists of two distinct groups, we reasoned that placing mean or median bars would not be appropriate. This was why no mean or median bars were shown in in the tight panel (The same is true for Fig. S5 A and B).

That said, for the reviewer’s reference, we have placed median bars in the right panel (see below). In the six cases of H2#2 (-/+IAA), Rad21#2 (-/+IAA), Double#2 (-IAA), and Double#3 (-IAA), the median bars are located at zero (note that in these cases the mean bars [black] completely overlap with the “bars” derived from the data points [blue and magenta]). In the two cases of Double#2 (+IAA) and Double#3 (+IAA), they are placed at values of ~0.15. Statistically significant differences between -IAA and +IAA are observed only in Double#2 and Double#3, as indicated by the P-value shown on the top of the panel. Thus, we are confident in our conclusion that CTs undergo severe deformation in the absence of both condensin II and cohesin.

Figure S1A: The two FACS profiles for Double-AID #3 Release-2 may be mixed up between -IAA and +IAA. The review is right. This inadvertent error has been corrected.

The method section explains that 'circularity' shows 'how closely the shape of an object approximates a perfect circle (with a value of 1 indicating a perfect circle), calculated from the segmented regions'. It would be helpful to provide further methodological details about it. We have added further explanations regarding the circularity in Materials and Methods together with a citation (two added sentences are underlined below):

To analyze the morphology of nuclei, CTs, and nucleoli, we measured “circularity,” a morphological index that quantifies how closely the shape of an object approximates a perfect circle (value =1). Circularity was defined as 4π x Area/Perimeter2, where both the area and perimeter of each segmented object were obtained using ImageJ. This index ranges from 0 to 1, with values closer to 1 representing more circular objects and lower values correspond to elongated or irregular shapes (Chen et al, 2017).

Chen, B., Y. Wang, S. Berretta and O. Ghita. 2017. Poly Aryl Ether Ketones (PAEKs) and carbon-reinforced PAEK powders for laser sintering. J Mater Sci 52:6004-6019.

Reviewer #1 (Significance (Required)):

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

See our reply above.

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

Summary:

Ono et al use a variety of imaging and genetic (AID) depletion approaches to examine the roles of condensin II and cohesin in the reformation of interphase genome architecture in human HCT16 cells. Consistent with previous literature, they find that condensin II is required for CENP-A dispersion in late mitosis/early G1. Using in situ FISH at the centromere/q arm of chromosome 12 they then establish that condensin II removal causes lengthwise elongation of chromosomes that, interestingly, can be suppressed by cohesin removal. To better understand changes in whole-chromosome morphology, they then use whole chromosome painting to examine chromosomes 18 and 19. In the absence of condensin II, cells effectively fail to reorganise their chromosomes from rod-like structures into spherical chromosome territories (which may explain why CENP-A dispersion is suppressed). Cohesin is not required for spherical CT formation, suggesting condensin II is the major initial driver of interphase genome structure. Double depletion results in complete disorganisation of chromatin, leading the authors to conclude that a typical cell cycle requires orderly 'handover' from the mitotic to interphase genome organising machinery. The authors then move on to G2 phase, where they use a variety of different FISH probes to assess alterations in chromosome structure at different scales. They thereby establish that perturbation of cohesin or condensin II influences local and longer range chromosome structure, respectively. The effects of condensin II depletion become apparent at a genomic distance of 20 Mb, but are negligible either below or above. The authors repeat the G1 depletion experiment in G2 and now find that condensin II and cohesin are individually dispensable for CT organisation, but that dual depletion causes CT collapse. This rather implies that there is cooperation rather than handover per se. Overall this study is a broadly informative multiscale investigation of the roles of SMC complexes in organising the genome of postmitotic cells, and solidifies a potential relationship between condensin II and cohesin in coordinating interphase genome structure. The deeper investigation of the roles of condensin II in establishing chromosome territories and intermediate range chromosome structure in particular is a valuable and important contribution, especially given our incomplete understanding of what functions this complex performs during interphase.

We sincerely appreciate the reviewer’s supportive comments. The reviewer has correctly acknowledged both the current gaps in our understanding of the role of condensin II in interphase chromosome organization and our new findings on the collaborative roles of condensin II and cohesin in establishing and maintaining interphase chromosome territories.

Major comments:

In general the claims and conclusions of the manuscript are well supported by multiscale FISH labelling. An important absent control is western blotting to confirm protein depletion levels. Currently only fluorescence is used as a readout for the efficiency of the AID depletion, and we know from prior literature that even small residual quantities of SMC complexes are quite effective in organising chromatin. I would consider a western blot a fairly straightforward and important technical control.

Let me explain why we used immunofluorescence measurements to evaluate the efficiency of depletion. In our current protocol for synchronizing at the M-to-G1 transition, ~60% of control and H2-depleted cells, and ~30% of Rad21-depleted and co-depleted cells, are successfully synchronized in G1 phase. The apparently lower synchronization efficiency in the latter two groups is attributable to the well-documented mitotic delay caused by cohesin depletion. From these synchronized populations, early G1 cells were selected based on their characteristic morphologies (see the legend of Fig. 1C). In this way, we analyzed an early G1 cell population that had completed mitosis without chromosome segregation defects. We acknowledge that this represents a technically challenging aspect of M-to-G1 synchronization in HCT116 cells, whose synchronization efficiency is limited compared with that of HeLa cells. Nevertheless, this approach constitutes the most practical strategy currently available. Hence, immunofluorescence provides the only feasible means to evaluate depletion efficiency under these conditions.

Although immunoblotting can, in principle, be applied to G2-arrested cell populations, we do not believe that information obtained from such experiments would affect the main conclusions of the current study. Please note that we carefully designed and performed all experiments with appropriate controls: H2 depletion, RAD21 depletion, and double depletion, with outcomes confirmed using independent cell lines (Double-AID#2 and Double-AID#3) whenever deemed necessary.

We fully acknowledge the technical limitations associated with the AID-mediated depletion techniques, which are now described in the section entitled “Limitations of the study” at the end of the Discussion. Nevertheless, we emphasize that these limitations do not compromise the validity of our findings.

I find the point on handover as a mechanism for maintaining CT architecture somewhat ambiguous, because the authors find that the dependence simply switches from condensin II to both condensin II and cohesin, between G1 and G2. To me this implies augmented cooperation rather than handover. I have two further suggestions, both of which I would strongly recommend but would consider desirable but 'optional' according to review commons guidelines.

First of all, we would like to clarify a possible misunderstanding regarding the phrase “handover as a mechanism for maintaining CT architecture somewhat ambiguous”. In the original manuscript, we proposed handover as a mechanism for establishing G1 chromosome territories, not for maintaining CTs.

That said, we take this comment very seriously, especially because Reviewer #1 also expressed the same concern. Please see our reply to Reviewer #1 (Major point).

In brief, we agree with the reviewer that the word “handover” may not be appropriate to describe the functional relationship between condensin II and cohesin during the M-to-G1 transition. In the revised manuscript, we have avoided the use of the word “handover”, replacing it with “interplay”. It should be emphasized, however, that given their distinct chromosome-binding kinetics, the cooperation of the two SMC complexes during the M-to-G1 transition is qualitatively different from that observed in G2. Therefore, the central conclusion of the present study remains unchanged.

For example, a sentence in Abstract has been changed as follows:

a functional interplay between condensin II and cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus.

Firstly, the depletions are performed at different stages of the cell cycle but have different outcomes. The authors suggest this is because handover is already complete, but an alternative possibility is that the phenotype is masked by other changes in chromosome structure (e.g. duplication/catenation). I would be very curious to see, for example, how the outcome of this experiment would change if the authors were to repeat the depletions in the presence of a topoisomerase II inhibitor.

The reviewer’s suggestion here is somewhat vague, and it is unclear to us what rationale underlies the proposed experiment or what meaningful outcomes could be anticipated. Does the reviewer suggest that we perform topo II inhibitor experiments both during the M-to-G1 transition and in G2 phase, and then compare the outcomes between the two conditions?

For the M-to-G1 transition, Hildebrand et at (2024) have already reported such experiments. They used a topo II inhibitor to provided evidence that mitotic chromatids are self-entangled and that the removal of these mitotic entanglements is required to establish a normal interphase nucleus. Our own preliminary experiments (not presented in the current manuscript) showed that ICRF treatment of cells undergoing the M-to-G1 transition did not affect post-mitotic centromere dispersion. The same treatment also had little effect on the suppression of centromere dispersion observed in condensin II-depleted cells.

Under G2-arrested condition, because chromosome territories are largely individualized, we would expect topo II inhibition to affect only the extent of sister catenation, which is not the focus of our current study. We anticipate that inhibiting topo II in G2 would have only a marginal, if any, effect on the maintenance of chromosome territories detectable by our current FISH approaches.

In any case, we consider the suggested experiment to be beyond the scope of the present manuscript, which focuses on the collaborative roles of condensin II and cohesin as revealed by multi-scale FISH analyses.

Secondly, if the author's claim of handover is correct then one (not exclusive) possibility is that there is a relationship between condensin II and cohesin loading onto chromatin. There does seem to be a modest co-dependence (e.g. fig S4 and S7), could the authors comment on this?

First of all, we wish to point out the reviewer’s confusion between the G2 experiments and the M-to-G1 experiments. Figs. S4 and S7 concern experiments using G2-arrested cells, not M-to-G1 cells in which a possible handover mechanism is discussed. Based on Fig. 1, in which the extent of depletion in M-to-G1 cells was tested, no evidence of “co-dependence” between H2 depletion and RAD21 depletion was observed.

That said, as the reviewer correctly points out, we acknowledge the presence of marginal yet statistically significant reductions in the RAD21 signal upon H2 depletion (and vice versa) in G2-arrested cells (Figs. S4 and S7).

Another control experiment here would be to treat fully WT cells with IAA and test whether non-AID labelled H2 or RAD21 dip in intensity. If they do not, then perhaps there's a causal relationship between condensin II and cohesin levels?

According to the reviewer’s suggestion, we tested whether IAA treatment causes an unintentional decreases in the H2 or RAD21 signals in G2-arrested cells, and found that it is not the case (see the attached figure below).

Thus, these data indicate that there is a modest functional interdependence between condensin II and cohesin in G2-arrested cells. For instance, condensin II depletion may modestly destabilize chromatin-bound cohesin (and vice versa). However, we note that these effects are minor and do not affect the overall conclusions of the study. In the revised manuscript, we have described these potentially interesting observations briefly as a note in the corresponding figure legends (Fig. S4).

I recognise this is something considered in Brunner et al 2025 (JCB), but in their case they depleted SMC4 (so all condensins are lost or at least dismantled). Might bear further investigation.

Methods:

Data and methods are described in reasonable detail, and a decent number of replicates/statistical analyses have been. Documentation of the cell lines used could be improved. The actual cell line is not mentioned once in the manuscript. Although it is referenced, I'd recommend including the identity of the cell line (HCT116) in the main text when the cells are introduced and also in the relevant supplementary tables. Will make it easier for readers to contextualise the findings.

We apologize for the omission of important information regarding the parental cell line used in the current study. The information has been added to Materials and Methods as well as the resource table.

Minor comments:

Overall the manuscript is well-written and well presented. In the introduction it is suggested that no experiment has established a causal relationship between human condensin II and chromosome territories, but this is not correct, Hoencamp et al 2021 (cell) observed loss of CTs after condensin II depletion. Although that manuscript did not investigate it in as much detail as the present study, the fundamental relationship was previously established, so I would encourage the authors to revise this statement.

We are somewhat puzzled by this comment. In the original manuscript, we explicitly cited Hoencamp et al (2021) in support of the following sentences:

• *

(Lines 78-83 in the original manuscript)

*Moreover, high-throughput chromosome conformation capture (Hi-C) analysis revealed that, under such conditions, chromosomes retain a parallel arrangement of their arms, reminiscent of the so-called Rabl configuration (Hoencamp et al., 2021). These findings indicate that the loss or impairment of condensin II during mitosis results in defects in post-mitotic chromosome organization. *

• *

That said, to make the sentences even more precise, we have made the following revision in the manuscript.

• *

(Lines 78- 82 in the revised manuscript)

*Moreover, high-throughput chromosome conformation capture (Hi-C) analysis revealed that, under such conditions, chromosomes retain a parallel arrangement of their arms, reminiscent of the so-called Rabl configuration (Hoencamp et al., 2021). These findings,together with cytological analyses of centromere distributions, indicate that the loss or impairment of condensin II during mitosis results in defects in post-mitotic chromosome organization. *

• *

The following statement was intended to explain our current understanding of the maintenance of chromosome territories. Because Hoencamp et al (2021) did not address the maintenance of CTs, we have kept this sentence unchanged.

• *

(Lines 100-102 in the original manuscript)

Despite these findings, there is currently no evidence that either condensin II, cohesin, or their combined action contributes to the maintenance of CT morphology in mammalian interphase cells (Cremer et al., 2020).

• *

• *

Reviewer #2 (Significance (Required)):

General assessment:

Strengths: the multiscale investigation of genome architecture at different stages of interphase allow the authors to present convincing and well-analysed data that provide meaningful insight into local and global chromosome organisation across different scales.

Limitations:

As suggested in major comments.

Advance:

Although the role of condensin II in generating chromosome territories, and the roles of cohesin in interphase genome architecture are established, the interplay of the complexes and the stage specific roles of condensin II have not been investigated in human cells to the level presented here. This study provides meaningful new insight in particular into the role of condensin II in global genome organisation during interphase, which is much less well understood compared to its participation in mitosis.

Audience:

Will contribute meaningfully and be of interest to the general community of researchers investigating genome organisation and function at all stages of the cell cycle. Primary audience will be cell biologists, geneticists and structural biochemists. Importance of genome organisation in cell/organismal biology is such that within this grouping it will probably be of general interest.

My expertise is in genome organization by SMCs and chromosome segregation.

We appreciate the reviewer’s supportive comments. As the reviewer fully acknowledges, this study is the first systematic survey of the collaborative role of condensin II and cohesin in establishing and maintaining interphase chromosome territories. In particular, multi-scale FISH analyses have enabled us to clarify how the two SMC protein complexes contribute to the maintenance of G2 chromosome territories through their actions at different genomic scales. As the reviewer notes, we believe that the current study will appeal to a broad readership in cell and chromosome biology. The limitations of the current study mentioned by the reviewer are addressed in our reply above.

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

Summary:

The manuscript “Condensin II collaborates with cohesin to establish and maintain interphase chromosome territories" investigates how condensin II and cohesin contribute to chromosome organization during the M-to-G1 transition and in G2 phase using published auxin-inducible degron (AID) cell lines which render the respective protein complexes nonfunctional after auxin addition. In this study, a novel degron cell line was established that enables the simultaneous depletion of both protein complexes, thereby facilitating the investigation of synergistic effects between the two SMC proteins. The chromosome architecture is studied using fluorescence in situ hybridization (FISH) and light microscopy. The authors reproduce a number of already published data and also show that double depletion causes during the M-to-G1 transition defects on chromosome territories, producing expanded, irregular shapes that obscure condensin II-specific phenotypes. Findings in G2 cells point to a new role of condensin II for chromosome conformation at a scale of ~20Mb. Although individual depletion has minimal effects on large-scale CT morphology in G2, combined loss of both complexes produces marked structural abnormalities, including irregular crescent-shaped CTs displaced toward the nucleolus and increased nucleolus-CT contact. The authors propose that condensin II and cohesin act sequentially and complementarily to ensure proper post-mitotic CT formation and maintain chromosome architecture across genomic scales.

We greatly appreciate the reviewer’s supportive comments. The reviewer has accurately recognized our new findings concerning the collaborative roles of condensin II and cohesin in the establishment and maintenance of interphase chromosome territories.

Concenrs about statistics:

• The authors provide the information on how many cells are analyzed but not the number of independent experiments. My concern is that there might variations in synchronization of the cell population and in the subsequent preparation (FISH) affecting the final result. We appreciate the reviewer’s important comment regarding the biological reproducibility of our experiments. As the reviewer correctly points out, variations in cell-cycle synchronization and FISH sample preparation can occur across experiments. To address this concern, we repeated the key experiments supporting our main conclusions (Figs. 3 and 6) two additional times, resulting in three independent biological replicas in total. All replicate experiments reproduced the major observations from the original analyses. These results further substantiated our original conclusion, despite the inevitable variability arising from cell synchronization or sample preparation in this type of experiments. In the revised manuscript, we have now explicitly indicated the number of biological replicates in the corresponding figures.

The analyses of chromosome-arm conformation shown in Fig. 5 were already performed in three independent rounds of experiments, as noted in the original submission. In addition, similar results were already obtained in other analyses reported in the manuscript. For example, centromere dispersion was quantified using an alternative centromere detection method (related to Fig. 1), and distances between specific chromosomal sites were measured using different locus-specific probes (related to Figs. 2 and 4). In both cases, the results were consistent with those presented in the manuscript.

• Statistically the authors analyze the effect of cells with induced degron vs. vehicle control (non-induced). However, the biologically relevant question is whether the data differ between cell lines when the degron system is induced. This is not tested here (cf. major concern 2 and 3). See our reply to major concerns 2 and 3.

• Some Journal ask for blinded analysis of the data which might make sense here as manual steps are involved in the data analysis (e.g. line 626 / 627the convex hull of the signals was manually delineated, line 635 / 636 Chromosome segmentation in FISH images was performed using individual thresholding). However personally I have no doubts on the correctness of the work. We thank the reviewer for pointing out that some steps in our data analysis were performed manually, such as delineating the convex hull of signals and segmenting chromosomes in FISH and IF images using individual thresholds. These manual steps were necessary because signal intensities vary among cells and chromosomes, making fully automated segmentation unreliable. To ensure objectivity, we confirmed that the results were consistent across two independently established double-depletion cell lines, which produced essentially identical findings. In addition, we repeated the key experiments underpinning our main conclusions (Figs. 3 and 6) two additional times, and the results were fully consistent with the original analyses. Therefore, we are confident that our current data analysis approach does not compromise the validity of our conclusions. Finally, we appreciate the reviewer’s kind remark that there is no doubt regarding the correctness of our work.

Major concerns:

• Degron induction appears to delay in Rad21-AID#1 and Double-AID#1 cells the transition from M to G1, as shown in Fig. S1. After auxin treatment, more cells exhibit a G2 phenotype than in an untreated population. What are the implications of this for the interpretation of the experiments? In our protocol shown in Fig. 1C, cells were released into mitosis after G2 arrest, and IAA was added 30 min after release. It is well established that cohesin depletion causes a prometaphase delay due to spindle checkpoint activation (e.g., Vass et al, 2003, Curr Biol; Toyoda and Yanagida, 2006, MBoC; Peters et al, 2008, Genes Dev), which explains why cells with 4C DNA content accumulated, as judged by FACS (Fig. S1). The same was true for doubly depleted cells. However, a fraction of cells that escaped this delay progressed through mitosis and enter the G1 phase of the next cell cycle. We selected these early G1 cells and used them for down-stream analyses. This experimental procedure was explicitly described in the legends of Fig. 1C and Fig. S1A as follows:

(Lines 934-937; Legend of Fig. 1C)

From the synchronized populations, early G1cells were selected based on their characteristic morphologies (i.e., pairs of small post-mitotic cells) and subjected to downstream analyses. Based on the measured nuclear sizes (Fig. S2 G), we confirmed that early G1 cells were appropriately selected.

(Lines 1114-1119; Legend of Fig. S1A)

In this protocol, ~60% of control and H2-depleted cells, and ~30% of Rad21-depleted and co-depleted cells, were successfully synchronized in G1 phase. The apparently lower synchronization efficiency in the latter two groups is attributable to the well documented mitotic delay caused by cohesin depletion (Hauf et al., 2005; Haarhuis et al., 2013; Perea-Resa et al., 2020). From these synchronized populations, early G1 cells were selected based on their characteristic morphologies (see the legend of Fig. 1 C).

• *

Thus, using this protocol, we analyzed an early G1 cell population that had completed mitosis without chromosome segregation defects. We acknowledge that this represents a technically challenging aspect of synchronizing cell-cycle progression from M to G1 in HCT116 cells, whose synchronization efficiency is limited compared with that of HeLa cells. Nevertheless, this approach constitutes the most practical strategy currently available.

• Line 178 "In contrast, cohesin depletion had a smaller effect on the distance between the two site-specific probes compared to condensin II depletion (Fig. 2, C and E)." The data in Fig. 2 E show both a significant effect of H2 and a significant effect of RAD21 depletion. Whether the absolute difference in effect size between the two conditions is truly relevant is difficult to determine, as the distribution of the respective control groups also appears to be different. This comment is well taken. Reviewer #1 has made a comment on the same issue. See our reply to Reviewer #1 (Other points, Figure 2E).

In brief, in the current study, we should focus on the differences between -IAA and +IAA within each cell line, rather than comparing the -IAA conditions across different cell lines. In this sense, a sentence in the original manuscript (lines 178-180) was misleading. In the revised manuscript, we have modified the corresponding and subsequent sentence as follows:

Although cohesin depletion had a marginal effect on the distance between the two site-specific probes (Fig.2, C and E), double depletion did not result in a significant change (Fig.2, D and E), consistent with the partial restoration of centromere dispersion (Fig. 1G).

• In Figures 3, S3 and related text in the manuscript I cannot follow the authors' argumentation, as H2 depletion alone leads to a significant increase in the CT area (Chr. 18, Chr. 19, Chr. 15). Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion). Here, too, appropriate statistical tests or more suitable parameters describing the effect should be used. I also cannot fully follow the argumentation regarding chromosome elongation, as double depletion in Chr. 18 and Chr. 19 also leads to a significantly reduced circularity. Therefore, the schematic drawing Fig. 3 H (double depletion) seems very suggestive to me. This comment is related to the comment above (Major comment #2). See our reply to Reviewer #1 (Other points, Figure 2E).

It should be noted that, in Figure 3 (unlike in Figure 2), we did not compare the different magnitudes of the effect observed between H2 depletion and double depletion. Thus, the reviewer’s comment that “Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion) ” does not accurately reflected our description.

Moreover, while the distance between two specific loci (Fig. 2E) and CT circularity (Fig. 3G) are intuitively related, they represent distinct parameters. Thus, it is not unexpected that double depletion resulted in apparently different outcomes for the two measurements. Thus, the reviewer’s counter-argument is not strictly applicable here.

That said, we agree with the reviewer that our descriptions here need to be clarified.

The differences between H2 depletion and double depletion are two-fold: (1) centromere dispersion is suppressed upon H2 depletion, but not upon double depletion (Fig 1G); (2) the distance between Cen 12 and 12q15 increased upon H2 depletion, but not upon double depletion (Fig 2E).

We have decided to remove the “homologous pair overlap” panel (formerly Fig. 3E) from the revised manuscript. Accordingly, the corresponding sentence has been deleted from the main text. Instead, we have added a new panel of “aspect ratio”, defined as the ratio of the major to the minor axis (new Fig. 3F). While this intuitive parameter was altered upon condensin II depletion and double depletion, again, we acknowledge that it is not sufficient to convincingly distinguish between the elongated and cloud-like phenotypes proposed in the original manuscript. For these reasons, in the revised manuscript, we have toned down our statements regarding the differences in CT morphology between the two conditions. Nonetheless, together with the data from Figs. 1 and 2, it is clear that the Rabl configuration observed upon condensin II depletion is further exacerbated in the absence of cohesin. Accordingly, we have modified the main text and the cartoon (Fig 3H) to more accurately depict the observations summarized above.

• 5 and accompanying text. I agree with the authors that this is a significant and very interesting effect. However, I believe the sharp bends is in most cases an artifact caused by the maximum intensity projection. I tried to illustrate this effect in two photographs: Reviewer Fig. 1, side view, and Reviewer Fig. 2, same situation top view (https://cloud.bio.lmu.de/index.php/s/77npeEK84towzJZ). As I said, in my opinion, there is a significant and important effect; the authors should simply adjust the description. This comment is well taken. We appreciate the reviewer’s effort to help clarify our original observations. We have therefore added a new section entitled “Limitations of the study” to explicitly describe the constrains of our current approach. That said, as the reviewer also acknowledges, our observations remain valid because all experiments were performed with appropriate controls.

Minor concerns:

• I would like to suggest proactively discussing possible artifacts that may arise from the harsh conditions during FISH sample preparation. We fully agree with the reviewer’s concerns. For FISH sample preparation, we used relatively harsh conditions, including (1) fixation under a hypotonic condition (0.3x PBS), (2) HCl treatment, and (3) a denaturation step. We recognize that these procedures inevitably affect the preservation of the original structure; however, they are unavoidable in the standard FISH protocol. We also acknowledge that our analyses were limited to 2D structures based on projected images, rather than full 3D reconstructions. These technical limitations are now explicitly described in a new section entitled “Limitations of the study”, and the technical details are provided in Materials and Methods.

• It would be helpful if the authors could provide the original data (microscopic image stacks) for download. We thank the reviewer for this suggestion and understand that providing the original image stacks could be of interest to readers. We agree that if the nuclei were perfectly spherical, as is the case for example in lymphocytes, 3D image stacks would contain much more information than 2D projections. However, as is typical for adherent cultured cells, including the HCT116-derived cells used in this study, the nuclei are flattened due to cell adhesion to the culture dish, with a thickness of only about one-tenth of the nuclear diameter (10–20 μm). Considering also the inevitable loss of structural preservation during FISH sample preparation, we were concerned that presenting 3D images might confuse rather than clarify. We therefore believe that representing the data as 2D projections, while explicitly acknowledging the technical limitations, provides the clearest and most interpretable presentation of our results. These limitations are now described in a new section of the manuscript.

• The authors use a blind deconvolution algorithm to improve image quality. It might be helpful to test other methods for this purpose (optional). We thank the reviewer for this valuable suggestion and fully agree that it is a valid point. We recognize that alternative image enhancement methods can offer advantages, particularly for smaller structures or when multiple probes are analyzed simultaneously. In our study, however, the focus was on detecting whole chromosome territories (CTs) and specific chromosomal loci, which can be visualized clearly with our current FISH protocol combined with blind deconvolution. We therefore believe that the image quality we obtained is sufficient to support the conclusions of this manuscript.

Reviewer #3 (Significance (Required)):

Advance:

Ono et al. addresses the important question on how the complex pattern of chromatin is reestablished after mitosis and maintained during interphase. In addition to affinity interactions (1,2), it is known that cohesin plays an important role in the formation and maintenance of chromosome organization interphase (3). However, current knowledge does not explain all known phenomena. Even with complete loss of cohesin, TAD-like structures can be recognized at the single-cell level (4), and higher structures such as chromosome territories are also retained (5). The function of condensin II during mitosis is another important factor that affects chromosome architecture in the following G1 phase (6). Although condensin II is present in the cell nucleus throughout interphase, very little is known about the role of this protein in this phase of the cell cycle. This is where the present publication comes in, with a new double degron cell line in which essential subunits of cohesin AND condensin can be degraded in a targeted manner. I find the data from the experiments in the G2 phase most interesting, as they suggest a previously unknown involvement of condensin II in the maintenance of larger chromatin structures such as chromosome territories.

The experiments regarding the M-G1 transition are less interesting to me, as it is known that condensin II deficiency in mitosis leads to elongated chromosomes (Rabl configuration)(6), and therefore the double degradation of condensin II and cohesin describes the effects of cohesin on an artificially disturbed chromosome structure.

For further clarification, we provide below a table summarizing previous studies relevant to the present work. We wish to emphasize three novel aspects of the present study. First, newly established cell lines designed for double depletion enabled us to address questions that had remained inaccessible in earlier studies. Second, to our knowledge, no study has previously reported condensin II depletion, cohesin depletion and double depletion in G2-arrested cells. Third, the present study represents the first systematic comparison of two different stages of the cell cycle using multiscale FISH under distinct depletion conditions. Although the M-to-G1 part of the present study partially overlaps with previous work, it serves as an important prelude to the subsequent investigations. We are confident that the reviewer will also acknowledge this point.

cell cycle

cond II depletion

cohesin depletion

double depletion

M-to-G1

Hoencamp et al (2021); Abramo et al (2019); Brunner et al (2025);

this study

Schwarzer et al (2017);

Wutz et al (2017);

this study

this study

G2

this study

this study

this study

Hoencamp et al (2021): Hi-C and imaging (CENP-A distribution)

Abramo et al (2019): Hi-C and imaging

Brunner et al (2025): mostly imaging (chromatin tracing)

Schwarzer et al (2017); Wutz et al (2017): Hi-C

this study: imaging (multi-scale FISH)

General limitations:

(1) Single cell imaging of chromatin structure typically shows only minor effects which are often obscured by the high (biological) variability. This holds also true for the current manuscript (cf. major concern 2 and 3).

See our reply above.

(2) A common concern are artefacts introduced by the harsh conditions of conventional FISH protocols (7). The authors use a method in which the cells are completely dehydrated, which probably leads to shrinking artifacts. However, differences between samples stained using the same FISH protocol are most likely due to experimental variation and not an artefact (cf. minor concern 1).

See our reply above.

• The anisotropic optical resolution (x-, y- vs. z-) of widefield microscopy (and most other light microscopic techniques) might lead to misinterpretation of the imaged 3D structures. This seems to be the cases in the current study (cf. major concern 4). See our reply above.

• In the present study, the cell cycle was synchronized. This requires the use of inhibitors such as the CDK1 inhibitor RO-3306. However, CDK1 has many very different functions (8), so unexpected effects on the experiments cannot be ruled out. The current approaches involving FISH inevitably require cell cycle synchronization. We believe that the use of the CDK1 inhibitor RO-3306 to arrest the cell cycle at G2 is a reasonable choice, although we cannot rule out unexpected effects arising from the use of the drug. This issue has now been addressed in the new section entitled “Limitations of the study”.

Audience:

The spatial arrangement of genomic elements in the nucleus and their (temporal) dynamics are of high general relevance, as they are important for answering fundamental questions, for example, in epigenetics or tumor biology (9,10). The manuscript from Ono et al. addresses specific questions, so its intended readership is more likely to be specialists in the field.

We are confident that, given the increasing interest in the 3D genome and its role in regulating diverse biological functions, the current manuscript will attract the broad readership of leading journals in cell biology.

About the reviewer:

By training I'm a biologist with strong background in fluorescence microscopy and fluorescence in situ hybridization. In recent years, I have been involved in research on the 3D organization of the cell nucleus, chromatin organization, and promoter-enhancer interactions.

We greatly appreciate the reviewer’s constructive comments on both the technical strengths and limitations of our fluorescence imaging approaches, which have been very helpful in revising the manuscript. As mentioned above, we have decided to add a special paragraph entitled “Limitations of the study” at the end of the Discussion section to discuss these issues.

All questions regarding the statistics of angularly distributed data are beyond my expertise. The authors do not correct their statistical analyses for "multiple testing". Whether this is necessary, I cannot judge.

We thank the reviewer for raising this important point. In our study, the primary comparisons were made between -IAA and +IAA conditions within the same cell line. Accordingly, the figures report P-values for these pairwise comparisons.

For the distance measurements, statistical evaluations were performed in PRISM using ANOVA (Kruskal–Wallis test), and the P-values shown in the figures are based on these analyses (Fig. 1, G and H; Fig. 2 E; Fig. 3 F and G; Fig. 4 F; Fig. 6 F [right]–H; Fig. S2 B and G; Fig. S3 D and H; Fig. S5 A [right] and B [right]; Fig. S8 B). While the manuscript focuses on pairwise comparisons between -IAA and +IAA conditions within the same cell line, we also considered potential differences across cell lines as part of the same ANOVA framework, thereby ensuring that multiple testing was properly addressed. Because cell line differences are not the focus of the present study, the corresponding results are not shown.

For the angular distribution analyses, we compared -IAA and +IAA conditions within the same cell line using the Mardia–Watson–Wheeler test; these analyses do not involve multiple testing (circular scatter plots; Fig. 5 C–E and Fig. S6 B, C, and E–H). In addition, to determine whether angular distributions exhibited directional bias under each condition, we applied the Rayleigh test to each dataset individually (Fig. 5 F and Fig. S6 I). As these tests were performed on a single condition, they are also not subject to the problem of multiple testing. Collectively, we consider that the statistical analyses presented in our manuscript appropriately account for potential multiple testing issues, and we remain confident in the robustness of the results.

Literature

Falk, M., Feodorova, Y., Naumova, N., Imakaev, M., Lajoie, B.R., Leonhardt, H., Joffe, B., Dekker, J., Fudenberg, G., Solovei, I. et al. (2019) Heterochromatin drives compartmentalization of inverted and conventional nuclei. Nature, 570, 395-399. Mirny, L.A., Imakaev, M. and Abdennur, N. (2019) Two major mechanisms of chromosome organization. Curr Opin Cell Biol, 58, 142-152. Rao, S.S.P., Huang, S.C., Glenn St Hilaire, B., Engreitz, J.M., Perez, E.M., Kieffer-Kwon, K.R., Sanborn, A.L., Johnstone, S.E., Bascom, G.D., Bochkov, I.D. et al. (2017) Cohesin Loss Eliminates All Loop Domains. Cell, 171, 305-320 e324. Bintu, B., Mateo, L.J., Su, J.H., Sinnott-Armstrong, N.A., Parker, M., Kinrot, S., Yamaya, K., Boettiger, A.N. and Zhuang, X. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science, 362. Cremer, M., Brandstetter, K., Maiser, A., Rao, S.S.P., Schmid, V.J., Guirao-Ortiz, M., Mitra, N., Mamberti, S., Klein, K.N., Gilbert, D.M. et al. (2020) Cohesin depleted cells rebuild functional nuclear compartments after endomitosis. Nat Commun, 11, 6146. Hoencamp, C., Dudchenko, O., Elbatsh, A.M.O., Brahmachari, S., Raaijmakers, J.A., van Schaik, T., Sedeno Cacciatore, A., Contessoto, V.G., van Heesbeen, R., van den Broek, B. et al. (2021) 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science, 372, 984-989. Beckwith, K.S., Ødegård-Fougner, Ø., Morero, N.R., Barton, C., Schueder, F., Tang, W., Alexander, S., Peters, J.-M., Jungmann, R., Birney, E. et al. (2023) Nanoscale 3D DNA tracing in single human cells visualizes loop extrusion directly in situ. BioRxiv 8 of 9https://doi.org/10.1101/2021.04.12.439407. Massacci, G., Perfetto, L. and Sacco, F. (2023) The Cyclin-dependent kinase 1: more than a cell cycle regulator. Br J Cancer, 129, 1707-1716. Bonev, B. and Cavalli, G. (2016) Organization and function of the 3D genome. Nat Rev Genet, 17, 661-678. Dekker, J., Belmont, A.S., Guttman, M., Leshyk, V.O., Lis, J.T., Lomvardas, S., Mirny, L.A., O'Shea, C.C., Park, P.J., Ren, B. et al. (2017) The 4D nucleome project. Nature, 549, 219-226.

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Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript „Condensin II collaborates with cohesin to establish and maintain interphase chromosome territories" investigates how condensin II and cohesin contribute to chromosome organization during the M-to-G1 transition and in G2 phase using published auxin-inducible degron (AID) cell lines which render the respective protein complexes nonfunctional after auxin addition. In this study, a novel degron cell line was established that enables the simultaneous depletion of both protein complexes, thereby facilitating the investigation of synergistic effects between the two SMC proteins. The chromosome architecture is studied using fluorescence in situ hybridization (FISH) and light microscopy. The authors reproduce a number of already published data and also show that double depletion causes during the M-to-G1 transition defects on chromosome territories, producing expanded, irregular shapes that obscure condensin II-specific phenotypes. Findings in G2 cells point to a new role of condensin II for chromosome conformation at a scale of ~20Mb. Although individual depletion has minimal effects on large-scale CT morphology in G2, combined loss of both complexes produces marked structural abnormalities, including irregular crescent-shaped CTs displaced toward the nucleolus and increased nucleolus-CT contact. The authors propose that condensin II and cohesin act sequentially and complementarily to ensure proper post-mitotic CT formation and maintain chromosome architecture across genomic scales.

Concerns about statistics:

(1) The authors provide the information on how many cells are analyzed but not the number of independent experiments. My concern is that there might variations in synchronization of the cell population and in the subsequent preparation (FISH) affecting the final result.

(2) Statistically the authors analyze the effect of cells with induced degron vs. vehicle control (non-induced). However, the biologically relevant question is whether the data differ between cell lines when the degron system is induced. This is not tested here (cf. major concern 2 and 3).

(3) Some Journal ask for blinded analysis of the data which might make sense here as manual steps are involved in the data analysis (e.g. line 626 / 627the convex hull of the signals was manually delineated, line 635 / 636 Chromosome segmentation in FISH images was performed using individual thresholding). However personally I have no doubts on the correctness of the work.

Major concerns:

(1) Degron induction appears to delay in Rad21-AID#1 an Double-AID#1 cells the transition from M to G1, as shown in Fig. S1. After auxin treatment, more cells exhibit a G2 phenotype than in an untreated population. What are the implications of this for the interpretation of the experiments?

(2) Line 178 "In contrast, cohesin depletion had a smaller effect on the distance between the two site-specific probes compared to condensin II depletion (Fig. 2, C and E)." The data in Fig. 2 E show both a significant effect of H2 and a significant effect of RAD21 depletion. Whether the absolute difference in effect size between the two conditions is truly relevant is difficult to determine, as the distribution of the respective control groups also appears to be different.

(3) In Figures 3, S3 and related text in the manuscript I cannot follow the authors' argumentation, as H2 depletion alone leads to a significant increase in the CT area (Chr. 18, Chr. 19, Chr. 15). Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion). Here, too, appropriate statistical tests or more suitable parameters describing the effect should be used. I also cannot fully follow the argumentation regarding chromosome elongation, as double depletion in Chr. 18 and Chr. 19 also leads to a significantly reduced circularity. Therefore, the schematic drawing Fig. 3 H (double depletion) seems very suggestive to me.

(4) Fig. 5 and accompanying text. I agree with the authors that this is a significant and very interesting effect. However, I believe the sharp bends is in most cases an artifact caused by the maximum intensity projection. I tried to illustrate this effect in two photographs: Reviewer Fig. 1, side view, and Reviewer Fig. 2, same situation top view (https://cloud.bio.lmu.de/index.php/s/77npeEK84towzJZ). As I said, in my opinion, there is a significant and important effect; the authors should simply adjust the description.

Minor concerns:

(1) I would like to suggest proactively discussing possible artifacts that may arise from the harsh conditions during FISH sample preparation..

(2) It would be helpful if the authors could provide the original data (microscopic image stacks) for download

(3) The authors use a blind deconvolution algorithm to improve image quality. It might be helpful to test other methods for this purpose (optional).

Significance

Advance:

Ono et al. addresses the important question on how the complex pattern of chromatin is reestablished after mitosis and maintained during interphase. In addition to affinity interactions (1,2), it is known that cohesin plays an important role in the formation and maintenance of chromosome organization interphase (3). However, current knowledge does not explain all known phenomena. Even with complete loss of cohesin, TAD-like structures can be recognized at the single-cell level (4), and higher structures such as chromosome territories are also retained (5). The function of condensin II during mitosis is another important factor that affects chromosome architecture in the following G1 phase (6). Although condensin II is present in the cell nucleus throughout interphase, very little is known about the role of this protein in this phase of the cell cycle. This is where the present publication comes in, with a new double degron cell line in which essential subunits of cohesin AND condensin can be degraded in a targeted manner. I find the data from the experiments in the G2 phase most interesting, as they suggest a previously unknown involvement of condensin II in the maintenance of larger chromatin structures such as chromosome territories. The experiments regarding the M-G1 transition are less interesting to me, as it is known that condensin II deficiency in mitosis leads to elongated chromosomes (Rabl configuration)(6), and therefore the double degradation of condensin II and cohesin describes the effects of cohesin on an artificially disturbed chromosome structure.

General limitations:

(1) Single cell imaging of chromatin structure typically shows only minor effects which are often obscured by the high (biological) variability. This holds also true for the current manuscript (cf. major concern 2 and 3).

(2) A common concern are artefacts introduced by the harsh conditions of conventional FISH protocols (7). The authors use a method in which the cells are completely dehydrated, which probably leads to shrinking artifacts. However, differences between samples stained using the same FISH protocol are most likely due to experimental variation and not an artefact (cf. minor concern 1).

(3) The anisotropic optical resolution (x-, y- vs. z-) of widefield microscopy (and most other light microscopic techniques) might lead to misinterpretation of the imaged 3D structures. This seems to be the cases in the current study (cf. major concern 4).

(4) In the present study, the cell cycle was synchronized. This requires the use of inhibitors such as the CDK1 inhibitor RO-3306. However, CDK1 has many very different functions (8), so unexpected effects on the experiments cannot be ruled out.

Audience:

The spatial arrangement of genomic elements in the nucleus and their (temporal) dynamics are of high general relevance, as they are important for answering fundamental questions, for example, in epigenetics or tumor biology (9,10). The manuscript from Ono et al. addresses specific questions, so its intended readership is more likely to be specialists in the field.

About the reviewer: By training I'm a biologist with strong background in fluorescence microscopy and fluorescence in situ hybridization. In recent years, I have been involved in research on the 3D organization of the cell nucleus, chromatin organization, and promoter-enhancer interactions.

All questions regarding the statistics of angularly distributed data are beyond my expertise. The authors do not correct their statistical analyses for "multiple testing". Whether this is necessary, I cannot judge.

Literature

1. Falk, M., Feodorova, Y., Naumova, N., Imakaev, M., Lajoie, B.R., Leonhardt, H., Joffe, B., Dekker, J., Fudenberg, G., Solovei, I. et al. (2019) Heterochromatin drives compartmentalization of inverted and conventional nuclei. Nature, 570, 395-399.
2. Mirny, L.A., Imakaev, M. and Abdennur, N. (2019) Two major mechanisms of chromosome organization. Curr Opin Cell Biol, 58, 142-152.
3. Rao, S.S.P., Huang, S.C., Glenn St Hilaire, B., Engreitz, J.M., Perez, E.M., Kieffer-Kwon, K.R., Sanborn, A.L., Johnstone, S.E., Bascom, G.D., Bochkov, I.D. et al. (2017) Cohesin Loss Eliminates All Loop Domains. Cell, 171, 305-320 e324.
4. Bintu, B., Mateo, L.J., Su, J.H., Sinnott-Armstrong, N.A., Parker, M., Kinrot, S., Yamaya, K., Boettiger, A.N. and Zhuang, X. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science, 362.
5. Cremer, M., Brandstetter, K., Maiser, A., Rao, S.S.P., Schmid, V.J., Guirao-Ortiz, M., Mitra, N., Mamberti, S., Klein, K.N., Gilbert, D.M. et al. (2020) Cohesin depleted cells rebuild functional nuclear compartments after endomitosis. Nat Commun, 11, 6146.
6. Hoencamp, C., Dudchenko, O., Elbatsh, A.M.O., Brahmachari, S., Raaijmakers, J.A., van Schaik, T., Sedeno Cacciatore, A., Contessoto, V.G., van Heesbeen, R., van den Broek, B. et al. (2021) 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science, 372, 984-989.
7. Beckwith, K.S., Ødegård-Fougner, Ø., Morero, N.R., Barton, C., Schueder, F., Tang, W., Alexander, S., Peters, J.-M., Jungmann, R., Birney, E. et al. (2023) Nanoscale 3D DNA tracing in single human cells visualizes loop extrusion directly in situ. BioRxiv https://doi.org/10.1101/2021.04.12.439407.
8. Massacci, G., Perfetto, L. and Sacco, F. (2023) The Cyclin-dependent kinase 1: more than a cell cycle regulator. Br J Cancer, 129, 1707-1716.
9. Bonev, B. and Cavalli, G. (2016) Organization and function of the 3D genome. Nat Rev Genet, 17, 661-678.
10. Dekker, J., Belmont, A.S., Guttman, M., Leshyk, V.O., Lis, J.T., Lomvardas, S., Mirny, L.A., O'Shea, C.C., Park, P.J., Ren, B. et al. (2017) The 4D nucleome project. Nature, 549, 219-226.

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Referee #1

Evidence, reproducibility and clarity

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

Major point:

They propose a functional 'handover' from condensin II to cohesin, for the organization of CTs at the M-to-G1 transition. However, the 'handover', i.e. difference in timing of executing their functions, was not experimentally substantiated. Ideally, they can deplete condensin II and cohesin at different times to prove the 'handover'. However, this would require the use of two different degron tags and go beyond the revision of this manuscript. At least, based on the literature, the authors should discuss why they think condensin II and cohesin should work at different timings in the CT organization.

Other points:

• Figure 2E: It seems that the chromosome length without IAA is shorter in Rad21-aid cells than H2-aid cells or H2-aid Rad21-aid cells. How can this be interpreted?

• Figure 3: Regarding the CT morphology, could they explain further the difference between 'elongated' and 'cloud-like (expanded)'? Is it possible to quantify the frequency of these morphologies?

• Figure 5: How did they assign C, P and D3 for two chromosomes? The assignment seems obvious in some cases, but not in other cases (e.g. in the image of H2-AID#2 +IAA, two D3s can be connected to two Ps in the other way). They may have avoided line crossing between two C-P-D3 assignments, but can this be justified when the CT might be disorganized e.g. by condensin II depletion?

• Figure 6F: The mean is not indicated on the right-hand side graph, in contrast to other similar graphs. Is this an error?

• Figure S1A: The two FACS profiles for Double-AID #3 Release-2 may be mixed up between -IAA and +IAA.

• The method section explains that 'circularity' shows 'how closely the shape of an object approximates a perfect circle (with a value of 1 indicating a perfect circle), calculated from the segmented regions'. It would be helpful to provide further methodological details about it.

Significance

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

In contrast, several high-skilled, traditionally well-paid fields, such as mechanical and electrical engineering, research and development, and events/hospitality management, have seen only modest nominal wage increases. Once inflation is factored in, these professions have experienced real income losses of 3-4%. Even IT and informatics, despite healthy nominal growth of 29%, saw purchasing power gains of just 3%.

Since 2016, the median monthly salary for geriatric nurses has risen 56% from €2,436 to €3,792. Although inflation ate away much of those gains, nurses today can still afford 24% more goods and services than eight years ago. This matters for housing: in cities where rents have remained stable or only moderately increased (Leipzig, Chemnitz, parts of Eastern Germany), these wage gains help essential workers keep up with local living costs. Despite receiving significant raises, care workers still struggle to afford to live in areas where their services are most needed. For instance, in Berlin, they lost 8 m² despite a 60% wage increase, while in Munich, they gained only 3 m² despite a 40% raise.

① (1) Children and adolescents, (1) Çocuklar ve ergenler, ② (2) Children with physical or mental disabilities, (2) Fiziksel veya zihinsel engeli olan çocuklar, ③ (3) Hospitalized patients, including older adults who need to have their teeth cleaned by caregivers, (3) Hastanede yatan hastalar, bakıcılar tarafından dişlerinin temizlenmesi gereken yaşlılar dahil, ④ (4) Patients with fixed orthodontic appliances. (4) Sabit ortodontik apareyleri olan hastalar. ⑤ (5) Patients who want to use powered toothbrushes should be encouraged to do so. (5) Elektrikli diş fırçası kullanmak isteyen hastalar teşvik edilmelidir.

① : Bristles should be 0.2 mm in diameter, 10 mm in length and have rounded tips. ① : Kıllar 0,2 mm çapında, 10 mm uzunluğunda ve yuvarlatılmış uçlu olmalıdır.

② : Handle of the brush should be straight and long enough for the palm to grasp. ② : Fırçanın sapı düz ve avucun kavrayabileceği kadar uzun olmalıdır.

③ : There should be 3-4 rows, each consisting of 5-12 bristle clusters. ③ : Her biri 5-12 kıl demetinden oluşan 3-4 sıra bulunmalıdır.

Effectively dealing with change

Synthèse de la Conférence : Les Chiffres Mesurent-ils l’Essentiel ?

Résumé

Cette conférence inaugurale du cycle "Mesurer la valeur de notre monde" explore la tension croissante entre la quantification omniprésente de la société et la perception d'une perte de valeur.

Les intervenants, issus des mathématiques, de la sondologie, de la comptabilité et de la philosophie, convergent vers une conclusion centrale :

les chiffres, en eux-mêmes, ne mesurent pas l'essentiel.

Leur véritable signification et leur pertinence dépendent entièrement des modèles, des conventions et des hypothèses qui les sous-tendent.

Loin d'être objectifs ou neutres, ces cadres de référence sont le fruit de choix conceptuels, sociaux et souvent politiques, qui méritent un examen critique approfondi.

Les principaux points à retenir sont les suivants :

• La primauté du modèle : Pour le mathématicien Cédric Villani, l'erreur la plus grave ne réside pas dans le calcul, mais dans le modèle de représentation du monde.

Les chiffres ne sont que le produit final d'un raisonnement, de formules et d'hypothèses qui constituent le véritable cœur de l'analyse.

• Le contexte est clé :

Le sondeur Jean-Daniel Lévy insiste sur le fait qu'un chiffre d'opinion isolé est dénué de sens.

La compréhension émerge de l'analyse des tendances ("un film plutôt qu'une photo"), de la segmentation des données et, crucialement, de l'articulation entre les mesures quantitatives et les études qualitatives qui révèlent les logiques profondes des individus.

• La comptabilité comme outil d'action : L'expert-comptable Alexandre Rambaud déconstruit l'idée d'une comptabilité comme miroir objectif de la réalité.

Il propose une vision instrumentale, notamment en comptabilité écologique, où les chiffres ne visent pas à "valoriser" la nature, mais à quantifier les moyens nécessaires à sa préservation pour guider l'action.

• La libération de la domination :

La philosophe Valérie Charolles appelle à se "libérer de la domination des chiffres" en prenant conscience de leur nature construite.

Elle met en lumière "l'innétrisme" (l'illettrisme numérique) qui nous rend vulnérables aux inférences trompeuses et plaide pour une réappropriation citoyenne des conventions (comptables, statistiques, électorales) qui façonnent notre monde.

1. Introduction : La Quantification du Monde

La conférence s'ouvre sur le constat d'une "quantification du monde" généralisée.

Bettina Laville, présidente de l'IEA de Paris, souligne le paradoxe contemporain : alors que tout est mesuré – des sondages d'opinion quotidiens au reporting extra-financier des entreprises, jusqu'aux indicateurs de bonheur – une impression de "perte de valeur" prédomine.

Ce sentiment naît de la crainte que le chiffre, en envahissant tous les domaines, n'efface "la valeur au sens de ce qui justement ne se compte pas".

Ce cycle de cinq conférences a pour ambition d'explorer ce phénomène à travers plusieurs thématiques :

1. Introduction générale (cette séance)

2. La mesure de la nature

3. La mesure des villes

4. La mesure de l'égalité

5. La mesure de la valeur elle-même (bonheur, etc.)

2. La Primauté du Modèle sur le Chiffre : La Perspective du Mathématicien

Cédric Villani, professeur de mathématiques et médaillé Fields, recadre d'emblée le débat en affirmant que l'essence des mathématiques réside dans le raisonnement et non dans le calcul.

Le Raisonnement avant le Calcul

Contrairement à l'image populaire du mathématicien comme "bon calculateur", la discipline, depuis la Grèce antique, se concentre sur "le raisonnement qui mène au calcul, pas dans le résultat lui-même".

À l'ère des ordinateurs, de nombreux mathématiciens excellent dans l'échafaudage de concepts et de relations logiques, même s'ils sont "des brêles en calcul".

Ce qui importe, ce sont les formules, les hypothèses et l'architecture intellectuelle sous-jacente.

Leçons de l'Histoire des Sciences

Cédric Villani illustre sa thèse par deux exemples historiques majeurs où l'erreur ne provenait pas du calcul mais du modèle :

Cas d'Étude

Le Modèle Sous-jacent

L'Erreur et sa Nature

Conclusion

La Définition du Mètre (Révolution Française)

Le mètre est défini comme la 40 millionième partie du tour de la Terre. Un projet scientifico-politique universaliste.

Une erreur de mesure de 0,2 millimètre, vécue comme une "honte" par ses auteurs (Delambre et Méchain).

L'erreur était minuscule, mais elle a tourmenté Méchain toute sa vie.

L'erreur était dans la précision de la mesure, mais le modèle conceptuel était révolutionnaire et a fondé le système d'unités universel.

Le Calcul de l'Âge de la Terre (19e siècle)

Un modèle de refroidissement d'une Terre supposée solide, basé sur les travaux de Fourier.

Une erreur monstrueuse. Le calcul de Lord Kelvin aboutissait à 24 millions d'années, alors que l'âge réel est de 4,5 milliards d'années.

L'erreur venait entièrement du modèle de départ.

La Terre possède un intérieur liquide générant de la convection, ce qui change radicalement les calculs.

Il cite à ce propos Thomas Huxley : "La mathématique peut se comparer à un moulin d'une facture exquise [...] cependant ce que l'on en tire dépend de ce que l'on y a mis [...] des pages de formule ne fourniront pas un résultat fiable à partir de données imprécises."

Les Hypothèses Politiques derrière les Chiffres

Les chiffres utilisés dans le débat public ne sont jamais neutres ; ils reposent sur des hypothèses et des choix, souvent politiques.

• L'objectif de 2 tonnes de carbone par an et par individu : Ce chiffre repose sur une hypothèse politique forte, celle d'une répartition "également à travers tous les citoyens de l'humanité" du droit à émettre du carbone.

• Le calcul de Jean-Marc Jancovici sur les vols en avion : L'idée que chaque personne ne devrait prendre l'avion que quatre ou cinq fois dans sa vie est le résultat d'un calcul basé sur des hypothèses scientifiques et politiques, notamment sur la répartition de cet effort.

• Le rapport Meadows (Club de Rome, 1972) : Ce célèbre modèle du monde reliait cinq grands compartiments (démographie, pollution, industrie, etc.) via 140 équations.

Ses auteurs reconnaissaient eux-mêmes l'impossibilité de modéliser des facteurs essentiels comme "la volonté politique d'agir" ou "le sentiment de justice".

Ce qu'il Reste à Mesurer

Interrogé sur ce qu'il regretterait de ne pas voir mesuré, Cédric Villani évoque le concept de "viscosité" de la société : "tout ce qui dans une société empêche d'agir".

Cela inclut les rapports de pouvoir établis, les lourdeurs administratives, les procédures dilatoires, etc.

Mesurer cette force d'inertie qui dissipe l'énergie du changement serait, selon lui, un indicateur fascinant.

3. L'Opinion en Chiffres : Entre Mesure et Compréhension

Jean-Daniel Lévy, directeur de l'institut Harris Interactive, apporte la perspective du sondeur, en soulignant la complexité cachée derrière les chiffres d'opinion.

L'Immense Partie Immergée des Sondages

Il révèle que les sondages publiés dans les médias représentent moins de 0,1 % de l'activité de son institut.

L'essentiel du travail (99,9 %) est confidentiel et concerne le marketing, l'évaluation de produits ou les études pour des acteurs publics et privés.

Nous sommes donc "sans le savoir entourés de formules mathématiques qui sont appelées à régir notre vie".

Dépasser le Chiffre Unique

Un chiffre de sondage ne doit jamais être considéré comme une "vérité absolue". Pour lui donner du sens, deux approches sont indispensables :

1. Faire un film, pas une photographie : Il est crucial de poser la même question à intervalles réguliers pour observer les dynamiques et les évolutions d'opinion, par exemple sur une réforme comme celle des retraites.

2. Analyser le détail des résultats : La véritable information se trouve dans la segmentation des données (selon le genre, l'âge, la catégorie sociale, la proximité politique, etc.), qui permet de comprendre les fractures et les logiques spécifiques à chaque groupe.

L'Articulation Essentielle du Quantitatif et du Qualitatif

Les chiffres mesurent, mais ne permettent pas toujours de comprendre.

Pour saisir les logiques profondes, il faut recourir à des méthodes qualitatives (groupes de discussion, entretiens).

• Exemple de la réforme des retraites : Les études qualitatives ont révélé que pour beaucoup de Français, le débat ne portait pas sur les retraites elles-mêmes, mais sur le sens et la pénibilité du travail.

• Exemple des valeurs fondamentales : Les enquêtes qualitatives montrent que les grandes éruptions sociales en France se structurent souvent autour de deux notions fondatrices non-explicites : l'égalité (héritage de 1789) et la solidarité/service public (héritage de 1945).

Les Chiffres Invisibles et la Subjectivité de la Mesure

• Les signaux faibles de 2017 : L'analyse des chiffres électoraux de 2017 aurait dû tempérer l'idée d'une adhésion massive au projet d'Emmanuel Macron.

Deux données clés ont été sous-estimées : la baisse de la participation entre les deux tours (un fait inédit hors 1969) et le record absolu de 4 millions de votes blancs ou nuls, signifiant que 12 % des votants présents au second tour refusaient le choix proposé.

• La formulation des questions : Le résultat d'un sondage dépend étroitement de la manière dont la question est posée.

Les cotes de confiance d'Emmanuel Macron peuvent ainsi varier de 29 % à 45 % selon l'institut, car les questions diffèrent subtilement ("faites-vous confiance pour...", "avoir de bonnes idées", "conduire le pays", etc.).

En conclusion, les chiffres sont une "condition nécessaire mais non suffisante". Ils fournissent des repères, mais se fier exclusivement à eux sans analyse contextuelle et qualitative mène à des "erreurs remarquables".

4. La Comptabilité comme Outil d'Action

Alexandre Rambaud, titulaire de la chaire de comptabilité écologique, propose de voir la comptabilité non pas comme une technique de calcul, mais comme un système de représentation et de gouvernance.

Les Quatre Fonctions de la Comptabilité

Le chiffre n'est que la dernière étape du processus comptable, qui repose sur trois fonctions fondamentales préalables :

1. Prendre en compte : Décider de ce qui est important, définir les objets à suivre et les classer dans des catégories.

C'est un acte de représentation et de modélisation.

2. Être comptable de ses actes : Lier les actions à des responsabilités (redevabilité) et en garder la trace.

3. Rendre des comptes : Établir des rapports et des codes pour permettre la discussion et la prise de décision au sein d'une gouvernance.

4. Compter : Utiliser des instruments chiffrés pour rendre la complexité d'une organisation assimilable et gérable.

Mesure Instrumentale contre "Juste Valeur"

La comptabilité est traversée par une opposition fondamentale :

• Le Measurement (la mesure) : Une approche instrumentale où les chiffres (quantitatifs et qualitatifs) sont des ordres de grandeur définis par des conventions internes pour piloter une organisation.

• La Valuation (la valorisation) : L'idée que le marché peut révéler une "juste valeur" objective d'un actif, y compris des ressources naturelles.

Cette approche vise une sorte de transparence, une représentation absolue du monde en chiffres.

La Proposition de la Comptabilité Écologique

La chaire de comptabilité écologique se positionne fermement du côté du measurement.

Elle rejette la tentation de "chiffrer un écosystème", ce qui n'aurait aucun sens.

Son projet est d'utiliser les chiffres pour accompagner et outiller l'action de préservation :

• Au lieu de "valoriser" un écosystème, elle cherche à calculer les coûts nécessaires pour le préserver ou le restaurer.

• Au lieu de chercher une "juste valeur" de la nature, elle se demande combien il faudrait payer un agriculteur pour qu'il puisse à la fois vivre décemment et garantir le bon état écologique de ses sols.

L'objectif n'est pas de mesurer l'essentiel dans l'absolu, mais de "mesurer ce qui est essentiel pour permettre de protéger ce que l'on a à protéger".

5. Se Libérer de la Domination des Chiffres

La philosophe Valérie Charolles conclut en appelant à une prise de distance critique face à l'hégémonie des chiffres.

Le Défi de l' "Innétrisme" et les Inférences Trompeuses

Nous sommes souvent mal armés pour interpréter les chiffres, ce qui conduit à des "inférences trompeuses".

La communication est asymétrique entre les experts qui produisent les chiffres et le public qui les reçoit.

• Exemple de la croissance : Annoncer un taux de croissance de 6,8 % en Éthiopie contre 1,7 % en France est trompeur.

Rapporté par habitant, le gain de richesse est 15 fois supérieur en France (705 $) qu'en Éthiopie (50 $).

• Présentation des données : Dire que la France a une croissance de 1,7 % est factuellement équivalent à dire que son PIB "est sur une tendance de doublement en 43 ans".

La seconde formulation change radicalement la perception de la situation.

Chiffres et Nombres : une Distinction Cruciale

Il faut distinguer :

• Les nombres : Des entités théoriques abstraites, opérant par raisonnement pur (domaine des mathématiques).

• Les chiffres : Des grandeurs mesurées ou des quantités calculées qui visent à rendre compte du réel.

Ils ne peuvent exister sans un ensemble de conventions (définitions, étalons de mesure, modèles).

L'Analyse Critique des Conventions : Là où Tout se Joue

La véritable analyse doit porter sur les normes, modèles et conventions qui servent à produire les chiffres, car "c'est là que tout se joue".

Ces conventions peuvent être datées, limitées ou biaisées.

• La comptabilité d'entreprise : Son cadre, hérité de la Renaissance, traite le travail comme une charge et non comme une valeur, et privilégie une perspective de liquidation à court terme.

• Les modèles financiers : Ils sous-estiment systématiquement la probabilité des événements extrêmes (crises, krachs), comme l'a montré Benoît Mandelbrot.

• Les systèmes électoraux : La manière de compter les voix (proportionnelle, majoritaire) détermine la composition des parlements et donc les politiques menées.

Le problème n'est donc pas de rejeter les chiffres, mais de "se libérer de leur domination".

Cela implique de comprendre que nous avons un pouvoir sur eux, car ce sont des représentations politiques et sociales qui décident des lois électorales, des normes comptables ou des modes de calcul du PIB.

La voie à suivre est de renforcer la culture statistique citoyenne et de soumettre les cadres de référence à un débat démocratique constant.

Synthèse : Le Côté Sombre de la Morale

Résumé

Cette synthèse examine les thèses présentées par Jean Decety sur ce qu'il nomme "le côté sombre de la morale".

L'argument central est que si la morale est un pilier de la coopération sociale, elle possède une facette destructrice.

Lorsque des croyances se transforment en convictions morales absolues, elles deviennent un puissant moteur de dogmatisme, d'intolérance et de violence.

Ces convictions, caractérisées par un sentiment d'objectivité, un consensus social perçu et une stabilité temporelle, transcendent les idéologies politiques et les causes spécifiques.

L'objectif de la recherche de Decety est de développer un modèle théorique unifié, en s'appuyant sur la psychologie, les neurosciences, l'anthropologie et la théorie de l'évolution, pour expliquer les mécanismes psychologiques universels qui sous-tendent ce phénomène.

Le processus clé est la "moralisation", qui convertit des préférences sociales en valeurs sacrées, engageant le système de récompense du cerveau.

Ce processus est souvent associé à une faible sensibilité métacognitive, où les individus les plus extrêmes sont paradoxalement les moins informés sur le sujet, mais les plus convaincus de leur savoir.

En moralisant une question, on la rend imperméable à l'analyse coûts-bénéfices et à tout compromis, ce qui conduit à une polarisation accrue et entrave le dialogue démocratique.

1. La Double Nature de la Morale

La morale est généralement perçue comme un produit de la co-évolution gènes-culture, spécifique à Homo sapiens, qui apporte des bénéfices clairs à la vie sociale.

• Le Côté Positif : La morale est un mécanisme essentiel qui :

◦ Régule les échanges interpersonnels.   

◦ Facilite la coexistence et la coopération.   

◦ Minimise ou canalise l'agression.   

◦ Équilibre les conflits entre les intérêts individuels et collectifs.   

◦ Motive les actions collectives pour le bien commun, comme le mouvement pour le droit de vote des femmes ou les droits civiques.

• Le Côté Sombre : C'est l'aspect qui intéresse principalement Jean Decety.

La morale, lorsqu'elle est poussée à l'extrême sous forme de convictions inébranlables, peut :

◦ Alimenter le dogmatisme et l'intolérance.  

◦ Motiver la violence et des actions collectives extrêmes.   

◦ Justifier le vigilantisme, où des individus s'arrogent le droit de rendre la justice eux-mêmes.

2. La Conviction Morale : Définition et Conséquences

La conviction morale est le concept central de l'analyse.

Elle est définie comme une croyance forte et absolue qu'une chose est intrinsèquement bonne ou mauvaise, morale ou immorale.

Caractéristiques

Une conviction morale est perçue par celui qui la détient comme :

• Absolue : Elle ne tolère aucune variation ou exception, quel que soit le contexte.

• Objective : Elle est considérée comme une vérité fondamentale de la réalité, applicable à tous, partout et à tout moment.

Conséquences Négatives

Lorsqu'une forte conviction morale est associée à la perception d'un large consensus au sein de sa communauté, elle peut conduire à :

• L'intolérance : Un refus d'accepter des points de vue divergents.

• Le dogmatisme : Un état d'esprit inflexible et un refus de l'analyse critique.

• La violence : L'histoire et l'actualité montrent que la violence est souvent utilisée pour maintenir un ordre moral perçu.

Les auteurs de génocides, de guerres ou de tortures pensent fréquemment que leurs actions sont légitimes.

Exemples Concrets Citées

Plusieurs cas illustrent comment des individus aux idéologies très différentes partagent des mécanismes psychologiques similaires fondés sur la conviction morale :

Cas

Description

Motivation Morale sous-jacente

Émeutes au Nigeria (2002)

Plus de 220 personnes tuées suite à la publication d'un article de journal jugé offensant envers le prophète Mahomet.

Défense de l'honneur religieux.

Lorna Green (Wyoming, USA)

Condamnée pour avoir incendié une clinique pratiquant l'avortement.

La vie est sacrée et l'avortement est un meurtre.

Activistes climatiques

Utilisation de "tactiques de choc" et de protestations violentes, comme celles contre un projet d'aéroport.

Urgence de lutter contre le réchauffement climatique.

Kathleen Stock (Angleterre)

Professeure de philosophie harcelée et contrainte à la démission par des activistes transgenres.

Conviction que l'affirmation selon laquelle le sexe est une réalité biologique est une attaque inacceptable.

Terrorisme

Les individus commettant des actes terroristes sont souvent fortement convaincus de la justesse de leur cause (divine ou politique).

Accomplissement d'un devoir moral supérieur.

3. L'Architecture Fonctionnelle de la Conviction Morale

Decety propose un modèle fonctionnel pour expliquer la formation et les effets des convictions morales, basé sur l'interaction de plusieurs composantes.

Composantes Clés

1. Objectivité : La croyance que ses propres valeurs sont des vérités objectives et universellement applicables.

2. Consensus Social : La perception que les membres de sa communauté ou de sa coalition partagent les mêmes croyances, ce qui renforce la conviction.

3. Stabilité Temporelle : Plus une croyance est perçue comme ayant une base morale, plus elle reste stable dans le temps.

Le Mécanisme Central : La Conversion des Préférences en Valeurs

Le moteur de la conviction morale est sa capacité à transformer des préférences sociales en valeurs sacrées.

• Préférence : "Je choisis de ne pas manger de viande issue de l'élevage industriel." (Problème personnel)

• Valeur Moralisée : "Personne ne devrait manger de viande issue de l'élevage industriel car c'est immoral." (Problème moral universel)

Les valeurs agissent comme des forces de motivation puissantes qui fixent des objectifs, guident les décisions et suscitent l'action.

Le Substrat Neurobiologique

• Les valeurs, y compris les valeurs morales, sont traitées par le système de récompense et de valuation du cerveau.

Il n'existe pas de circuit cérébral spécifique à la morale ; celle-ci utilise les mêmes mécanismes que ceux qui attribuent une valeur à la nourriture ou à un partenaire.

• La spécificité humaine réside dans la capacité unique de notre espèce à attribuer une valeur à des objets abstraits et arbitraires, comme des idéologies, des symboles (drapeau), une religion ou une cause politique.

4. Mécanismes Psychologiques : Métacognition et Dogmatisme

Les convictions morales fortes sont souvent associées à une faible capacité de réflexion critique.

• Métacognition : La capacité de réfléchir à ses propres processus de pensée.

La sensibilité métacognitive mesure la corrélation entre la confiance d'une personne en sa réponse et la justesse réelle de cette réponse.

• Faible Sensibilité Métacognitive : Les recherches montrent que les individus dogmatiques et moralement convaincus ont souvent une faible sensibilité métacognitive.

Il y a un décalage entre leur niveau de confiance (très élevé) et leurs connaissances réelles (souvent faibles).

• L'Exemple des OGM : Une étude menée aux États-Unis, en Allemagne et en France a montré que les opposants les plus extrêmes aux OGM étaient ceux qui avaient le moins de connaissances en biologie, mais qui pensaient en savoir le plus.

C'est une illustration du principe : "Moins ils en savent, plus ils pensent savoir".

5. Les Défis de la "Moralisation" et l'Analyse Coûts-Bénéfices

Une fois qu'une question est "moralisée", elle devient extrêmement difficile à débattre rationnellement.

• Échec de l'Analyse Coûts-Bénéfices : Les convictions morales, en devenant des valeurs sacrées, empêchent toute forme de compromis ou d'analyse pragmatique des coûts et des bénéfices.

Par exemple, pour un militant anti-avortement absolu, aucun argument contextuel (viol, âge de la mère, malformation du fœtus) ne peut justifier une exception.

• Polarisation et Démocratie : La moralisation excessive des débats publics conduit à une polarisation extrême, rendant le dialogue constructif et la recherche de compromis – essentiels à la vie en société – presque impossibles.

• Approche Proposée : Decety suggère que, même pour des sujets moralisés, encourager une analyse coûts-bénéfices est une voie pour progresser en tant que société, plutôt que de rester figé dans des positions irréconciliables.

6. Points Clés de la Discussion (Q&A)

• Distinction entre Morale et Éthique : Pour les besoins de sa recherche sur les mécanismes psychologiques, Decety ne fait pas de distinction fondamentale.

Il ne s'intéresse pas à ce que les gens devraient faire (éthique prescriptive), mais aux mécanismes qui transforment une préférence en une croyance absolue.

• Signification du terme "Absolu" : Une valeur est absolue lorsqu'elle est insensible au contexte, aux preuves factuelles ou aux circonstances atténuantes.

L'exemple de l'avortement montre que même face à des scénarios extrêmes, la position morale reste inchangée.

• Perspective sur le Terrorisme : Decety est en accord avec l'idée que les terroristes sont hautement convaincus moralement.

Cependant, il conteste le terme de "lavage de cerveau" (brainwashed), arguant que leurs actions sont souvent rationnelles au sein de leur propre système de valeurs, de leur histoire et des normes de leur groupe.

L'Illusion du Contrôle à l'Ère de l'Intelligence Artificielle : Synthèse de la Présentation de Helga Nowotny

Résumé

Cette note de synthèse analyse les thèmes centraux de la présentation de Helga Nowotny sur l'intelligence artificielle (IA), axée sur le concept de "l'illusion du contrôle".

L'émergence spectaculaire de l'IA générative, illustrée par ChatGPT, a non seulement surpris les experts par ses performances, mais a également exacerbé une anxiété sociétale profonde liée à la perte de contrôle.

Ce sentiment est alimenté par les craintes concernant l'automatisation de l'emploi, la désinformation via les "deepfakes", la persistance des biais algorithmiques et la fragmentation de la réalité commune.

Un point central de l'analyse est la tendance humaine à l'anthropomorphisme, qui consiste à attribuer des intentions à l'IA, un phénomène qui peut avoir des conséquences tragiques.

La notion de contrôle technologique elle-même est en pleine évolution : après s'être étendue de la simple fonctionnalité de la machine à la sécurité des travailleurs, puis à la protection de l'environnement, elle doit maintenant intégrer l'impact de l'IA sur les capacités cognitives et émotionnelles humaines.

La présentation met en lumière une concentration économique et de pouvoir sans précédent entre les mains de quelques entreprises technologiques, qui financent 90% de la recherche et du développement en IA, orientant ainsi sa trajectoire au détriment de la recherche publique et fondamentale.

Face à un monde de plus en plus complexe et potentiellement incompréhensible que nous créons nous-mêmes, la conclusion insiste sur la nécessité d'adopter le scepticisme, une vertu scientifique essentielle, comme antidote aux illusions et pour naviguer de manière éclairée dans cette nouvelle ère.

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1. L'Avènement de l'IA Générative et la Fin de la Hype

L'intervention de Helga Nowotny s'inscrit dans le prolongement de son livre de 2021, In AI We Trust, et se concentre sur l'illusion du contrôle face aux développements récents de l'IA.

• L'Expérience ChatGPT : Le lancement de ChatGPT fin 2022 est qualifié d'« expérience déchaînée sans le consentement de personne ».

Son principal avantage a été de mettre un grand nombre de personnes en contact direct avec une technologie numérique avancée.

• Une Performance Surprenante : La performance de l'IA générative a surpris même les experts, bien qu'ils s'attendaient à son arrivée.

• Plus qu'une Simple Hype : Nowotny soutient que l'engouement actuel pour l'IA n'est pas passager pour deux raisons principales :

1. Investissements Massifs : Des investissements colossaux sont engagés, créant un pari sur le principe du "trop gros pour faire faillite" (too big to fail).   

2. Adoption Scientifique : L'IA est déjà en train de transformer la science.

Des outils comme AlphaFold de DeepMind sont devenus des instruments fantastiques pour les biologistes, et des applications similaires émergent en science des matériaux, en découverte de médicaments, et dans d'autres domaines.

2. Inquiétudes Sociétales et la Peur Fondamentale de la Perte de Contrôle

Un malaise généralisé persiste face à l'IA, reposant sur plusieurs craintes interconnectées.

• Automatisation et Emploi : Au-delà de la question de la destruction et de la création d'emplois, le véritable enjeu, selon Nowotny, est notre capacité à inventer de nouvelles tâches à réaliser en collaboration avec l'IA.

• Menaces sur la Démocratie : Les "deepfakes" et les campagnes de désinformation délibérée posent un risque majeur pour les démocraties libérales, créant une situation où personne ne semble avoir le contrôle, sauf ceux qui lancent ces campagnes.

• Biais Algorithmiques : Les biais présents dans les données d'entraînement sont perpétués et amplifiés par les algorithmes.

Lorsque les gens commencent à croire aux prédictions de ces algorithmes, les biais s'ancrent profondément dans la société.

• Fragmentation Sociale : La personnalisation extrême risque de nous enfermer dans des "réalités personnalisées", nous faisant perdre le terrain commun nécessaire au débat et à la cohésion sociale.

• Tendance à l'Anthropomorphisme : Nous avons une tendance naturelle à attribuer des intentions humaines aux machines.

◦ La "Posture Intentionnelle" : Le philosophe Daniel Dennett a longuement écrit sur ce qu'il appelle la "posture intentionnelle".   

◦ Croyances Dangereuses : Cette tendance culmine dans des affirmations choquantes comme « L'IA me connaît mieux que je ne me connais moi-même », transférant un pouvoir quasi métaphysique à la machine.  

◦ Cas Extrême : Un cas tragique en Belgique a vu une personne souffrant de problèmes mentaux être encouragée au suicide par une application thérapeutique non réglementée, illustrant les dangers extrêmes de cette confusion.

3. L'Évolution du Concept de Contrôle Technologique

Le concept de "contrôle" d'une technologie a évolué au fil de l'histoire et fait face aujourd'hui à un défi sans précédent avec l'IA.

1. Contrôle Opérationnel : Initialement, le contrôle signifiait s'assurer que la technologie fonctionne correctement (maintenance, réparations).

2. Contrôle de la Sécurité Humaine : Avec l'industrialisation, le contrôle s'est étendu à la protection de la santé et de la sécurité des travailleurs opérant les machines.

3. Contrôle Sociétal et Environnemental : L'État-providence a ajouté des législations et des assurances.

Plus récemment (ces 20-25 dernières années), le contrôle a été étendu pour limiter les dommages environnementaux causés par la technologie.

4. **Le Nouveau Défi - Le Contrôle Cognitif et Émotionnel :

** Le défi actuel est d'étendre ce contrôle à l'impact que l'IA a sur nos capacités cognitives et émotionnelles.

Cela est particulièrement visible avec les algorithmes prédictifs qui, en extrapolant le passé, façonnent nos choix et nous font oublier que le futur reste incertain.

4. Concentration du Pouvoir et Dynamiques Géopolitiques

Derrière les avancées de l'IA se cache une énorme concentration de pouvoir économique qui influence sa trajectoire et sa régulation.

• Déséquilibre du Financement :

◦ Dans les pays de l'OCDE, la R&D générale est financée à environ deux tiers par le secteur privé et un tiers par le secteur public.  

◦ Pour l'IA, le rapport est de 90 % de financement privé contre seulement 10 % de financement public.

• Conséquences du Déséquilibre :

◦ Les universités sont désavantagées, manquant d'accès à la puissance de calcul et aux données détenues par les grandes entreprises.  

◦ Les entreprises n'ont aucune obligation de rendre publics leurs algorithmes ou leurs données.   

◦ La direction de la recherche est dictée par des objectifs de profit, bien que les entreprises affirment travailler pour le bien de l'humanité.

Il est nécessaire de financer davantage la recherche publique pour explorer des voies alternatives.

• Paysage de la Régulation :

◦ Union Européenne : À l'avant-garde avec un ensemble de législations, dont l'AI Act.  

◦ États-Unis : Réticents à réguler par crainte d'étouffer l'innovation, et sous l'influence du lobbying de la Big Tech.  

◦ Chine : L'autre acteur majeur de cette configuration géopolitique.

5. Questions Philosophiques et Épistémologiques

L'IA nous confronte à des questions profondes sur notre rapport au monde et à la connaissance.

• Comprendre ce que nous créons : Citant Giambattista Vico ("Nous ne comprenons que ce que nous faisons"), Nowotny se demande si nous ne nous dirigeons pas vers un monde créé par l'homme que nous ne comprenons plus.

L'IA permet de créer des "jumeaux numériques" et des systèmes complexes dont les propriétés émergentes sont impossibles à prédire.

• De Nouvelles Formes de Raisonnement : L'exemple d'un mathématicien dont le problème a été résolu par une IA d'une manière différente de celle d'un humain soulève des questions fondamentales :

notre cerveau fonctionne-t-il différemment, ou l'IA révèle-t-elle de nouvelles facettes de la mathématique, elle-même une "technologie culturelle" ?

• Co-évolution avec des "Autres Numériques" : Faisant une analogie spéculative avec les travaux de l'anthropologue Marshall Sahlins sur le "cosmos immanent" (un monde où les humains partageaient leur existence avec des esprits et des dieux), Nowotny suggère que nous pourrions être au début d'une trajectoire co-évolutive où nous devrons apprendre à vivre avec des "autres numériques".

6. Le Scepticisme Scientifique comme Antidote

Face à ces illusions et à ces complexités, la démarche scientifique offre une méthode pour ne pas se tromper soi-même.

• La Leçon de Feynman : Citant le physicien Richard Feynman, "La science est ce que nous avons appris sur la manière de ne pas nous tromper nous-mêmes."

• Une Vertu pour la Société : Le scepticisme est une vertu scientifique qui doit être diffusée dans toute la société et auprès des politiciens.

Il est crucial d'éviter le déterminisme technologique, qui est le revers de l'illusion du contrôle.

Le sentiment d'impuissance mène à la peur, à la passivité et au repli, ce qui constitue le pire scénario possible.

7. Thèmes Abordés dans la Session de Questions-Réponses

• IA et Sciences Sociales : L'IA offre une opportunité de lier la recherche qualitative (la "connaissance épaisse" de Clifford Geertz) et quantitative en analysant de vastes corpus de données qualitatives.

De plus, comme pour le transistor qui a permis l'émergence de la radio portable, des usages sociaux imprévus de l'IA apparaîtront.

• Les "Objectifs" de l'IA : Une IA n'a que les objectifs qui lui sont inscrits par ses créateurs.

La vraie question est : "Quels sont les objectifs des personnes qui développent, possèdent et investissent dans l'IA ?"

• Armes Autonomes : Le développement se dirige rapidement vers des armes autonomes.

Atteindre un accord international de non-prolifération, similaire à celui sur les armes nucléaires, sera très difficile car les composants de l'IA sont beaucoup plus complexes à tracer que les substances nucléaires.

• Langage, Traduction et Culture : L'IA facilite énormément la traduction instantanée.

Cela pourrait entraîner la fermeture de départements universitaires de traduction et décourager l'apprentissage des langues.

Un marché ségrégué pourrait émerger pour les livres : une production de masse par l'IA et un marché de "luxe" pour les auteurs humains.

• Communiquer sur l'IA : Il faut aller au-delà de la simple "littératie numérique" pour développer une véritable conscience du fait que l'IA est une technologie créée et dirigée par des humains. Ceci est essentiel pour éviter la peur et la passivité face à un prétendu déterminisme technologique.

Dossier d'Information : Les Dynamiques de la Négociation de Paix selon Alberto Fergusson

Synthèse

Ce document de synthèse analyse les réflexions et les expériences d'Alberto Fergusson, un acteur clé du processus de paix colombien, qui allie une expertise en médecine, psychiatrie et psychanalyse à une pratique intensive des négociations.

Ses observations, issues de plus d'une décennie d'implication, notamment dans les pourparlers avec l'ELN, révèlent les dynamiques psychologiques et sociales complexes qui sous-tendent les processus de paix.

Les points à retenir sont les suivants :

• Le Paradoxe de l'Accord (Individu vs. Groupe) :

L'observation la plus frappante de Fergusson est qu'un accord est quasi systématiquement possible lors de discussions individuelles et privées avec les membres de la partie adverse, y compris les dirigeants.

Cependant, cet accord devient impossible à atteindre une fois que les discussions retournent à la table de négociation formelle, avec ses dynamiques de groupe et ses impératifs de représentation.

• L'Importance Capitale des Canaux Parallèles ("Back Channels") : Contrairement à l'idée reçue, la majorité des décisions cruciales ne sont pas prises lors des sessions officielles, mais dans le cadre de discussions informelles et de réunions secrètes.

La maîtrise de ces canaux parallèles est un art qui requiert l'identification des bons interlocuteurs et la gestion précise du format et de la durée des échanges.

• L'Application de la Psychopathologie à la Négociation : Fergusson tire ses principaux outils d'analyse de son travail avec des sans-abri atteints de maladies mentales graves.

Il postule que les mécanismes de défense et les perturbations émotionnelles observées dans la "folie" éclairent les comportements, parfois irrationnels, des acteurs dans des situations de haute tension comme les négociations de paix.

• La Question Fondamentale sur l'Impact Réel des Négociations : Fergusson s'interroge de manière critique sur la capacité des négociations à modifier durablement les processus sociaux.

Il se demande si les accords de paix réussis sont le fruit d'une habileté de négociation ou s'ils ne font que formaliser une évolution déjà inéluctable des dynamiques sociales, soulevant le risque de parvenir à des accords "artificiels" et prématurés.

Contexte et Objectifs du Chercheur

Alberto Fergusson, fort d'une formation en médecine, psychiatrie et psychanalyse, a consacré une part importante de sa carrière à des activités psychosociales.

Son travail initial auprès de sans-abri atteints de schizophrénie en Colombie lui a permis de développer un modèle, l'"auto-analyse accompagnée", pour comprendre et accompagner les personnes souffrant de troubles émotionnels sévères.

Depuis près de vingt ans, il applique les connaissances acquises dans ce domaine au processus de paix colombien.

Il a été directement impliqué dans les pourparlers, notamment en tant que membre de la délégation gouvernementale du président Santos lors des discussions avec l'ELN en Équateur et à Cuba.

Il a également été membre de la Commission de la Vérité en Colombie.

Actuellement professeur à l'Université du Rosaire, il consacre un mois à l'IEA de Paris (en mode virtuel) pour organiser, synthétiser et repenser une décennie d'expériences.

Ce travail de réflexion est crucial car il s'apprête à réintégrer le processus de paix colombien avec une perspective académique, visant à analyser la situation d'un point de vue plus large et moins partisan.

Thèmes Centraux et Observations Clés

De la "Folie" à la "Normalité" : Une Approche Inversée

Fergusson qualifie son approche de "confession" : il reconnaît que l'essentiel de sa compréhension des processus de négociation provient de son expérience avec des personnes atteintes de maladies mentales graves.

Sa présentation est intitulée "La normalité à la lumière de la folie" (Normality in the light of Madness), signifiant que les mécanismes psychologiques extrêmes observés chez ses patients offrent une grille de lecture pertinente pour les dynamiques apparemment "normales" des négociations politiques.

Le Paradoxe de l'Accord : Individu contre Groupe

L'observation la plus puissante et la plus récurrente de Fergusson est la dichotomie radicale entre les interactions individuelles et les dynamiques de groupe.

• En tête-à-tête : Fergusson affirme que, sans exception, lors de conversations approfondies et individuelles avec n'importe quel membre de la partie adverse (y compris les plus hauts dirigeants de l'ELN), il a toujours été possible de parvenir à un consensus.

Il déclare : "nous aurions toujours pu signer l'accord individuellement, en tête-à-tête."

• À la table de négociation : Dès que la discussion est portée à la table formelle, où les dynamiques de groupe, les hiérarchies (nécessité d'obtenir l'approbation du leader suprême, comme "Gabino" pour l'ELN) et les pressions de représentation entrent en jeu, l'accord devient compliqué, voire impossible.

Ce paradoxe constitue le cœur de son questionnement actuel : pourquoi ce qui est mutuellement acceptable en privé devient-il inacceptable en public ?

L'Irrationalité Apparente : Agir Contre Ses Propres Intérêts

Une autre observation centrale est que, dans le cadre des négociations, les individus et les groupes adoptent fréquemment des positions qui vont manifestement à l'encontre de leurs propres intérêts, ou du moins partiellement.

Fergusson cherche à dépasser la simple explication des "facteurs émotionnels et psychologiques" pour analyser en détail les mécanismes qui conduisent à ces décisions contre-productives.

Le Rôle Crucial des Canaux de Négociation Parallèles ("Back Channels")

Fergusson affirme sans équivoque que la plupart des décisions importantes ne sont pas prises à la table officielle de négociation.

• Lieu de décision réel : Les véritables avancées se produisent lors de réunions informelles, en marge des sessions officielles.

• L'art du "Back Channel" : Le succès de ces canaux parallèles dépend d'une stratégie fine :

1. Identifier l'interlocuteur clé : Il faut savoir repérer la personne de l'autre camp avec qui un accord de principe peut être trouvé.   

2. Rassembler les décideurs : Dans un exemple réussi, Fergusson et son homologue de l'ELN, après s'être mis d'accord, ont organisé une réunion privée entre leurs deux dirigeants respectifs pour leur présenter leur solution commune.

Ce fut le moment où les négociations ont le plus progressé.  

3. Maîtriser la durée : La longueur d'une réunion est un facteur critique. Fergusson note que si des êtres humains continuent de parler après avoir trouvé un accord, ils finiront par trouver un désaccord.

Savoir quand s'arrêter est essentiel.

La Question Fondamentale : Négociation et Évolution Sociale

La principale question de recherche de Fergusson, qu'il explore durant sa résidence, est la suivante :

"Jusqu'à quel point peut-on changer les processus sociaux par le biais des négociations ?"

Il illustre ce dilemme avec une analogie : celle d'une personne qui, toute la nuit, pousse de toutes ses forces pour faire venir le soleil et qui, à 6 heures du matin, lorsque le soleil se lève, s'écrie : "J'ai réussi !".

• Négociateur : Agent du changement ou simple facilitateur ?

Les négociateurs sont-ils les artisans d'un accord, ou leur intervention se contente-t-elle de faciliter ou d'accélérer une trajectoire que les dynamiques sociales et les conflits auraient de toute façon suivie ?

• Le risque des "lois sociales naturelles" : Il se demande si les négociateurs, en tentant de forcer un accord, ne vont pas à l'encontre des "lois sociales naturelles", créant ainsi des arrangements artificiels et prématurés.

• Le critère du succès : Pour Fergusson, un accord réussi n'est pas celui qui tient six mois ou deux ans.

Sa question porte sur les accords de paix durables et leur véritable origine : l'habileté des négociateurs ou l'évolution inéluctable de la société.

Perspectives Issues de la Discussion

Les échanges avec les autres chercheurs ont enrichi et précisé plusieurs points :

• Légitimer le Changement de Position sans "Perdre la Face" :

◦ Un participant a suggéré que le rôle du négociateur est de créer un cadre où les parties peuvent légitimement changer de position sans "perdre la face".  

◦ Cette idée est illustrée par une expérience de dégustation de vin : des dégustateurs ont radicalement changé leur évaluation d'un vin après avoir vu l'étiquette, mais n'ont jamais admis avoir changé d'avis.

Ils ont prétendu que c'était le vin qui avait "changé" (il s'était "ouvert").  

◦ Leçon pour le négociateur : Il ne s'agit pas de convaincre l'autre partie de changer d'avis, mais de présenter la situation différemment (par exemple, en invoquant de "nouveaux événements" ou de "nouveaux aspects") afin que l'adoption d'une nouvelle position apparaisse comme une réponse logique à un contexte modifié, et non comme une capitulation.

• L'Équilibre entre Secret et Public :

◦ Même les processus de paix qui semblent secrets, comme celui avec les FARC, sont en réalité un mélange complexe d'échanges publics et de canaux parallèles.  

◦ Fergusson confirme que l'accord final avec les FARC a été le résultat d'une "chaîne de canaux parallèles", souvent au grand dam des dirigeants qui n'apprécient pas ces manœuvres.

Mesurer les Inégalités : Synthèse et Perspectives du Débat

Résumé Exécutif

Ce document de synthèse analyse les thèmes centraux d'un débat d'experts sur la mesure des inégalités et son lien avec leur réduction.

Trois perspectives complémentaires émergent :

1. Les indicateurs comme conventions socio-politiques:

Florence Jany-Catrice, économiste, soutient que toute mesure des inégalités est le fruit de conventions socio-politiques et non une vérité objective.

Les indicateurs sont des instruments à double face, servant à la fois la connaissance et la gouvernance.

Elle critique les mesures standards comme le rapport interdécile (D9/D1) qui masquent les réalités aux extrêmes de la distribution et occulte des inégalités fondamentales comme le partage capital/travail.

Mesurer n'entraîne pas automatiquement une réduction, car il existe une chaîne complexe entre savoir et agir.

2. La communication et l'action citoyenne :

Cécile Duflot, directrice d'Oxfam France, présente l'approche de son organisation, qui consiste à utiliser des données robustes (notamment de Crédit Suisse/UBS) pour produire des "killer facts" :

des comparaisons choc conçues pour rendre visible l'ampleur de la concentration extrême des richesses.

L'objectif est de mobiliser l'opinion publique et de plaider pour une régulation politique, en arguant que les niveaux actuels d'inégalité de patrimoine créent des fractures sociales, privent l'action publique de ressources et posent un problème démocratique fondamental.

3. L'expérience vécue comme révélateur : Nicolas Duvoux, sociologue, propose de dépasser le décalage entre la stabilité relative des indicateurs officiels et la forte tension sociale ressentie.

En s'appuyant sur l'analogie de la "température ressentie", il affirme que la mesure de la perception subjective des inégalités n'est pas une alternative à la mesure objective, mais un moyen de l'affiner.

Cette approche révèle le rôle central du patrimoine dans le sentiment de sécurité et la capacité à se projeter dans l'avenir.

Elle met en lumière des fractures que les indicateurs monétaires traditionnels ne captent pas, de la précarité des classes populaires à la capacité des ultra-riches de façonner l'avenir collectif via la philanthropie.

En conclusion, le débat converge sur l'idée que si mesurer les inégalités ne suffit pas à les réduire, mesurer autrement — en critiquant les conventions, en rendant visibles les extrêmes et en intégrant l'expérience vécue — est le premier pas indispensable pour poser un diagnostic partagé et engager une action politique et sociale efficace.

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Thème 1 : Les Indicateurs comme Conventions Socio-Politiques (Florence Jany-Catrice)

L'économiste Florence Jany-Catrice pose le cadre conceptuel du débat en affirmant que la quantification des faits sociaux, et en particulier des inégalités, est une opération complexe qui repose sur des conventions.

Reprenant les travaux d'Alain Desrosières, elle insiste sur le duo "convenir et mesurer", soulignant que derrière chaque chiffre se cache une part de normativité et une théorie de la justice, consciente ou non.

La Double Face des Indicateurs : Connaissance et Gouvernance

Les indicateurs d'inégalité ne sont pas de simples outils de connaissance neutres. Ils possèdent une double nature :

• Instruments de connaissance : Ils permettent de se représenter l'état de la société.

• Instruments de gouvernance : Ils servent de marqueurs pour évaluer l'efficacité des politiques publiques de redistribution et reflètent l'état des rapports de force sociaux.

Cependant, le lien entre l'observation d'un phénomène et sa prise en charge politique n'est ni linéaire ni automatique.

Comme le démontre l'exemple de la commission Stiglitz-Sen-Fitousi (2008), dont la recommandation d'adjoindre au PIB un indicateur de répartition des richesses a été largement ignorée, "on peut très bien savoir mais ne pas vouloir".

L'impact d'un diagnostic dépend de la capacité des acteurs sociaux (experts, chercheurs, ONG) à le rendre suffisamment partagé et à défendre des visions politiques alternatives.

Les Limites des Mesures Conventionnelles

Florence Jany-Catrice met en évidence les faiblesses et les angles morts des indicateurs les plus couramment utilisés.

Indicateur / Concept

Description et Critique

Rapport Capital/Travail

Considéré comme la "première inégalité" du capitalisme, il mesure le partage de la valeur ajoutée entre la rémunération du travail (salaires) et celle du capital (dividendes, intérêts).

Cet indicateur, bien qu'existant, est de moins en moins visible dans le débat public, illustrant un glissement des intérêts et des expertises.

Rapport Interdécile (D9/D1)

Rapport entre le revenu des 10 % les plus riches et celui des 10 % les plus pauvres.

Bien qu'il semble stable en France (autour de 3,5), cet indicateur est critiqué car il exclut volontairement les "valeurs aberrantes", c'est-à-dire les très hauts et très bas revenus. Il masque ainsi l'aggravation des inégalités aux "queues de la distribution".

Pauvreté Monétaire Relative

En France, elle est définie par le seuil de 60 % du revenu médian. F. Jany-Catrice souligne qu'il s'agit avant tout d'un indicateur d'inégalité de répartition, et non de pauvreté absolue.

Vers des Indicateurs Alternatifs et le "Statactivisme"

Face aux limites des outils officiels, des initiatives de la société civile émergent pour proposer d'autres manières de compter.

• Le BIP 40 (Baromètre des Inégalités et de la Pauvreté) :

Créé dans les années 2000 par le Réseau d'alerte sur les inégalités, cet indicateur composite et multidimensionnel (revenu, travail, éducation, santé, logement, justice) montrait une "explosion" des inégalités entre 1980 et 1995, à rebours de l'indicateur officiel de l'INSEE qui indiquait une régression de la pauvreté.

L'objectif n'était pas d'opposer un "vrai" chiffre à un "faux", mais de démontrer que "selon les lunettes que l'on chausse, on peut raconter des histoires" très différentes sur l'état de la société.

• Le "Statactivisme" : Ce néologisme désigne les stratégies statistiques utilisées par des acteurs sociaux pour critiquer une autorité et s'en émanciper.

Il s'agit d'une réappropriation du "pouvoir émancipateur" des statistiques pour fournir des données sur les angles morts de la production publique (ex: les plus riches) ou des visions alternatives.

Thème 2 : Le Rôle d'Oxfam dans le Débat Public (Cécile Duflot)

Cécile Duflot explique comment Oxfam, une organisation historiquement dédiée à la lutte contre la pauvreté, s'est concentrée sur les causes de celle-ci, arrivant "assez rapidement sur la question des inégalités".

L'approche d'Oxfam est décrite comme éminemment politique et militante, visant à mobiliser le pouvoir citoyen.

Méthodologie et Stratégie de Communication

Le rapport annuel d'Oxfam, publié symboliquement pendant le Forum de Davos, repose sur une méthodologie précise et une stratégie de communication percutante.

• Source des données : Le rapport s'appuie principalement sur les données du Crédit Suisse (aujourd'hui UBS) et de Forbes, utilisant la "méthode la plus robuste pour calculer le patrimoine", la même que celle utilisée par des institutions comme la Haute autorité pour la transparence de la vie publique.

• Les "Killer Facts" : La stratégie d'Oxfam consiste à traduire des données brutes en comparaisons frappantes et intuitivement compréhensibles, car les ordres de grandeur comme le milliard d'euros sont "nébuleux" pour le grand public.

◦ Exemple cité : "Les 8 premiers milliardaires du monde possédaient ce que possède la moitié des plus pauvres".   

◦ Illustration de l'effet de moyenne : L'entrée de Carlos Tavares (PDG de Stellantis) dans une pièce de 99 smicards ferait passer le revenu moyen de 16 000 € à environ 400 000 €, masquant le fait que 99 % des personnes sont toujours au SMIC.

Même le ratio D9/D1 (écart de 1 à 229 dans ce cas) reste trompeur, car il y a "plus d'écart au sein des 10 % les plus riches [...] que entre les 10 % les plus pauvres et les 10 % les plus riches".

Au-delà du Revenu : La Concentration du Patrimoine et ses Conséquences

Oxfam se concentre sur les inégalités de patrimoine, considérées comme plus fondamentales que celles de revenu.

• L'injustice perçue : La majorité des grandes fortunes sont héritées. En France, "plus de 70 % de la fortune des milliardaires est une fortune héritée".

C. Duflot cite un milliardaire danois parlant de "gagner à la loterie du sperme".

• Conséquences des inégalités extrêmes :

1. Fracturation sociale : Elles sont vécues comme injustes et fragilisent la cohésion sociale.  

2. Privation de ressources publiques : La concentration du patrimoine chez les ultra-riches, qui bénéficient de taux d'imposition effectifs plus faibles, réduit la base taxable.   

3. Problème démocratique : L'accumulation extrême de richesse se traduit par l'achat du pouvoir.

C. Duflot cite un interlocuteur : "Le premier milliard, on peut le dépenser. [...] À partir du 2e milliard, [...] on achète le pouvoir", notamment via l'achat de médias et la pression sur les dirigeants politiques.

Une Démarche Militante pour la Régulation

La finalité du travail d'Oxfam n'est pas de "ne pas aimer les riches", mais de plaider pour une plus grande régulation, arguant que les sociétés plus égalitaires sont en meilleure santé globale (travaux de Wilkinson) et plus stables.

Le rapport d'Oxfam de septembre 2017, qui analysait le premier budget du gouvernement Macron (baisse des APL, suppression de l'ISF), est présenté comme ayant anticipé la colère sociale qui a mené au mouvement des "gilets jaunes", car "les gens [...] comprennent très bien le message politique".

Thème 3 : L'Objectivité Supérieure du Subjectif (Nicolas Duvoux)

Le sociologue Nicolas Duvoux part d'une énigme : le contraste entre la relative stabilité des indicateurs macroéconomiques d'inégalité en France et le niveau très élevé de "tension, de colère, d'insatisfaction".

Son travail vise à réconcilier la mesure objective et l'expérience vécue sans renoncer à la scientificité.

La "Température Ressentie" des Inégalités

Nicolas Duvoux propose de ne pas opposer l'objectif et le subjectif, mais d'utiliser la subjectivité comme une clé d'entrée pour "raffiner, mieux comprendre, mieux saisir l'objectivité des rapports sociaux".

• Analogie : Tout comme la température ressentie affine la température ambiante en y ajoutant des facteurs comme le vent ou l'humidité, la mesure du statut social subjectif donne une information plus fine que le statut objectif, car elle intègre la synthèse cognitive que fait l'individu de sa propre situation.

• Récusation du "subjectivisme" : Il insiste sur le fait que sa démarche n'isole pas le point de vue subjectif, mais l'intègre à l'analyse des structures objectives (ressources économiques, patrimoine) pour obtenir une vision plus riche. L'objectif est de "contextualiser la subjectivité".

Le Patrimoine comme Clé de Lecture de la Sécurité Sociale

La mesure subjective fait systématiquement ressortir le poids du patrimoine comme facteur déterminant de la sécurité ou de l'insécurité sociale.

• La pauvreté ressentie : Elle touche des groupes qui ne sont pas nécessairement pauvres au sens monétaire (petits indépendants, retraités locataires).

Elle révèle une "impossibilité de rendre soutenable une situation" où les revenus stagnent face à des charges qui augmentent (ex: loyers).

La pauvreté est alors vécue comme un "enfermement" et un manque de liberté dans l'affectation de ses ressources.

• L'avenir confisqué : L'inégalité est redéfinie comme une "inégalité de temps vécu", c'est-à-dire une différence dans la "capacité à se projeter" dans l'avenir.

Cette capacité est directement indexée sur la dotation en ressources, et particulièrement en patrimoine.

• La philanthropie des ultra-riches : À l'autre extrême du spectre social, le don philanthropique est analysé non pas comme un simple acte de générosité, mais comme un levier permettant aux plus fortunés d'assurer la transmission dynastique de leur patrimoine et d'exercer un contrôle sur les choix collectifs, se saisissant ainsi de "l'avenir collectif".

Changer la Représentation de la Hiérarchie Sociale

Cette approche conduit à une vision de la société structurée par des "franchissements de paliers de sécurité" plutôt que par une échelle linéaire et monétaire.

Elle réintroduit de la discontinuité entre les groupes sociaux et permet de donner une représentation statistique à des phénomènes comme la mobilisation des "gilets jaunes", en validant la difficulté exprimée par de larges pans de la population.

DOI: 10.17912/micropub.biology.001065

Resource: None

Curator: @Apiekniewska

SciCrunch record: RRID:WB-STRAIN:WBStrain00040855

What is this?

DOI: 10.1101/2024.02.15.580526

Resource: Caenorhabditis Genetics Center (RRID:SCR_007341)

Curator: @Apiekniewska

SciCrunch record: RRID:SCR_007341

What is this?

DOI: 10.1016/j.ydbio.2024.02.007

Resource: Caenorhabditis Genetics Center (RRID:SCR_007341)

Curator: @Apiekniewska

SciCrunch record: RRID:SCR_007341

What is this?

DOI: 10.1016/j.ydbio.2024.02.007

Resource: Caenorhabditis Genetics Center (RRID:SCR_007341)

Curator: @Apiekniewska

SciCrunch record: RRID:SCR_007341

What is this?

DOI: 10.1016/j.celrep.2025.116552

Resource: (BioLegend Cat# 118206, RRID:AB_1134172)

Curator: @scibot

SciCrunch record: RRID:AB_1134172

What is this?

Figure 3 shows the sea ice concentration anomalies for each climate scenario.

Application avec une portée extra territoriale (cf Article 3 LPD)

Un traitement à risque élevé est un traitement qui, par sa nature, son ampleur, sa finalité ou le type de données, peut causer des conséquences graves aux personnes si quelque chose se passe mal (mauvaise utilisation, fuite, décision automatique injuste, etc.).

**Exemples de risques élevés ** 1. Surveillance systématique et à grande échelle Ex. vidéosurveillance intelligente, traçage des employés. 2. Profilage et décisions automatisées qui ont un effet juridique ou important Ex. refus automatique d’un crédit bancaire basé sur un algorithme. 3. Traitement de données sensibles (art. 9 RGPD) Santé, orientation sexuelle, convictions religieuses, données biométriques, etc. 4. Traitement de données à grande échelle Bases de données de millions de clients avec informations personnelles détaillées. 5. Traitement concernant des personnes vulnérables Enfants, patients, personnes âgées dépendantes. 6. Utilisation de nouvelles technologies intrusives Reconnaissance faciale, IA de surveillance, géolocalisation en temps réel.

A reading of childhood rearing conditions of british working class households post-WWII can give a fair idea of the adverse circumstances operating against any such "well-intentioned efforts" at that time & place. Furthermore, such efforts likely failed to account for early (educational) developmental windows, combined nutritional differences, & given wide-spread racism & classism at the time. The efforts would be unlikely to counteract disparity in opportunity. Thus it is likely Fisher's (1951) assertion would not stand scrutiny today & may not be the best statement to be cited for Lynn's argument.

For a somewhat entertaining account of 1950s working class conditions & social mores, see Jennifer Worth's book (or tv series?) Call the Midwife.

This is a very common concern for new Tailwind users, and the answer is twofold:

1. No, you do not have to memorize all the classes. The developer workflow is built around powerful code editor tools that make this unnecessary.
2. Yes, interactive editors like you described absolutely exist. They are excellent for certain workflows, especially for building pages quickly.

Here is a detailed breakdown of the solutions available.

Solution 1: The Standard Developer Workflow (Code Editor Extensions)

This is how the vast majority of developers use Tailwind CSS. Instead of memorizing classes, you rely on an intelligent plugin in your code editor (like VS Code).

The most essential tool is the Tailwind CSS IntelliSense extension for Visual Studio Code.

This plugin solves the "memorization" problem in three specific ways:

1. Autocomplete: You rarely type the full class name. You start typing a prefix, and the editor shows you all possible options.
   • If you type bg-, it will pop up a list of all available colors (bg-blue-500, bg-red-700, etc.), complete with a color swatch.
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2. Hover-to-Preview: If you are unsure what a class does, you can hover your mouse over it. The plugin will show you the exact CSS it generates.
   • Hovering over p-4 will show a popup that says padding: 1rem;.
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   • This feature turns the editor into a powerful learning tool.
3. Linting (Error Checking): The plugin will underline conflicting classes, helping you avoid mistakes. For example, if you accidentally type p-2 and p-4 on the same element, it will flag this as an issue because you are applying two different padding values.

This workflow is not based on memory, but on a logical, discoverable system. The class names are consistent: * p is padding, m is margin. * t is top, b is bottom, l is left, r is right. * Therefore, pt-4 is padding-top of 1rem. This logic becomes second nature very quickly.

Solution 2: Visual (WYSIWYG) Editors

For the "PowerPoint-like" scenario you described, several tools provide a full graphical user interface (GUI) for styling with Tailwind. These are often called visual builders or page builders.

With these tools, you would click an element, and then use a properties panel on the side to adjust its padding, color, or margin. The tool then writes the correct Tailwind HTML for you.

Examples of these tools include:

• Windframe: A visual editor and AI tool designed specifically for Tailwind CSS. It features a drag-and-drop interface and a properties panel to adjust styles, then exports production-ready code.
• Pinegrow: A professional desktop web editor that has a dedicated Tailwind Visual Editor add-on. It allows you to visually edit your project and provides controls for all Tailwind properties.
• Shuffle (and Tailwind.build): An online editor with a large library of pre-built UI components. It allows you to drag components onto a canvas, customize their styles with visual controls, and export the final HTML.
• GrayGrids: Another online tool that functions as a "Tailwind CSS Website UI Builder" with drag-and-drop functionality.

These tools are excellent for rapidly building landing pages or prototyping. The primary trade-off is that for complex, dynamic applications, many developers find it faster and more precise to work directly in the code using the IntelliSense plugin (Solution 1).

Solution 3: Component Libraries (The Middle Ground)

There is a third option that also reduces the need to "memorize" individual classes: using pre-built component libraries.

The official Tailwind UI is the most popular example.

This is not a visual editor, but a paid library of over 500+ professionally designed components (navbars, forms, buttons, page sections, etc.).

• Your Workflow: Instead of building a complex form from 100 different utility classes, you find the form you need in the Tailwind UI library, copy its HTML, and paste it into your project.
• How it Helps: This solves the problem by giving you large, complete, and perfectly-styled blocks, so you only need to make minor adjustments (like changing bg-blue-500 to bg-indigo-500) rather than building everything from scratch.

Would you like me to elaborate on how to install and configure the Tailwind CSS IntelliSense plugin for VS Code?

This section explains why the MLA works cited page is important. It shows that giving credit builds credibility and helps readers find your sources. it also reminds you to put the works cited on its own page at the end of your paper.

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

Reviewer #1 (Evidence, reproducibility and clarity (Required)): * Summary: * In this manuscript, Turner AH. et al. demonstrated the viral replication in cells depleting Rab11B small GTPase, which is a paralogue of Rab11A. It has been reported that Rab11A is responsible for the intracellular transport of viral RNP via recycling endosomes. The authors showed that Rab11B knockdown reduced the viral protein expression and viral titer. This may be caused by reduced attachment of viral particles on Rab11B knockdown cells.

• Major comments:*
• Comment 1 Fig 2-4: The authors should provide Western blot results with equal amount of loading control (GAPDH). The bands shown in these figures lack quantifiability and are not reliable as data.*

We have rerun these western blots with more equal loading, and included a second loading control (beta-actin) in addition to the GAPDH. These blots can be seen in new Figures 2 and 3, and the quantification against both GAPDH (Figure 2/3) as well as actin (Fig S2) is now included. We have also included additional biological replicates for Fig 2 B-D. These additional experiments have strengthened our conclusion that Rab11B is required for efficient protein production in cells infected with recent H3N2, but not H1N1, isolates.

Comment 2 Fig 2-4: Why are the results different between Rab11B knockdown alone and Rab11A/B double knockdown? If the authors claims are correct, the results of Rab11B knockdown should be reproducible in Rab11A/B double knockdown cells.

Prior literature indicates that the Rab11A and Rab11B isoforms can play opposing roles in the trafficking of some cargos (ie, with one isoform transporting a molecule to the cell surface, while the other isoform takes it off again). In this scenario, it is possible that removing both 'halves' of the trafficking loop can ablate a phenotype. However, since our double knockdown used half the amount of siRNA for each isoform (for the same total amount), it is also possible this observation is simply the result of less efficient knockdown. In order to distinguish between these possibilities we depleted Rab11A or Rab11B individually, with this same 'half dose' of siRNA (see new Figure S3). We observed that Rab11B was still robustly required for H3N2 viral protein production. These results suggest that Rab11A and Rab11B could be playing mutually opposing roles in this case, which is consistent with prior Rab11 literature.

Comment 3 Fig 6: For better understanding, please provide a schematic illustration of experimental setting.

We have added a new graphical overview to this figure (see new Figure 6A).

Comment 4: It is necessary to test other siRNA sequences or perform a rescue experiment by expressing an siRNA-resistant clone in the knockdown cells. There seems to be an activation of host defense system, such as IFN pathways.

In order to rule out the possibility of off-target effects we created a novel cell line that inducibly expresses a Rab11B shRNA sequence (see new Fig 4). This knockdown strategy used a completely different method (shRNA delivered by lentiviral vector vs transient transfection of siRNA), in a different cellular background (H441 "club like" cells vs A549 lung adenocarcinoma). This new depletion strategy showed that the Rab11B dependent H3N2 protein production phenotype is seen across multiple knockdown strategies and cellular backgrounds.

**Referees cross-commenting**

I agree with other reviewers' comments in part.

Reviewer #1 (Significance (Required)):

The authors propose a novel role for Rab11B in modulating attachment pathway of H3N2 influenza A virus by unknown mechanism. Although previous studies focus on the function of Rab11A on endocytic transport, the function and specificity of Rab11B has remained less clear. The findings may be of interest to a broad audience, including researchers in cell biology, immunology, and host-pathogen interactions. However, the study remains at a superficial level of analysis and does not lead to a deeper understanding of the underlying mechanisms.

We agree with the reviewer that a strength of this manuscript is its multi-disciplinary nature, particularly with regard to advances in our understanding of Rab11B function. We have added a significant number of experiments and new figures to bolster the rigor and reproducibility of our findings. We have also added a new figure (Fig 7) that uses reverse genetics to map the Rab11B phenotype to the HA gene of the H3N2 isolate under study. By creating '7+1' reassortant viruses with the H3 HA or the N2 NA on a PR8 (H1N1) background (see Fig 7E-H) we were able to demonstrate that Rab11B is acting specifically on one of the HA-mediated entry steps. This provides additional mechanistic insight, by mapping the Rab11B-phenotype to a step at or prior to fusion. Fundamentally, we believe the novelty and rigor of our observation that recent H3N2 viruses enter through a different route than H1N1 isolates is worthy of observation in this updated form, so that the field can begin follow up studies.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Summary: The authors compare the effect of RAB11A and RAB11B knockdown on replication of contemporary H1N1 and H3N2 influenza A virus strains in A549 cells (human lung epithelials cells). They find a reduction in viral protein expression for tested H3N2 but not for H1N1 isolates. Mechanistically they suggest that RAB11A affects virion attachment to the cell surface.

Major comments: The provided data do not conclusively support the suggested mechanism of action and essential controls are missing to substantiate the authors claims: • Knockdown efficacy has to be confirmed on protein level, showing reduced levels of RAB11A and B by Western blot. This is a standard in the field. Off target effects cannot be avoided by RNAi approaches and are usually ruled out by using multiple siRNAs or by complementing the targeted protein in trans.

We have verified knockdown efficacy at the protein level in new Fig 1A/B. However, due to the high degree of protein level conservation between Rab11A and Rab11B it is very difficult to develop isoform specific antibodies, and we were unable to obtain a Rab11B-specific antibody that can detect endogenous protein (despite testing 6 commercially available antibodies for specificity). Using an antibody that detects both 11A and 11B (Fig1A) we were able to observe very slight changes in the molecular weight of the Rab11 band(s) detected upon knockdown of 11A vs 11B (suggestive of the two isoforms running as a dimer, with Rab11A the lower band and Rab11B the upper band). Cells depleted of both isoforms simultaneously showed a near complete loss of signal. Using a Rab11A antibody (that we confirmed as specific) we were able to observe loss of the Rab11A signal in both the 11A and 11A+B knockdowns (Fig 1B).

• Viral titers should be presented as absolute titers not as % (here the labelling is actually misleading in all graphs indicating pfu/ml)

This data is now shown in new Figure S1, where it is clear that the trends remain consistent across biological replicates. The axis labels of Fig 1D/E and Fig 3A have been corrected as requested to make clear we are normalizing to account for experiment-to-experiment variation in peak titer.

• Reduction of viral protein expression goes hand in hand with a reduction in GAPDH. While this is accounted for in the quantification a general block of protein expression cannot be ruled out since the stability of house keeper proteins and viral proteins might be different. Testing multiple house keeping proteins could overcome this issue.

We have included a second loading control (beta-actin) in addition to the GAPDH for new Figure 2 and 3. The quantification of viral protein production compared to beta actin is now included in new Fig S2. We have also included additional biological replicates for Fig 2 B-D. These additional experiments have strengthened our conclusion that Rab11B is required for efficient protein production in cells infected with recent H3N2, but not H1N1, isolates.

• The FACS data in Fig 5 are not convincing. The previous figures showed modest reduction in viral protein expression and the fluorescence is indicated here on a logarithmic scale. Quantification and indication of mean fluorescence intensity from the same data would be a better readout to convincingly show that less cells are infected.

We have reanalyzed the existing data to quantify the geometric mean of viral protein expression in the infected cell populations (new Figure 5D, E). This analysis shows no significant difference in geometric mean of HA (Fig 5D) or M2 (Fig 5E) expression between cells treated with NT, 11A or 11B siRNA. This additional analysis strengthens our original conclusion that when Rab11B is knocked down, fewer cells get infected, but those that do produce the same level of viral proteins.

• During the time of addition experiment in Fig 6, the authors are testing for HA/M2 positive cells after 16h of infection. This is a multicycle scnario so in a second round they would measure the effect of knockdown in absence of amonium chloride. Shorter infections up to 8h with higher MOI would overcome this problem.

By maintaining cells in ammonium chloride throughout the infection we are preventing endosomal acidification at any point in the infection period, so this experiment should be measuring solely the effect of one round of infection. The 16 hr timepoint was chosen to allow for optimized staining and analysis of samples by flow cytometry, within the available hours of the flow cytometry facility.

• Standard error of mean is not an appropriate way of representing experimental error for the provided results and should be replaced by SD. Correct labeling of axis with units is required.

We have updated the axes throughout the manuscript as requested. We have obtained additional statistical expertise (reflected in the updated author list) regarding the issue of SD vs SEM. Standard deviation (SD) would show a measure of the spread of the data, however the full distribution can be clearly seen as we plotted every individual data point. Standard error of the mean (SEM) is a measure of confidence for the mean of the population which takes into account SD and also sample size. SEM is not obvious to estimate by eye in the same way as SD, and we feel is more helpful to the reader to understand how likely the two population means differ from each other on a given graph.

Minor comments: • The authors show a rescue of viral replication upon double knockdown of RAB11A and B. Maybe this is just a consequence of inefficient knockdown since only half of the siRNAs were used?

In order to determine if this was the case we depleted Rab11A or Rab11B individually, with this same 'half dose' of siRNA (see new Figure S3). We observed that Rab11B was still robustly required for H3N2 viral protein production. These results suggest that Rab11A and Rab11B could be playing mutually opposing roles in this case (ie, Rab11B transporting a molecule to the surface, while Rab11A recycles it off), which is consistent with prior Rab11 literature.

• Specific experimental issues that are easily addressable. • Are prior studies referenced appropriately? • Are the text and figures clear and accurate? • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

• Reviewer #2 (Significance (Required)): Significance The authors claim an H3N2 specific dependency on RAB11B for early steps of infection. While this is per se interesting the provided data do not fully support the claims and lack a mechanistic explanation. What is the difference between H1 and H3 strains (virion shape, HA load per virion, attachment force of H1 vs H3). The readouts used are not close enough to the events with regards to timing and could be supported by established entry assays in the field.

We have provided additional discussion of the differences between H1s and H3s, including sialic acid binding preferences and changes in the HA-sialic acid avidity (lines 76-84). Notably, we have included a new assay (new Fig 7) that provides additional mechanistic insight into the observation that recent H3N2 but not H1N1 isolates depend on Rab11B early in infection. Using reverse genetics we were able to map the Rab11B phenotype to the HA gene of the H3N2 isolate under study. By creating '7+1' reassortant viruses with either the H3 HA or the N2 NA on a PR8 (H1N1) background (see Fig 7E) we are able to demonstrate that Rab11B is acting specifically at one of the HA-mediated entry steps. This excludes several non-HA dependent steps early in the life cycle (uncoating, RNP transport to the nucleus, nuclear import), thus providing additional confirmation that Rab11B acts at one of the earliest steps in the viral life cycle (and by definition, at or prior to fusion). Fundamentally, we believe the novelty and rigor of our observation that recent H3N2 viruses enter through a different route than H1N1 isolates is worthy of observation in this updated form, so that the field can begin follow up studies.

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

Manuscript Reference: RC-2025-03007 TITLE: Rab11B is required for binding and entry of recent H3N2, but not H1N1, influenza A isolates Allyson Turner, Sara Jaffrani, Hannah Kubinski, Deborah Ajayi, Matthew Owens, Madeline McTigue, Conor Fanuele, Cailey Appenzeller, Hannah Despres, Madaline Schmidt, Jessica Crothers, and Emily Bruce

Summary Here, Turner et al. build upon existing knowledge of Influenza A virus (IAV) dependence on the Rab11 family of proteins and provide insights into the specific role of Rab11B isoform in H3N2 virus binding and entry. The introduction is clearly written and provides sufficient background on prior research involving Rab11. It effectively identifies the current gap in knowledge and justifies the investigation of more clinically relevant, circulating strains of IAV. The methods section provides sufficient detail to ensure reproducibility. Similarly, the discussion is well structured, aligns with the introduction, and thoughtfully outlines relevant follow-up experiments. The authors present data from a series of experiments which suggest that the reduced H3N2 infection and viral protein production in Rab11B-depleted cells is due to impaired virus binding. While the evidence supports a Rab11B-specific phenotype in the context of H3N2 infection, we recommend additional experiments (outlined below), to further validate and strengthen these findings. These would help solidify the mechanistic link between Rab11B depletion and the observed phenotype for H3N2 strains of IAV.

Major comments Figure 1. (B) & (C) The authors normalise viral titers to the non-targeting control (NTC) siRNA set at 100. While this approach allows for relative comparisons, we recommend including the corresponding raw PFU/ml values, at least in the supplementary materials. This will better illustrate the biological significance of gene depletion and variability of the results.

We have included the raw PFU/mL values in new Figure S1, while peak viral production varied by biological replicate (pasted below, with each biological replicate having a differently shaped data point). While the depletion-induced trends are clearly visible across biological replicates, normalization to average titer in the NT condition for each replicate allows for cleaner visualization.

In addition, the current protocol uses a high MOI (1), and a relatively short infection period (16 hours) to capture single-cycle replication. However, to better assess the impact of gene knockdown on virus production and spread, we suggest performing a multicycle replication assay using a lower MOI (e.g, 0.01-0.001) over an extended time period, such as 48 hours before titration, provided that cell viability under these conditions is acceptable.

We appreciate this suggestion and repeatedly attempted to carry out a multicycle growth curve to obtain this data. Unfortunately, out of four independent biological replicates we attempted, we were only able to maintain cell viability and adherence in one biological replicate (shown below). We have not included this data in the revised manuscript due to the limited replicates we were able to obtain, though we can add it in a further revision if the reviewer feels it is warranted.

Figure 7. (B) & (C) The authors present interesting data showing that siRNA-mediated depletion of Rab11B reduces virion binding of a recently circulating strain of H3N2, but not H1N1, suggesting a subtype-specific role. However, we strongly recommend complementing this assay with a single-cell resolution approach such as immunofluorescence detection of surface-bound viruses through HA staining and image quantification. This would allow the authors to directly assess virion binding per cell and visualise the phenotype, strengthening the mechanistic insight on H3N2 binding in Rab11B-depleted cells. Furthermore, the data, particularly for H1N1 (Figure 7.C), shows substantial variance, which suggests a suboptimal assay sensitivity and limits the strength of the conclusion that the knockdown does not affect H1N1 binding, this limitation may be overcome by implementing the above experimental suggestion.

We have made substantial efforts to include this data, but were ultimately unable to include this assay due to technical difficulties in implementation (NA stripping caused cells to lift off coverslips, difficulties in antibody sensitivity and specificity, among other issues). We also piloted single cell-based flow cytometry assays to attempt to measure signal from bound virions, but were unable to achieve sufficient differentiation between mock and bound samples with the antibodies we could obtain. However, we have included a new experimental approach that is able to genetically map the 11B-dependent phenotype to the HA gene, thus providing additional mechanistic insight and confirming that Rab11B acts on one of the earliest steps in the viral life cycle (prior to or at fusion).

Minor comments General The authors should state which statistical test was used for each dataset in the respective figure legends.

This information is now included in each figure legend.

Figure 1. Suggest changing Y axis title to PFU/ml [relative to NTC]

We have changed the axis titles of normalized data to "PFU as % of NT" throughout.

The co-depletion of Rab11A and Rab11B appears to be less efficient than individual knockdowns, based on RT- qPCR data (Figure 1.A). It is possible that the partial 'rescue' phenotype observed in Figures 2-4 is due to incomplete knockdown, rather than a true biological interaction. This possibility should be acknowledged.

In order to distinguish between a partial 'rescue' and inefficient knockdown, we depleted Rab11A or Rab11B individually, with the same 'half dose' of siRNA used in the double knockdown (see new Figure S3). We observed that Rab11B was still robustly required for H3N2 viral protein production. These results suggest that Rab11A and Rab11B could be playing mutually opposing roles in this case, which is consistent with prior Rab11 literature, rather than simply inefficient knockdown.

Furthermore, knockdown efficiency is assessed only at the mRNA level. To strengthen the conclusions, the authors are encouraged to provide western blot data confirming protein-level depletion of Rab11A and Rab11B, particularly in the double knockdown condition. This would help clarify whether co-transfection of siRNAs affect the efficiency of each individual knockdown at the protein level.

We have verified knockdown efficacy at the protein level in new Fig 1A/B. However, due to the high degree of protein level conservation between Rab11A and Rab11B it is very difficult to develop isoform specific antibodies, and we were unable to obtain a Rab11B-specific antibody that can detect endogenous protein (despite testing 6 commercially available antibodies for specificity). Using an antibody that detects both 11A and 11B (Fig1A) we were able to observe very slight changes in the molecular weight of the Rab11 band(s) detected upon knockdown of 11A vs 11B (suggestive of the two isoforms running as a dimer, with Rab11A the lower band and Rab11B the upper band). Cells depleted of both isoforms simultaneously showed a near complete loss of signal. Using a Rab11A antibody (that we confirmed as specific) we were able to observe loss of the Rab11A signal in both the 11A and 11A+B knockdowns (Fig 1B).

Figure 6. (A) & (B) are missing error bars, particularly the Rab11B knockdown data points.

Error bars are plotted in each graph, but due to very limited experimental variation these error bars are too small to appear on the graph (11B points in Fig 6B, D).

Figure 7. If including any repeats in the binding assay, authors are encouraged to use appropriate controls in each experiment such as exogenous neuraminidase treatment or sialidase treatment.

When attempting to establish a microscopy based binding assay we included exogenous neuraminidase in each experiment. Unfortunately, the combination of glass coverslips and treatment with exogenous neuraminidase at incubation times sufficient to strip virus also removed cells from the coverslips.

Reviewer #3 (Significance (Required)):

General assessment: Provides a conceptual advancement of subtype specific receptor preferences.

Advance: The study raises interesting observations regarding influenza virus subtype differences in cell surface receptor binding, in a Rab11B-dependent manner.

Audience: Influenza virologists, respiratory virologists

Expertise: Virus entry, Virus cell biology

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Referee #3

Evidence, reproducibility and clarity

Title: Rab11B is required for binding and entry of recent H3N2, but not H1N1, influenza A isolates

Allyson Turner, Sara Jaffrani, Hannah Kubinski, Deborah Ajayi, Matthew Owens, Madeline McTigue, Conor Fanuele, Cailey Appenzeller, Hannah Despres, Madaline Schmidt, Jessica Crothers, and Emily Bruce

Summary

Here, Turner et al. build upon existing knowledge of Influenza A virus (IAV) dependence on the Rab11 family of proteins and provide insights into the specific role of Rab11B isoform in H3N2 virus binding and entry. The introduction is clearly written and provides sufficient background on prior research involving Rab11. It effectively identifies the current gap in knowledge and justifies the investigation of more clinically relevant, circulating strains of IAV. The methods section provides sufficient detail to ensure reproducibility. Similarly, the discussion is well structured, aligns with the introduction, and thoughtfully outlines relevant follow-up experiments. The authors present data from a series of experiments which suggest that the reduced H3N2 infection and viral protein production in Rab11B-depleted cells is due to impaired virus binding. While the evidence supports a Rab11B-specific phenotype in the context of H3N2 infection, we recommend additional experiments (outlined below), to further validate and strengthen these findings. These would help solidify the mechanistic link between Rab11B depletion and the observed phenotype for H3N2 strains of IAV.

Major comments

Figure 1. (B) & (C)

The authors normalise viral titers to the non-targeting control (NTC) siRNA set at 100. While this approach allows for relative comparisons, we recommend including the corresponding raw PFU/ml values, at least in the supplementary materials. This will better illustrate the biological significance of gene depletion and variability of the results. In addition, the current protocol uses a high MOI (1), and a relatively short infection period (16 hours) to capture single-cycle replication. However, to better assess the impact of gene knockdown on virus production and spread, we suggest performing a multicycle replication assay using a lower MOI (e.g, 0.01-0.001) over an extended time period, such as 48 hours before titration, provided that cell viability under these conditions is acceptable.

Figure 7. (B) & (C)

The authors present interesting data showing that siRNA-mediated depletion of Rab11B reduces virion binding of a recently circulating strain of H3N2, but not H1N1, suggesting a subtype-specific role. However, we strongly recommend complementing this assay with a single-cell resolution approach such as immunofluorescence detection of surface-bound viruses through HA staining and image quantification. This would allow the authors to directly assess virion binding per cell and visualise the phenotype, strengthening the mechanistic insight on H3N2 binding in Rab11B-depleted cells. Furthermore, the data, particularly for H1N1 (Figure 7.C), shows substantial variance, which suggests a suboptimal assay sensitivity and limits the strength of the conclusion that the knockdown does not affect H1N1 binding, this limitation may be overcome by implementing the above experimental suggestion.

Minor comments

General

The authors should state which statistical test was used for each dataset in the respective figure legends.

Figure 1.

Suggest changing Y axis title to PFU/ml [relative to NTC] The co-depletion of Rab11A and Rab11B appears to be less efficient than individual knockdowns, based on RT- qPCR data (Figure 1.A). It is possible that the partial 'rescue' phenotype observed in Figures 2-4 is due to incomplete knockdown, rather than a true biological interaction. This possibility should be acknowledged. Furthermore, knockdown efficiency is assessed only at the mRNA level. To strengthen the conclusions, the authors are encouraged to provide western blot data confirming protein-level depletion of Rab11A and Rab11B, particularly in the double knockdown condition. This would help clarify whether co-transfection of siRNAs affect the efficiency of each individual knockdown at the protein level.

Figure 6.

(A) & (B) are missing error bars, particularly the Rab11B knockdown data points.

Figure 7.

If including any repeats in the binding assay, authors are encouraged to use appropriate controls in each experiment such as exogenous neuraminidase treatment or sialidase treatment.

Significance

General assessment: Provides a conceptual advancement of subtype specific receptor preferences.

Advance: The study raises interesting observations regarding influenza virus subtype differences in cell surface receptor binding, in a Rab11B-dependent manner.

Audience: Influenza virologists, respiratory virologists

Expertise: Virus entry, Virus cell biology

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this manuscript, Turner AH. et al. demonstrated the viral replication in cells depleting Rab11B small GTPase, which is a paralogue of Rab11A. It has been reported that Rab11A is responsible for the intracellular transport of viral RNP via recycling endosomes. The authors showed that Rab11B knockdown reduced the viral protein expression and viral titer. This may be caused by reduced attachment of viral particles on Rab11B knockdown cells.

Major comments:

Comment 1 Fig 2-4: The authors should provide Western blot results with equal amount of loading control (GAPDH). The bands shown in these figures lack quantifiability and are not reliable as data.

Comment 2 Fig 2-4: Why are the results different between Rab11B knockdown alone and Rab11A/B double knockdown? If the authors claims are correct, the results of Rab11B knockdown should be reproducible in Rab11A/B double knockdown cells.

Comment 3 Fig 6: For better understanding, please provide a schematic illustration of experimental setting.

Comment 4: It is necessary to test other siRNA sequences or perform a rescue experiment by expressing an siRNA-resistant clone in the knockdown cells. There seems to be an activation of host defense system, such as IFN pathways.

Referees cross-commenting

I agree with other reviewers' comments in part.

Significance

The authors propose a novel role for Rab11B in modulating attachment pathway of H3N2 influenza A virus by unknown mechanism. Although previous studies focus on the function of Rab11A on endocytic transport, the function and specificity of Rab11B has remained less clear. The findings may be of interest to a broad audience, including researchers in cell biology, immunology, and host-pathogen interactions. However, the study remains at a superficial level of analysis and does not lead to a deeper understanding of the underlying mechanisms.

I think it might be useful to add an asterisk to this statement. In principle by tracking the cascade of G->B->P one might construct a more powerful model through biologically informed sparsity. However, it is crucial to keep in mind that while expression variation is partly driven by genotype, in a field setting realistically a significant fraction of expression might be attributable to environmental effects. (This tracks with the performance of expression only models in Sup fig 3/4). E.g. a stressed plant likely has a unique transcriptomic profile, and an easier to predict silking phenotype. However, these relationships won't translate to the G->B->P model since transcript abundance itself is not used during model inference. This effect is what likely explain the difference in performance between the expression RR models in sup fig3/4 and the G2B2P model.

The results from Fig 2 in the main text do not seem to suggest that the B2P model is substantially more powerful than the G2P model which might suggest that this could be an issue in the maize dataset. On the other hand the Fig 3/4 in the supplement do show (the expected) pattern that expression is the most powerful predictor of phenotype. I have struggled to reconcile the results from these two figures while reading the manuscript.

Here is my annotation...

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Reply to the reviewers

We will provide the revised manuscript as a PDF with highlighted changes, the Word file with tracked changes linked to reviewer comments, and all updated figures.

To address the reviewers' suggestions, we have conducted additional experiments that are now incorporated into new figures, or we have added new images to several existing figures where appropriate.

Please note that all figures have been renumbered to improve clarity and facilitate cross-referencing throughout the text. As recommended by Referee #3, all figure legends have been thoroughly revised to reflect these updates and are now labeled following the standard A-Z panel format, enhancing readability and ensuring easier identification. In addition, all figure legends now include the sample size for each statistical analysis.

For clarity and ease of reference, we provide below a comprehensive list of all figures included in the revised version. Figures that have undergone modifications are underlined.

Figure 1____. The first spermatogenesis wave in prepuberal mice.

This figure now includes amplified images of representative spermatocytes and a summary schematic illustrating the timeline of spermatogenesis. In addition, it now presents the statistical analysis of spermatocyte quantification to support the visual data.

__Figure 2.____ Cilia emerge across all stages of prophase I in spermatocytes during the first spermatogenesis wave. __

The images of this figure remain unchanged from the original submission, but all the graphs present now the statistical analysis of spermatocyte quantification.

Figure 3. Ultrastructure and markers of prepuberal meiotic cilia.

This figure remains unchanged from the original submission; however, we have replaced the ARL3-labelled spermatocyte image (A) with one displaying a clearer and more representative signal.

__Figure 4. Testicular tissue presents spermatocyte cysts in prepuberal mice and adult humans. __

This figure remains unchanged from the original submission.

__Figure 5. Cilia and flagella dynamics are correlated during prepuberal meiosis. __

This figure remains unchanged from the original submission.

__Figure 6. Comparative proteomics identifies potential regulators of ciliogenesis and flagellogenesis. __

This figure remains unchanged from the original submission.

Figure 7.____ Deciliation induces persistence of DNA damage in meiosis.

This figure has been substantially revised and now includes additional experiments analyzing chloral hydrate treatment, aimed at more accurately assessing DNA damage under both control and treated conditions. Images F-I and graph J are new.

Figure 8____. Aurora kinase A is a regulator of cilia disassembly in meiosis.

This figure is remodelled as the original version contained a mistake in previous panel II, for this, graph in new Fig.8 I has been corrected. In addition, it now contains additional data of αTubulin staining in arrested ciliated metaphases I after AURKA inhibition (new panel L1´).

__Figure 9. Schematic representation of the prepuberal versus adult seminiferous epithelium. __

This figure remains unchanged from the original submission.

__Supplementary Figure 1. Meiotic stages during the first meiotic wave. __

This figure remains unchanged from the original submission.

__Supplementary Figure 2 (new)____. __

This is a new figure that includes additional data requested by the reviewers. It includes additional markers of cilia in spermatocytes (glutamylated Tubulin/GT335), and the control data of cilia markers in non-ciliated spermatocytes. It also includes now the separated quantification of ciliated spermatocytes for each stage, as requested by reviewers, complementing graphs included in Figure 2.

Please note that with the inclusion of this new Supplementary Figure 2, the numbering of subsequent supplementary figures has been updated accordingly.

Supplementary Figure 3 (previously Suppl. Fig. 2)__. Ultrastructure of prophase I spermatocytes. __

This figure is equal in content to the original submission, but some annotations have been included.

Supplementary Figure 4 (previously Suppl. Fig. 3).__ Meiotic centrosome under the electron microscope. __

This figure remains unchanged from the original submission, but additional annotations have been included.

Supplementary Figure 5 (previously Suppl. Fig. 4)__. Human testis contains ciliated spermatocytes. __

This figure has been revised and now includes additional H2AX staining to better determine the stage of ciliated spermatocytes and improve their identification.

Supplementary Figure 6 (previously Suppl. Fig. 5). GLI1 and GLI3 readouts of Hedgehog signalling are not visibly affected in prepuberal mouse testes.

This figure has been remodeled and now includes the quantification of GLI1 and GLI3 and its corresponding statistical analysis. It also includes the control data for Tubulin, instead of GADPH.

Supplementary Figure 7 (previously Suppl. Fig. 6)__. CH and MLN8237 optimization protocol. __

This figure has been remodeled to incorporate control experiments using 1-hour organotypic culture treatment.

Supplementary Figure 8 (previously Suppl. Fig. 7)__. Tracking first meiosis wave with EdU pulse injection during prepubertal meiosis. __This figure remains unchanged from the original submission.

Supplementary Figure 9 (previously Suppl. Fig. 8)__. PLK1 and AURKA inhibition in cultured spermatocytes. __

This figure has been remodeled and now includes additional data on spindle detection in control and AURKA-inhibited spermatocytes (both ciliated and non ciliated).

__Response to the reviewers __

We will submit both the PDF version of the revised manuscript and the Word file with tracked changes relative to the original submission. Each modification made in response to reviewers' suggestions is annotated in the Word document within the corresponding section of the text.

A detailed, point-by-point response to each reviewer's comments is provided in the following section.

Response to the Referee #1

In this manuscript by Perez-Moreno et al., titled "The dynamics of ciliogenesis in prepubertal mouse meiosis reveal new clues about testicular maturation during puberty", the authors characterize the development of primary cilia during meiosis in juvenile male mice. The authors catalog a variety of testicular changes that occur as juvenile mice age, such as changes in testis weight and germ cell-type composition. They next show that meiotic prophase cells initially lack cilia, and ciliated meiotic prophase cells are detected after 20 days postpartum, coinciding with the time when post-meiotic spermatids within the developing testes acquire flagella. They describe that germ cells in juvenile mice harbor cilia at all substages of meiotic prophase, in contrast to adults where only zygotene stage meiotic cells harbor cilia. The authors also document that cilia in juvenile mice are longer than those in adults. They characterize cilia composition and structure by immunofluorescence and EM, highlighting that cilia polymerization may initially begin inside the cell, followed by extension beyond the cell membrane. Additionally, they demonstrate ciliated cells can be detected in adult human testes. The authors next perform proteomic analyses of whole testes from juvenile mice at multiple ages, which may not provide direct information about the extremely small numbers of ciliated meiotic cells in the testis, and is lacking follow up experiments, but does serve as a valuable resource for the community. Finally, the authors use a seminiferous tubule culturing system to show that chemical inhibition of Aurora kinase A likely inhibits cilia depolymerization upon meiotic prophase I exit and leads to an accumulation of metaphase-like cells harboring cilia. They also assess meiotic recombination progression using their culturing system, but this is less convincing.

Author response: We sincerely thank Ref #1 for the thorough and thoughtful evaluation of our manuscript. We are particularly grateful for the reviewer's careful reading and constructive feedback, which have helped us refine several sections of the text and strengthen our discussion. All comments and suggestions have been carefully considered and addressed, as detailed below.

__Major comments: __

1. There are a few issues with the experimental set up for assessing the effects of cilia depolymerization on DNA repair (Figure 7-II). First, how were mid pachytene cells identified and differentiated from early pachytene cells (which would have higher levels of gH2AX) in this experiment? I suggest either using H1t staining (to differentiate early/mid vs late pachytene) or the extent of sex chromosome synapsis. This would ensure that the authors are comparing similarly staged cells in control and treated samples. Second, what were the gH2AX levels at the starting point of this experiment? A more convincing set up would be if the authors measure gH2AX immediately after culturing in early and late cells (early would have higher gH2AX, late would have lower gH2AX), and then again after 24hrs in late cells (upon repair disruption the sampled late cells would have high gH2AX). This would allow them to compare the decline in gH2AX (i.e., repair progression) in control vs treated samples. Also, it would be informative to know the starting gH2AX levels in ciliated vs non-ciliated cells as they may vary.

Response:

We thank Ref #1 for this valuable comment, which significantly contributed to improving both the design and interpretation of the cilia depolymerization assay.

Following this suggestion, we repeated the experiment including 1-hour (immediately after culturing), and 24-hour cultures for both control and chloral hydrate (CH)-treated samples (n = 3 biological replicates). To ensure accurate staging, we now employ triple immunolabelling for γH2AX, SYCP3, and H1T, allowing clear distinction of zygotene (H1T−), early pachytene (H1T−), and late pachytene (H1T+) cells. The revised data (Figure 7) now provide a more complete and statistically robust analysis of DNA damage dynamics. These results confirm that CH-induced deciliation leads to persistence of the γH2AX signal at 24 hours, indicating impaired DNA repair progression in pachytene spermatocytes. The new images and graphs are included in the revised Figure 7.

Regarding the reviewer's final point about the comparison of γH2AX levels between ciliated and non-ciliated cells, we regret that direct comparison of γH2AX levels between ciliated and non-ciliated cells is not technically feasible. To preserve cilia integrity, all cilia-related imaging is performed using the squash technique, which maintains the three-dimensional structure of the cilia but does not allow reliable quantification of DNA damage markers due to nuclear distortion. Conversely, the nuclear spreading technique, used for DNA damage assessment, provides optimal visualization of repair foci but results in the loss of cilia due to cytoplasmic disruption during the hypotonic step. Given that spermatocytes in juvenile testes form developmentally synchronized cytoplasmic cysts, we consider that analyzing a statistically representative number of spermatocytes offers a valid and biologically meaningful measure of tissue-level effects.

In conclusion, we believe that the additional experiments and clarifications included in revised Figure 7 strengthen our conclusion that cilia depolymerization compromises DNA repair during meiosis. Further functional confirmation will be pursued in future works, since we are currently generating a conditional genetic model for a ciliopathy in our laboratory.

The authors analyze meiotic progression in cells cultured with/without AURKA inhibition in Figure 8-III and conclude that the distribution of prophase I cells does not change upon treatment. Is Figure 8-III A and B the same data? The legend text is incorrect, so it's hard to follow. Figure 8-III A shows a depletion of EdU-labelled pachytene cells upon treatment. Moreover, the conclusion that a higher proportion of ciliated zygotene cells upon treatment (Figure 8-II C) suggests that AURKA inhibition delays cilia depolymerization (page 13 line 444) does not make sense to me.

Response:

We thank Ref#1 for identifying this issue and for the careful examination of Figure 8. We discovered that the submitted version of Figure 8 contained a mismatch between the figure legend and the figure panels. The legend text was correct; however, the figure inadvertently included a non-corresponding graph (previously panel II-A), which actually belonged to Supplementary Figure 7 in the original submission. We apologize for this mistake.

This error has been corrected in the revised version. The updated Figure 8 now accurately presents the distribution of EdU-labelled spermatocytes across prophase I substages in control and AURKA-inhibited cultures (previously Figure 8-II B, now Figure 8-A). The corrected data show no significant differences in the proportions of EdU-labelled spermatocytes among prophase I substages after 24 hours of AURKA inhibition, confirming that meiotic progression is not delayed and that no accumulation of zygotene cells occurs under this treatment. Therefore, the observed increase in ciliated zygotene spermatocytes upon AURKA inhibition (new Figure 8 H-I) is best explained by a delay in cilia disassembly, rather than by an arrest or slowdown in meiotic progression. The figure legend and main text have been revised accordingly.

How do the authors know that there is a monopolar spindle in Figure 8-IV treated samples? Perhaps the authors can use a different Tubulin antibody (that does not detect only acetylated Tubulin) to show that there is a monopolar spindle.

Response:

We appreciate Ref#1 for this excellent suggestion. In the original submission (lines 446-447), we described that ciliated metaphase I spermatocytes in AURKA-inhibited samples exhibited monopolar spindle phenotypes. This description was based on previous reports showing that AURKA or PLK1 inhibition produces metaphases with monopolar spindles characterized by aberrant yet characteristic SYCP3 patterns, abnormal chromatin compaction, and circular bivalent alignment around non-migrated centrosomes (1). In our study, we observed SYCP3 staining consistent with these characteristic features of monopolar metaphases I.

However, we agree with Ref #1 that this could be better sustained with data. Following the reviewer's suggestion, we performed additional immunostaining using α-Tubulin, which labels total microtubules rather than only the acetylated fraction. For clarity purposes, the revised Figure 8 now includes α-Tubulin staining in the same ciliated metaphase I cells shown in the original submission, confirming the presence of defective microtubule polymerization and defective spindle organization. For clarity, we now refer to these ciliated metaphases I as "arrested MI". This new data further support our conclusion that AURKA inhibition disrupts spindle bipolarization and prevents cilia depolymerization, indicating that cilia maintenance and bipolar spindle organization are mechanistically incompatible events during male meiosis. The abstract, results, and discussion section has been expanded accordingly, emphasizing that the persistence of cilia may interfere with microtubule polymerization and centrosome separation under AURKA inhibition. The Discussion has been expanded to emphasize that persistence of cilia may interfere with centrosome separation and microtubule polymerization, contrasting with invertebrate systems -e.g. Drosophila (2) and P. brassicae (3)- in which meiotic cilia persist through metaphase I without impairing bipolar spindle assembly.

1. Alfaro, et al. EMBO Rep 22, (2021). DOI: 15252/embr.202051030 (PMID: 33615693)
2. Riparbelli et al . Dev Cell (2012) DOI: 1016/j.devcel.2012.05.024 (PMID: 22898783)
3. Gottardo et al, Cytoskeleton (Hoboken) (2023) DOI: 1002/cm.21755 (PMID: 37036073)

The authors state in the abstract that they provide evidence suggesting that centrosome migration and cilia depolymerization are mutually exclusive events during meiosis. This is not convincing with the data present in the current manuscript. I suggest amending this statement in the abstract.

Response:

We thank Ref#1 for this valuable observation, with which we fully agree. To avoid overstatement, the original statement has been removed from the Abstract, Results, and Discussion, and replaced with a more accurate formulation indicating that cilia maintenance and bipolar spindle formation are mutually exclusive events during mouse meiosis.

This revised statement is now directly supported by the new data presented in Figure 8, which demonstrate that AURKA inhibition prevents both spindle bipolarization and cilia depolymerization. We are grateful to the reviewer for highlighting this important clarification.

Minor comments:

The presence of cilia in all stages of meiotic prophase I in juvenile mice is intriguing. Why is the cellular distribution and length of cilia different in prepubertal mice compared to adults (where shorter cilia are present only in zygotene cells)? What is the relevance of these developmental differences? Do cilia serve prophase I functions in juvenile mice (in leptotene, pachytene etc.) that are perhaps absent in adults?

Related to the above point, what is the relevance of the absence of cilia during the first meiotic wave? If cilia serve a critical function during prophase I (for instance, facilitating DSB repair), does the lack of cilia during the first wave imply differing cilia (and repair) requirements during the first vs latter spermatogenesis waves?

In my opinion, these would be interesting points to discuss in the discussion section.

Response:

We thank the reviewer for these thoughtful observations, which we agree are indeed intriguing.

We believe that our findings likely reflect a developmental role for primary cilia during testicular maturation. We hypothesize that primary cilia at this stage might act as signaling organelles, receiving cues from Sertoli cells or neighboring spermatocytes and transmitting them through the cytoplasmic cysts shared by spermatocytes. Such intercellular communication could be essential for coordinating tissue maturation and meiotic entry during puberty. Although speculative, this hypothesis aligns with the established role of primary cilia as sensory and signaling hubs for GPCR and RTK pathways regulating cell differentiation and developmental patterning in multiple tissues (e.g., 1, 2). The Discussion section has been expanded to include these considerations.

1. Goetz et al, Nat Rev Genet (2010)- DOI: 1038/nrg2774 (PMID: 20395968)
2. Naturky et al , Cell (2019) DOI: 1038/s41580-019-0116-4 (PMID: 30948801) Our study focuses on the first spermatogenic wave, which represents the transition from the juvenile to the reproductive phase. It is therefore plausible that the transient presence of longer cilia during this period reflects a developmental requirement for external signaling that becomes dispensable in the mature testis. Given that this is only the second study to date examining mammalian meiotic cilia, there remains a vast area of research to explore. We plan to address potential signaling cascades involved in these processes in future studies.

On the other hand, while we cannot confirm that the cilia observed in zygotene spermatocytes persist until pachytene within the same cell, it is reasonable to speculate that they do, serving as longer-lasting signaling structures that facilitate testicular development during the critical pubertal window. In addition, the observation of ciliated spermatocytes at all prophase I substages at 20 dpp, together with our proteomic data, supports the idea that the emergence of meiotic cilia exerts a significant developmental impact on testicular maturation.

In summary, although we cannot yet define specific prophase I functions for meiotic cilia in juvenile spermatocytes, our data demonstrate that the first meiotic wave differs from later waves in cilia dynamics, suggesting distinct regulatory requirements between puberty and adulthood. These findings underscore the importance of considering developmental context when using the first meiotic wave as a model for studying spermatogenesis.

The authors state on page 9 lines 286-288 that the presence of cytoplasmic continuity via intercellular bridges (between developmentally synchronous spermatocytes) hints towards a mechanism that links cilia and flagella formation. Please clarify this statement. While the correlation between the timing of appearance of cilia and flagella in cells that are located within the same segment of the seminiferous tubule may be hinting towards some shared regulation, how would cytoplasmic continuity participate in this regulation? Especially since the cytoplasmic continuity is not between the developmentally distinct cells acquiring the cilia and flagella?

Response:

We thank Ref#1 for this excellent question and for the opportunity to clarify our statement.

The presence of intercellular bridges between spermatocytes is well known and has long been proposed to support germ cell communication and synchronization (1,2) as well as sharing mRNA (3) and organelles (4). A classic example is the Akap gene, located on the X chromosome and essential for the formation of the sperm fibrous sheath; cytoplasmic continuity through intercellular bridges allows Akap-derived products to be shared between X- and Y-bearing spermatids, thereby maintaining phenotypic balance despite transcriptional asymmetry (5). In addition, more recent work has further demonstrated that these bridges are critical for synchronizing meiotic progression and for processes such as synapsis, double-strand break repair, and transposon repression (6).

In this context, and considering our proteomic data (Figure 6), our statement did not intend to imply direct cytoplasmic exchange between ciliated and flagellated cells. Although our current methods do not allow comprehensive tracing of cytoplasmic continuity from the basal to the luminal compartment of the seminiferous epithelium, we plan to address this limitation using high-resolution 3D and ultrastructural imaging approaches in future studies.

Based on our current data, we propose that cytoplasmic continuity within developmentally synchronized spermatocyte cysts could facilitate the coordinated regulation of ciliogenesis, and similarly enable the sharing of regulatory factors controlling flagellogenesis within spermatid cysts. This coordination may occur through the diffusion of centrosomal or ciliary proteins, mRNAs, or signaling intermediates involved in the regulation of microtubule dynamics. However, we cannot exclude the possibility that such cytoplasmic continuity extends across all spermatocytes derived from the same spermatogonial clone, potentially providing a larger regulatory network.]] This mechanism could help explain the temporal correlation we observe between the appearance of meiotic cilia and the onset of flagella formation in adjacent spermatids within the same seminiferous segment.

We have revised the Discussion to explicitly clarify this interpretation and to note that, although hypothetical, it is consistent with established literature on cytoplasmic continuity and germ cell coordination.

1. Dym, et al. * Reprod.*(1971) DOI: 10.1093/biolreprod/4.2.195 (PMID: 4107186)
2. Braun et al. Nature. (1989) DOI: 1038/337373a0 (PMID: 2911388)
3. Greenbaum et al. * Natl. Acad. Sci. USA*(2006). DOI: 10.1073/pnas.0505123103 (PMID: 16549803)
4. Ventelä et al. Mol Biol Cell. (2003) DOI: 1091/mbc.e02-10-0647 (PMID: 12857863)
5. Turner et al. Journal of Biological Chemistry (1998). DOI: 1074/jbc.273.48.32135 (PMID: 9822690)
6. Sorkin, et al. Nat Commun (2025). DOI: 1038/s41467-025-56742-9 (PMID: 39929837)
7. *note: due to manuscript-length limitations, not all cited references can be included in the text; they are listed here to substantiate our response.*

Individual germ cells in H&E-stained testis sections in Figure 1-II are difficult to see. I suggest adding zoomed-in images where spermatocytes/round spermatids/elongated spermatids are clearly distinguishable.

Response:

Ref#1 is very right in this suggestion. We have revised Figure 1 to improve the quality of the H&E-stained testis sections and have added zoomed-in panels where spermatocytes, round spermatids, and elongated spermatids are clearly distinguishable. These additions significantly enhance the clarity and interpretability of the figure.

In Figure 2-II B, the authors document that most ciliated spermatocytes in juvenile mice are pachytene. Is this because most meiotic cells are pachytene? Please clarify. If the data are available (perhaps could be adapted from Figure 1-III), it would be informative to see a graph representing what proportions of each meiotic prophase substages have cilia.

Response:

We thank the reviewer for this valuable observation. Indeed, the predominance of ciliated pachytene spermatocytes reflects the fact that most meiotic cells in juvenile testes are at the pachytene stage (Figure 1). We have clarified this point in the text and have added a new supplementary figure (Supplementary Figure 2, new figure) presenting a graph showing the proportion of spermatocytes at each prophase I substage that possess primary cilia. This visualization provides a clearer quantitative overview of ciliation dynamics across meiotic substages.

I suggest annotating the EM images in Sup Figure 2 and 3 to make it easier to interpret.

Response:

We thank the reviewer for this helpful suggestion. We have now added annotations to the EM images in Supplementary Figures 3 and 4 to facilitate their interpretation. These visual guides help readers more easily identify the relevant ultrastructural features described in the text.

The authors claim that the ratio between GLI3-FL and GLI3-R is stable across their analyzed developmental window in whole testis immunoblots shown in Sup Figure 5. Quantifying the bands and normalizing to the loading control would help strengthen this claim as it hard to interpret the immunoblot in its current form.

Response:

We thank the reviewer for this valuable suggestion. Following this recommendation, Supplementary Figure 5 has been revised to include quantification of GLI1 and GLI3 protein levels, normalized to the loading control.

After quantification, we observed statistically significant differences across developmental stages. Specifically, GLI1 expression is slightly higher at 21 dpp compared to 8 dpp. For GLI3, we performed two complementary analyses:

• Total GLI3 protein (sum of full-length and repressor forms normalized to loading control) shows a progressive decrease during development, with the lowest levels at 60 dpp (Supplementary Figure 5D).
• GLI3 activation status, assessed as the GLI3-FL/GLI3-R ratio, is highest during the 19-21 dpp window, compared to 8 dpp and 60 dpp. Although these results suggest a possible transient activation of GLI3 during testicular maturation, we caution that this cannot automatically be attributed to increased Hedgehog signaling, as GLI3 processing can also be affected by other processes, such as changes in ciliogenesis. Furthermore, because the analysis was performed on whole-testis protein extracts, these changes cannot be specifically assigned to ciliated spermatocytes.

We have expanded the Discussion to address these findings and to highlight the potential involvement of the Desert Hedgehog (DHH) pathway, which plays key roles in testicular development, Sertoli-germ cell communication, and spermatogenesis (1, 2, 3). We plan to investigate these pathways further in future studies.

1. Bitgood et al. Curr Biol. (1996). DOI: 1016/s0960-9822(02)00480-3 (PMID: 8805249)
2. Clark et al. Biol Reprod. (2000) DOI: 1095/biolreprod63.6.1825 (PMID: 11090455)
3. O'Hara et al. BMC Dev Biol. (2011) DOI: 1186/1471-213X-11-72 (PMID: 22132805) *note: due to manuscript-length limitations, not all cited references can be included in the text; they are listed here to substantiate our response.

There are a few typos throughout the manuscript. Some examples: page 5 line 172, Figure 3-I legend text, Sup Figure 5-II callouts, Figure 8-III legend, page 15 line 508, page 17 line 580, page 18 line 611.

Response:

We thank the reviewer for detecting this. All typographical errors have been corrected, and figure callouts have been reviewed for consistency.

__ ____Response to the Referee #2__

__ __This study focuses on the dynamic changes of ciliogenesis during meiosis in prepubertal mice. It was found that primary cilia are not an intrinsic feature of the first wave of meiosis (initiating at 8 dpp); instead, they begin to polymerize at 20 dpp (after the completion of the first wave of meiosis) and are present in all stages of prophase I. Moreover, prepubertal cilia (with an average length of 21.96 μm) are significantly longer than adult cilia (10 μm). The emergence of cilia coincides temporally with flagellogenesis, suggesting a regulatory association in the formation of axonemes between the two. Functional experiments showed that disruption of cilia by chloral hydrate (CH) delays DNA repair, while the AURKA inhibitor (MLN8237) delays cilia disassembly, and centrosome migration and cilia depolymerization are mutually exclusive events. These findings represent the first detailed description of the spatiotemporal regulation and potential roles of cilia during early testicular maturation in mice. The discovery of this phenomenon is interesting; however, there are certain limitations in functional research.

We thank Ref#2 for taking the time to evaluate our manuscript and for summarizing its main findings. We regret that the reviewer did not find the study sufficiently compelling, but we respectfully clarify that the strength of our work lies precisely in addressing a largely unexplored aspect of mammalian meiosis for which virtually no prior data exist. Given the extremely limited number of studies addressing cilia in mammalian meiosis (only five to date, including our own previous publication on adult mouse spermatogenesis) (1-5), we consider that the present work provides the first robust and integrative evidence on the emergence, morphology, and potential roles of primary cilia during prepubertal testicular development. The study combines histology, high-resolution microscopy, proteomics, and pharmacological perturbations, supported by quantitative analyses, thereby establishing a solid and much-needed reference framework for future functional studies.

We emphasize that this manuscript constitutes the first comprehensive characterization of ciliogenesis during prepubertal mouse meiosis, complemented by functional in vitro assays that begin to address potential roles of these cilia. For this reason, we want to underscore the importance of this study in providing a solid framework that will support and guide future research

Major points:

1. The prepubertal cilia in spermatocytes discovered by the authors lack specific genetic ablation to block their formation, making it impossible to evaluate whether such cilia truly have functions. Because neither in the first wave of spermatogenesis nor in adult spermatogenesis does this type of cilium seem to be essential. In addition, the authors also imply that the formation of such cilia appears to be synchronized with the formation of sperm flagella. This suggests that the production of such cilia may merely be transient protein expression noise rather than a functionally meaningful cellular structure.

Response:

We agree that a genetic ablation model would represent the ideal approach to directly test cilia function in spermatogenesis. However, given the complete absence of prior data describing the dynamics of ciliogenesis during testis development, our priority in this study was to establish a rigorous structural and temporal characterization of this process in the main mammalian model organism, the mouse. This systematic and rigorous phenotypic characterization is a necessary first step before any functional genetics could be meaningfully interpreted.

To our knowledge, this study represents the first comprehensive analysis of ciliogenesis during prepubertal mouse meiosis, extending our previous work on adult spermatogenesis (1). Beyond these two contributions, only four additional studies have addressed meiotic cilia-two in zebrafish (2, 3), with Mytlys et al. also providing preliminary observations relevant to prepubertal male meiosis that we discuss in the present work, one in Drosophila (4) and a recent one in butterfly (5). No additional information exists for mammalian gametogenesis to date.

1. López-Jiménez et al. Cells (2022) DOI: 10.3390/cells12010142 (PMID: 36611937)
2. Mytlis et al. Science (2022) DOI: 10.1126/science.abh3104 (PMID: 35549308)
3. Xie et al. J Mol Cell Biol (2022) DOI: 10.1093/jmcb/mjac049 (PMID: 35981808)
4. Riparbelli et al . Dev Cell (2012) DOI: 10.1016/j.devcel.2012.05.024 (PMID: 22898783)
5. Gottardo et al, Cytoskeleton (Hoboken) (2023) DOI: 10.1002/cm.21755 (PMID: 37036073) We therefore consider this descriptive and analytical foundation to be essential before the development of functional genetic models. Indeed, we are currently generating a conditional genetic model for a ciliopathy in our laboratory. These studies are ongoing and will directly address the type of mechanistic questions raised here, but they extend well beyond the scope and feasible timeframe of the present manuscript.

We thus maintain that the present work constitutes a necessary and timely contribution, providing a robust reference dataset that will facilitate and guide future functional studies in the field of cilia and meiosis.

Taking this into account, we would be very pleased to address any additional, concrete suggestions from Ref#2 that could further strengthen the current version of the manuscript

The high expression of axoneme assembly regulators such as TRiC complex and IFT proteins identified by proteomic analysis is not particularly significant. This time point is precisely the critical period for spermatids to assemble flagella, and TRiC, as a newly discovered component of flagellar axonemes, is reasonably highly expressed at this time. No intrinsic connection with the argument of this paper is observed. In fact, this testicular proteomics has little significance.

Response:

We appreciate this comment but respectfully disagree with the reviewer's interpretation of our proteomic data. To our knowledge, this is the first proteomic study explicitly focused on identifying ciliary regulators during testicular development at the precise window (19-21 dpp) when both meiotic cilia and spermatid flagella first emerge.

While Piprek et al (1) analyzed the expression of primary cilia in developing gonads, proteomic data specifically covering the developmental transition at 19-21 dpp were not previously available. Furthermore, a recent cell-sorting study (2), detected expression of cilia proteins in pachytene spermatocytes compared to round spermatids, but did not explore their functional relevance or integrate these data with developmental timing or histological context.

In contrast, our dataset integrates histological staging, high-resolution microscopy, and quantitative proteomics, revealing a set of candidate regulators (including DCAF7, DYRK1A, TUBB3, TUBB4B, and TRiC) potentially involved in cilia-flagella coordination. We view this as a hypothesis-generating resource that outlines specific proteins and pathways for future mechanistic studies on both ciliogenesis and flagellogenesis in the testis.

Although we fully agree that proteomics alone cannot establish causal function, we believe that dismissing these data as having little significance overlooks their value as the first molecular map of the testis at the developmental window when axonemal structures arise. Our dataset provides, for the first time, an integrated view of proteins associated with ciliary and flagellar structures at the developmental stage when both axonemal organelles first appear. We thus believe that our proteomic dataset represents an important and novel contribution to the understanding of testicular development and ciliary biology.

Considering this, we would again welcome any specific suggestions from Ref#2 on additional analyses or clarifications that could make the relevance of this dataset even clearer to readers.

1. Piprek et al. Int J Dev Biol. (2019) doi: 10.1387/ijdb.190049rp (PMID: 32149371).
2. Fang et al. Chromosoma. (1981) doi: 10.1007/BF00285768 (PMID: 7227045).

Response to the Referee #3

In "The dynamics of ciliogenesis in prepubertal mouse meiosis reveals new clues about testicular development" Pérez-Moreno, et al. explore primary cilia in prepubertal mouse spermatocytes. Using a combination of microscopy, proteomics, and pharmacological perturbations, the authors carefully characterize prepubertal spermatocyte cilia, providing foundational work regarding meiotic cilia in the developing mammalian testis.

Response: We sincerely thank Ref#3 for their positive assessment of our work and for the thoughtful suggestions that have helped us strengthen the manuscript. We are pleased that the reviewer recognizes both the novelty and the relevance of our study in providing foundational insights into meiotic ciliogenesis during prepubertal testicular development. All specific comments have been carefully considered and addressed as detailed below.

Major concerns:

1. The authors provide evidence consistent with cilia not being present in a larger percentage of spermatocytes or in other cells in the testis. The combination of electron microscopy and acetylated tubulin antibody staining establishes the presence of cilia; however, proving a negative is challenging. While acetylated tubulin is certainly a common marker of cilia, it is not in some cilia such as those in neurons. The authors should use at least one additional cilia marker to better support their claim of cilia being absent.

Response:

We thank the reviewer for this helpful suggestion. In the revised version, we have strengthened the evidence for cilia identification by including an additional ciliary marker, glutamylated tubulin (GT335), in combination with acetylated tubulin and ARL13B (which were included in the original submission). These data are now presented in the new Supplementary Figure 2, which also includes an example of a non-ciliated spermatocyte showing absence of both ARL13B and AcTub signals.

Taken together, these markers provide a more comprehensive validation of cilia detection and confirm the absence of ciliary labelling in non-ciliated spermatocytes.

The conclusion that IFT88 localizes to centrosomes is premature as key controls for the IFT88 antibody staining are lacking. Centrosomes are notoriously "sticky", often sowing non-specific antibody staining. The authors must include controls to demonstrate the specificity of the staining they observe such as staining in a genetic mutant or an antigen competition assay.

Response:

We appreciate the reviewer's concern and fully agree that antibody specificity is critical when interpreting centrosomal localization. The IFT88 antibody used in our study is commercially available and has been extensively validated in the literature as both a cilia marker (1, 2), and a centrosome marker in somatic cells (3). Labelling of IFT88 in centrosomes has also been previously described using other antibodies (4, 5). In our material, the IFT88 signal consistently appears at one of the duplicated centrosomes and at both spindle poles-patterns identical to those reported in somatic cells. We therefore consider the reported meiotic IFT88 staining as specific and biologically reliable.

That said, we agree that genetic validation would provide the most definitive confirmation. We would like to inform that we are currently since we are currently generating a conditional genetic model for a ciliopathy in our laboratory that will directly assess both antibody specificity and functional consequences of cilia loss during meiosis. These experiments are in progress and will be reported in a follow-up study.

1. Wong et al. Science (2015). DOI: 1126/science.aaa5111 (PMID: 25931445)
2. Ocbina et al. Nat Genet (2011). DOI: 1038/ng.832 (PMID: 21552265)
3. Vitre et al. EMBO Rep (2020). DOI: 15252/embr.201949234 (PMID: 32270908)
4. Robert A. et al. J Cell Sci (2007). DOI: 1242/jcs.03366 (PMID: 17264151)
5. Singla et al, Developmental Cell (2010). DOI: 10.1016/j.devcel.2009.12.022 (PMID: 20230748) *note: due to manuscript-length limitations, not all cited references can be included in the text; they are listed here to substantiate our response.

There are many inconsistent statements throughout the paper regarding the timing of the first wave of spermatogenesis. For example, the authors state that round spermatids can be detected at 21dpp on line 161, but on line 180, say round spermatids can be detected a 19dpp. Not only does this lead to confusion, but such discrepancies undermine the validity of the rest of the paper. A summary graphic displaying key events and their timing in the first wave of spermatogenesis would be instrumental for reader comprehension and could be used by the authors to ensure consistent claims throughout the paper.

Response:

We thank the reviewer for identifying this inconsistency and apologize for the confusion. We confirm that early round spermatids first appear at 19 dpp, as shown in the quantitative data (Figure 1J). This can be detected in squashed spermatocyte preparations, where individual spermatocytes and spermatids can be accurately quantified. The original text contained an imprecise reference to the histological image of 21 dpp (previous line 161), since certain H&E sections did not clearly show all cell types simultaneously. However, we have now revised Figure 1, improving the image quality and adding a zoomed-in panel highlighting early round spermatids. Image for 19 dpp mice in Fig 1D shows early, yet still aflagellated spermatids. The first ciliated spermatocytes and the earliest flagellated spermatids are observed at 20 dpp. This has been clarified in the text.

In addition, we also thank the reviewer for the suggestion of adding a summary graphic, which we agree greatly facilitates reader comprehension. We have added a new schematic summary (Figure 1K) illustrating the key stages and timing of the first spermatogenic wave.

In the proteomics experiments, it is unclear why the authors assume that changes in protein expression are predominantly due to changes within the germ cells in the developing testis. The analysis is on whole testes including both the somatic and germ cells, which makes it possible that protein expression changes in somatic cells drive the results. The authors need to justify why and how the conclusions drawn from this analysis warrant such an assumption.

Response:

We agree with the reviewer that our proteomic analysis was performed on whole testis samples, which contain both germ and somatic cells. Although isolation of pure spermatocyte populations by FACS would provide higher resolution, obtaining sufficient prepubertal material for such analysis would require an extremely large number of animals. To remain compliant with the 3Rs principle for animal experimentation, we therefore used whole-testis samples from three biological replicates per age.

We acknowledge that our assumption-that the main differences arise from germ cells-is a simplification. However, germ cells constitute the vast majority of testicular cells during this developmental window and are the population undergoing major compositional changes between 15 dpp and adulthood. It is therefore reasonable to expect that a substantial fraction of the observed proteomic changes reflects alterations in germ cells. We have clarified this point in the revised text and have added a statement noting that changes in somatic cells could also contribute to the proteomic profiles.

The authors should provide details on how proteins were categorized as being involved in ciliogenesis or flagellogenesis, specifically in the distinction criteria. It is not clear how the categorizations were determined or whether they are valid. Thus, no one can repeat this analysis or perform this analysis on other datasets they might want to compare.

Response:

We thank the reviewer for this opportunity to clarify our approach. The categorization of protein as being involved in ciliogenesis or flagellogenesis was based on their Gene Ontology (GO) cellular component annotations obtained from the PANTHER database (Version 19.0), using the gene IDs of the Differentially Expressed Proteins (DEPs). Specifically, we used the GO terms cilium (GO:0005929) and motile cilium (GO:0031514). Since motile cilium is a subcategory of cilium, proteins annotated only with the general cilium term, but not included under motile cilium, were considered to be associated with primary cilia or with shared structural components common to different types of cilia. These GO terms are represented in the bottom panel of the Figure 6.

This information has been added to the Methods section and referenced in the Results for transparency and reproducibility.

In the pharmacological studies, the authors conclude that the phenotypes they observe (DNA damage and reduced pachytene spermatocytes) are due to loss of or persistence of cilia. This overinterprets the experiment. Chloral hydrate and MLN8237 certainly impact ciliation as claimed, but have additional cellular effects. Thus, it is possible that the observed phenotypes were not a direct result of cilia manipulation. Either additional controls must address this or the conclusions need to be more specific and toned down.

Response:

We thank the reviewer for this fair observation and have taken steps to strengthen and refine our interpretation. In the revised version, we now include data from 1-hour and 24-hour cultures for both control and chloral hydrate (CH)-treated samples (n = 3 biological replicates). The triple immunolabelling with γH2AX, SYCP3, and H1T allows accurate staging of zygotene (H1T⁻), early pachytene (H1T⁻), and late pachytene (H1T⁺) spermatocytes.

The revised Figure 7 now provides a more complete and statistically supported analysis of DNA damage dynamics, confirming that CH-induced deciliation leads to persistent γH2AX signal at 24 hours, indicative of delayed or defective DNA repair progression. We have also toned down our interpretation in the Discussion, acknowledging that CH could affect other cellular pathways.

As mentioned before, the conditional genetic model that we are currently generating will allow us to evaluate the role of cilia in meiotic DNA repair in a more direct and specific way.

Assuming the conclusions of the pharmacological studies hold true with the proper controls, the authors still conflate their findings with meiotic defects. Meiosis is not directly assayed, which makes this conclusion an overstatement of the data. The conclusions need to be rephrased to accurately reflect the data.

Response:

We agree that this aspect required clarification. As noted above, we have refined both the Results and Discussion sections to make clear that our assays specifically targeted meiotic spermatocytes.

We now present data for meiotic stages at zygotene, early pachytene and late pachytene. This is demonstrated with the labelling for SYCP3 and H1T, both specific marker for meiosis that are not detectable in non meiotic cells. We believe that this is indeed a way to assay the meiotic cells, however, we have specified now in the text that we are analysing potential defects in meiosis progression. We are sorry if this was not properly explained in the original manuscript: it is now rephrased in the new version both in the results and discussion section.

It is not clear why the authors chose not to use widely accepted assays of Hedgehog signaling. Traditionally, pathway activation is measured by transcriptional output, not GLI protein expression because transcription factor expression does not necessarily reflect transcription levels of target genes.

Response:

We agree with the reviewer that measuring mRNA levels of Hedgehog pathway target genes, typically GLI1 and PTCH1, is the most common method for measuring pathway activation, and is widely accepted by researchers in the field. However, the methods we use in this manuscript (GLI1 and GLI3 immunoblots) are also quite common and widely accepted:

Regarding GLI1 immunoblot, many articles have used this method to monitor Hedgehog signaling, since GLI1 protein levels have repeatedly been shown to also go up upon pathway activation, and down upon pathway inhibition, mirroring the behavior of GLI1 mRNA. Here are a few publications that exemplify this point:

• Banday et al. 2025 Nat Commun. DOI: 10.1038/s41467-025-56632-0 (PMID: 39894896)
• Shi et al 2022 JCI Insight DOI: 10.1172/jci.insight.149626 (PMID: 35041619)
• Deng et al. 2019 eLife, DOI: 10.7554/eLife.50208 (PMID: 31482846)
• Zhu et al. 2019 Nat Commun, DOI: 10.1038/s41467-019-10739-3 (PMID: 31253779)
• Caparros-Martin et al 2013 Hum Mol Genet, DOI: 10.1093/hmg/dds409 (PMID: 23026747) *note: due to manuscript-length limitations, not all cited references can be included in the text; they are listed here to substantiate our response.

As for GLI3 immunoblot, Hedgehog pathway activation is well known to inhibit GLI3 proteolytic processing from its full length form (GLI3-FL) to its transcriptional repressor (GLI3-R), and such processing is also commonly used to monitor Hedgehog signal transduction, of which the following are but a few examples:

• Pedraza et al 2025 eLife, DOI: 10.7554/eLife.100328 (PMID: 40956303)
• Somatilaka et al 2020 Dev Cell, DOI: 10.1016/j.devcel.2020.06.034 (PMID: 32702291)
• Infante et al 2018, Nat Commun, DOI: 10.1038/s41467-018-03339-0 (PMID: 29515120)
• Wang et al 2017 Dev Biol DOI: 10.1016/j.ydbio.2017.08.003 (PMID: 28800946)
• Singh et al 2015 J Biol Chem DOI: 10.1074/jbc.M115.665810 (PMID: 26451044)
• *note: due to manuscript-length limitations, not all cited references can be included in the text; they are listed here to substantiate our response.*

In summary, we think that we have used two well established markers to look at Hedgehog signaling (three, if we include the immunofluorescence analysis of SMO, which we could not detect in meiotic cilia).

These Hh pathway analyses did not provide any convincing evidence that the prepubertal cilia we describe here are actively involved in this pathway, even though Hh signaling is cilia-dependent and is known to be active in the male germline (Sahin et al 2014 Andrology PMID: 24574096; Mäkelä et al 2011 Reproduction PMID: 21893610; Bitgood et al 1996 Curr Biol. PMID: 8805249).

That said, we fully agree that our current analyses do not allow us to draw definitive conclusions regarding Hedgehog pathway activity in meiotic cilia, and we now state this explicitly in the revised Discussion.

Also in the Hedgehog pathway experiment, it is confusing that the authors report no detection of SMO yet detect little to no expression of GLIR in their western blot. Undetectable SMO indicates Hedgehog signaling is inactive, which results in high levels of GLIR. The impact of this is that it is not clear what is going on with Hh signaling in this system.

Response:

It is true that, when Hh signaling is inactive (and hence SMO not ciliary), the GLI3FL/GLI3R ratio tends to be low.

Although our data in prepuberal mouse testes show a strong reduction in total GLI3 protein levels (GLI3FL+GLI3R) as these mice grow older, this downregulation of total GLI3 occurs without any major changes in the GLI3FL/GLI3R ratio, which is only modestly affected (suppl. Figure 6).

Hence, since it is the ratio that correlates with Hh signaling rather than total levels, we do not think that the GLI3R reduction we see is incompatible with our non-detection of SMO in cilia: it seems more likely that overall GLI3 expression is being downregulated in developing testes via a Hh-independent mechanism.

Also potentially relevant here is the fact that some cell types depend more on GLI2 than on GLI3 for Hh signaling. For instance, in mouse embryos, Hh-mediated neural tube patterning relies more heavily on GLI2 processing into a transcriptional activator than on the inhibition of GLI3 processing into a repressor. In contrast, the opposite is true during Hh-mediated limb bud patterning (Nieuwenhuis and Hui 2005 Clin Genet. PMID: 15691355). We have not looked at GLI2, but it is conceivable that it could play a bigger role than GLI3 in our model.

Moreover, several forms of GLI-independent non-canonical Hh signaling have been described, and they could potentially play a role in our model, too (Robbins et al 2012 Sci Signal. PMID: 23074268).

We have revised the discussion to clarify some of these points.

All in all, we agree that our findings regarding Hh signaling are not conclusive, but we still think they add important pieces to the puzzle that will help guide future studies.

There are multiple instances where it is not clear whether the authors performed statistical analysis on their data, specifically when comparing the percent composition of a population. The authors need to include appropriate statistical tests to make claims regarding this data. While the authors state some impressive sample sizes, once evaluated in individual categories (eg specific cell type and age) the sample sizes of evaluated cilia are as low as 15, which is likely underpowered. The authors need to state the n for each analysis in the figures or legends.

We thank the reviewer for highlighting this important issue. We have now included the sample size (n) for every analysis directly in the figure legends. Although this adds length, it improves transparency and reproducibility.

Regarding the doubts of Ref#3 about the different sample sizes, the number of spermatocytes quantified in each stage is in agreement with their distribution in meiosis (example, pachytene lasts for 10 days this stage is widely represented in the preparations, while its is much difficult to quantify metaphases I that are less present because the stage itself lasts for less than 24hours). Taking this into account, we ensured that all analyses remain statistically valid and representative, applying the appropriate statistical tests for each dataset. These details are now clearly indicated in the revised figures and legends.

Minor concerns:

1. The phrase "lactating male" is used throughout the paper and is not correct. We assume this term to mean male pups that have yet to be weaned from their lactating mother, but "lactating male" suggests a rare disorder requiring medical intervention. Perhaps "pre-weaning males" is what the authors meant.

Response:

We thank the reviewer for noticing this terminology error. The expression has been corrected to "pre-weaning males" throughout the manuscript.

The convention used to label the figures in this paper is confusing and difficult to read as there are multiple panels with the same letter in the same figure (albeit distinct sections). Labeling panels in the standard A-Z format is preferred. "Panel Z" is easier to identify than "panel III-E".

Response:

We thank the reviewer for this suggestion. All figures have been relabelled using the standard A-Z panel format, ensuring consistency and easier readability across the manuscript.

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Referee #3

Evidence, reproducibility and clarity

Summary:

In "The dynamics of ciliogenesis in prepubertal mouse meiosis reveals new clues about testicular development" Pérez-Moreno, et al. explore primary cilia in prepubertal mouse spermatocytes. Using a combination of microscopy, proteomics, and pharmacological perturbations, the authors carefully characterize prepubertal spermatocyte cilia, providing foundational work regarding meiotic cilia in the developing mammalian testis.

Major concerns:

1. The authors provide evidence consistent with cilia not being present in a larger percentage of spermatocytes or in other cells in the testis. The combination of electron microscopy and acetylated tubulin antibody staining establishes the presence of cilia; however, proving a negative is challenging. While acetylated tubulin is certainly a common marker of cilia, it is not in some cilia such as those in neurons. The authors should use at least one additional cilia marker to better support their claim of cilia being absent.

2. The conclusion that IFT88 localizes to centrosomes is premature as key controls for the IFT88 antibody staining are lacking. Centrosomes are notoriously "sticky", often sowing non-specific antibody staining. The authors must include controls to demonstrate the specificity of the staining they observe such as staining in a genetic mutant or an antigen competition assay.

3. There are many inconsistent statements throughout the paper regarding the timing of the first wave of spermatogenesis. For example, the authors state that round spermatids can be detected at 21dpp on line 161, but on line 180, say round spermatids can be detected a 19dpp. Not only does this lead to confusion, but such discrepancies undermine the validity of the rest of the paper. A summary graphic displaying key events and their timing in the first wave of spermatogenesis would be instrumental for reader comprehension and could be used by the authors to ensure consistent claims throughout the paper.

4. In the proteomics experiments, it is unclear why the authors assume that changes in protein expression are predominantly due to changes within the germ cells in the developing testis. The analysis is on whole testes including both the somatic and germ cells, which makes it possible that protein expression changes in somatic cells drive the results. The authors need to justify why and how the conclusions drawn from this analysis warrant such an assumption.

5. The authors should provide details on how proteins were categorized as being involved in ciliogenesis or flagellogenesis, specifically in the distinction criteria. It is not clear how the categorizations were determined or whether they are valid. Thus, no one can repeat this analysis or perform this analysis on other datasets they might want to compare.

6. In the pharmacological studies, the authors conclude that the phenotypes they observe (DNA damage and reduced pachytene spermatocytes) are due to loss of or persistence of cilia. This overinterprets the experiment. Chloral hydrate and MLN8237 certainly impact ciliation as claimed, but have additional cellular effects. Thus, it is possible that the observed phenotypes were not a direct result of cilia manipulation. Either additional controls must address this or the conclusions need to be more specific and toned down.

7. Assuming the conclusions of the pharmacological studies hold true with the proper controls, the authors still conflate their findings with meiotic defects. Meiosis is not directly assayed, which makes this conclusion an overstatement of the data. The conclusions need to be rephrased to accurately reflect the data.

8. It is not clear why the authors chose not to use widely accepted assays of Hedgehog signaling. Traditionally, pathway activation is measured by transcriptional output, not GLI protein expression because transcription factor expression does not necessarily reflect transcription levels of target genes.

9. Also in the Hedgehog pathway experiment, it is confusing that the authors report no detection of SMO yet detect little to no expression of GLIR in their western blot. Undetectable SMO indicates Hedgehog signaling is inactive, which results in high levels of GLIR. The impact of this is that it is not clear what is going on with Hh signaling in this system.

10. There are multiple instances where it is not clear whether the authors performed statistical analysis on their data, specifically when comparing the percent composition of a population. The authors need to include appropriate statistical tests to make claims regarding this data. While the authors state some impressive sample sizes, once evaluated in individual categories (eg specific cell type and age) the sample sizes of evaluated cilia are as low as 15, which is likely underpowered. The authors need to state the n for each analysis in the figures or legends.

Minor concerns:

1. The phrase "lactating male" is used throughout the paper and is not correct. We assume this term to mean male pups that have yet to be weaned from their lactating mother, but "lactating male" suggests a rare disorder requiring medical intervention. Perhaps "pre-weaning males" is what the authors meant.

2. The convention used to label the figures in this paper is confusing and difficult to read as there are multiple panels with the same letter in the same figure (albeit distinct sections). Labeling panels in the standard A-Z format is preferred. "Panel Z" is easier to identify than "panel III-E".

Significance

Overall, this is a well-done body of work that deserves recognition for the novel and implicative discoveries it presents. Assuming the conclusions hold true following appropriate statistical analysis and rephrasing, this paper would report the first documented evidence of meiotic cilia in the developing mammalian testis with sufficient rigor to become the foundational work on this topic.

This paper will be of interest to communities focused on germ cell development, cilia, and Hedgehog signaling. It may prompt a new perspective on Desert Hedgehog signaling as it pertains to spermatogenesis. Further, this work will be of interest to those studying male fertility, as it highlights the potential role of cilia in spermatogenesis.

Further, the proteomic analysis presented has the potential to invoke hypotheses and experimentation investigating the role of several proteins with previously uncharacterized roles in ciliogenesis, flagellogenesis, and/or spermatogenesis. The finding that the onset of ciliogenesis and flagellogenesis appear to be temporally linked has the potential to prompt research regarding shared molecular mechanisms dictating axonemal formation. We believe this paper has the potential to have an impact in its respective field, underscored by the exquisite microscopy and detailed characterization of meiotic cilia.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript by Perez-Moreno et al., titled "The dynamics of ciliogenesis in prepubertal mouse meiosis reveal new clues about testicular maturation during puberty", the authors characterize the development of primary cilia during meiosis in juvenile male mice. The authors catalog a variety of testicular changes that occur as juvenile mice age, such as changes in testis weight and germ cell-type composition. They next show that meiotic prophase cells initially lack cilia, and ciliated meiotic prophase cells are detected after 20 days postpartum, coinciding with the time when post-meiotic spermatids within the developing testes acquire flagella. They describe that germ cells in juvenile mice harbor cilia at all substages of meiotic prophase, in contrast to adults where only zygotene stage meiotic cells harbor cilia. The authors also document that cilia in juvenile mice are longer than those in adults. They characterize cilia composition and structure by immunofluorescence and EM, highlighting that cilia polymerization may initially begin inside the cell, followed by extension beyond the cell membrane. Additionally, they demonstrate ciliated cells can be detected in adult human testes. The authors next perform proteomic analyses of whole testes from juvenile mice at multiple ages, which may not provide direct information about the extremely small numbers of ciliated meiotic cells in the testis, and is lacking follow up experiments, but does serve as a valuable resource for the community. Finally, the authors use a seminiferous tubule culturing system to show that chemical inhibition of Aurora kinase A likely inhibits cilia depolymerization upon meiotic prophase I exit and leads to an accumulation of metaphase-like cells harboring cilia. They also assess meiotic recombination progression using their culturing system, but this is less convincing.

Few suggestions/comments are listed below:

Major comments

1. There are a few issues with the experimental set up for assessing the effects of cilia depolymerization on DNA repair (Figure 7-II). First, how were mid pachytene cells identified and differentiated from early pachytene cells (which would have higher levels of gH2AX) in this experiment? I suggest either using H1t staining (to differentiate early/mid vs late pachytene) or the extent of sex chromosome synapsis. This would ensure that the authors are comparing similarly staged cells in control and treated samples. Second, what were the gH2AX levels at the starting point of this experiment? A more convincing set up would be if the authors measure gH2AX immediately after culturing in early and late cells (early would have higher gH2AX, late would have lower gH2AX), and then again after 24hrs in late cells (upon repair disruption the sampled late cells would have high gH2AX). This would allow them to compare the decline in gH2AX (i.e., repair progression) in control vs treated samples. Also, it would be informative to know the starting gH2AX levels in ciliated vs non-ciliated cells as they may vary.

2. The authors analyze meiotic progression in cells cultured with/without AURKA inhibition in Figure 8-III and conclude that the distribution of prophase I cells does not change upon treatment. Is Figure 8-III A and B the same data? The legend text is incorrect, so it's hard to follow. Figure 8-III A shows a depletion of EdU-labelled pachytene cells upon treatment. Moreover, the conclusion that a higher proportion of ciliated zygotene cells upon treatment (Figure 8-II C) suggests that AURKA inhibition delays cilia depolymerization (page 13 line 444) does not make sense to me.

3. How do the authors know that there is a monopolar spindle in Figure 8-IV treated samples? Perhaps the authors can use a different Tubulin antibody (that does not detect only acetylated Tubulin) to show that there is a monopolar spindle.

4. The authors state in the abstract that they provide evidence suggesting that centrosome migration and cilia depolymerization are mutually exclusive events during meiosis. This is not convincing with the data present in the current manuscript. I suggest amending this statement in the abstract.

Minor comments

1. The presence of cilia in all stages of meiotic prophase I in juvenile mice is intriguing. Why is the cellular distribution and length of cilia different in prepubertal mice compared to adults (where shorter cilia are present only in zygotene cells)? What is the relevance of these developmental differences? Do cilia serve prophase I functions in juvenile mice (in leptotene, pachytene etc.) that are perhaps absent in adults?

Related to the above point, what is the relevance of the absence of cilia during the first meiotic wave? If cilia serve a critical function during prophase I (for instance, facilitating DSB repair), does the lack of cilia during the first wave imply differing cilia (and repair) requirements during the first vs latter spermatogenesis waves?

In my opinion, these would be interesting points to discuss in the discussion section.

1. The authors state on page 9 lines 286-288 that the presence of cytoplasmic continuity via intercellular bridges (between developmentally synchronous spermatocytes) hints towards a mechanism that links cilia and flagella formation. Please clarify this statement. While the correlation between the timing of appearance of cilia and flagella in cells that are located within the same segment of the seminiferous tubule may be hinting towards some shared regulation, how would cytoplasmic continuity participate in this regulation? Especially since the cytoplasmic continuity is not between the developmentally distinct cells acquiring the cilia and flagella?

2. Individual germ cells in H&E-stained testis sections in Figure 1-II are difficult to see. I suggest adding zoomed-in images where spermatocytes/round spermatids/elongated spermatids are clearly distinguishable.

3. In Figure 2-II B, the authors document that most ciliated spermatocytes in juvenile mice are pachytene. Is this because most meiotic cells are pachytene? Please clarify. If the data are available (perhaps could be adapted from Figure 1-III), it would be informative to see a graph representing what proportions of each meiotic prophase substages have cilia.

4. I suggest annotating the EM images in Sup Figure 2 and 3 to make it easier to interpret.

5. The authors claim that the ratio between GLI3-FL and GLI3-R is stable across their analyzed developmental window in whole testis immunoblots shown in Sup Figure 5. Quantifying the bands and normalizing to the loading control would help strengthen this claim as it hard to interpret the immunoblot in its current form.

6. There are a few typos throughout the manuscript. Some examples: page 5 line 172, Figure 3-I legend text, Sup Figure 5-II callouts, Figure 8-III legend, page 15 line 508, page 17 line 580, page 18 line 611.

Significance

This work provides new information about an important but poorly understood cellular structure present in meiotic cells, the primary cilium. More generally, this work expands on our understanding of testis development in juvenile mice. The microscopy images presented here are beautiful. The work is mostly descriptive but lays the groundwork for future investigations. I believe that this study would of interest to the germ cell, meiosis, and spermatogenesis communities, and with a few modifications, is suitable for publication.

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Reply to the reviewers

We would like to thank the three reviewers for their careful reading of our manuscript and suggested modifications. We have incorporated their suggestions as described below; these changes have significantly improved the structure and focus of the manuscript.

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

The possibility of observing 3D cellular organisation in tissues at nanometre resolution is a hope for many cell biologists. Here, the authors have combined two volume electron microscopy approaches with scanning electron microscopy: Focused Ion Beam (FIB-SEM) and Array Tomography (AT-SEM) to study the evolution of the shape and organisation of cytoplasmic bridges, the 'ring canals' (RCs) in the Drosophila ovarian follicle that connect nurse cells and oocyte. This type of cytoplasmic link, found in insects and humans, is essential for oocyte development.

RCs have mainly been studied using light microscopy with various markers that constitute them, but this approach does not fully capture an overall view of their organization. Due to their three-dimensional arrangement within the ovarian follicle, characterizing their organization using transmission electron microscopy (TEM) has been very limited until now. This v-EM study allows the authors to document the evolution of RC size and thickness during the development of germline cysts, from the germarium to stage 4, and potentially beyond. This study confirmed previous findings, namely that RC size correlates with lineage: the largest RC is formed after the first division, while the smallest is formed during the last division.

Furthermore, this work allowed a better characterisation of the membrane interdigitation surrounding the RCs. In addition, the authors highlight the important potential of v-EM for further structural analysis of the fusome, migrating border cells and the stem cell niche.

Majors comment

The output of this work can be divided into two parts. First, this work presents a technical challenge, involving image acquisition by volume electron microscopy and manual 3D reconstruction of the contours of the membranes, nuclei, RCs, and fusome in different cysts at different stages.

Secondly, this work is based on a structural study of the RCs and their associated membranes. This work is descriptive but important, although the results largely confirm previous findings, both for the structure of the RCs and their relationship to the division sequence of the cyst cells, and for the organisation of the membranes around the RCs.

Very interestingly, the authors report the spatial characterisation of membrane structures associated with and close to CRs that have already been identified (Loyer et al.). However, their characterisation is somewhat incomplete, as it lacks quantified data - how many CRs were analysed? and, above all, the characteristics of these membranes, their length and orientation according to their position and their connection in the lineage - these data could be obtained from the VEM data already collected and would be an important addition to the RC structural analysis in this work.

*Following the suggestions of this reviewer, we have reduced the emphasis on the technical approach to better highlight the ring canal data. We have summarized the ring canal measurements in graphs presented in Fig. 4B, C and included the sample sizes for these measurements in the figure legend. *

• To gain further insight into the membrane interdigitations, we have developed a detailed model of the oocyte and four ring canals that connect to the posterior nurse cells of the stage 4 egg chamber (Fig. 5). From this model, we see that the interdigitations are longer and more abundant that in the germarium (Fig. S5), but not as extensive as in the stage 8 egg chamber (Fig. 6). The interdigitations were not all oriented in the same direction, and we did not observe an obvious correlation between interdigitation number, orientation, and lineage. We plan to continue to explore these structures in future studies. *

In line with this, the authors importantly report the presence of an ER-like membrane structure lining the RCs. First, it would be nice to have statistics to support the observation of how many RCs..? Secondly, does this ER membrane structure vary according to the position of the RC in the cyst, are they related to the RC lineage?

*We appreciate the reviewer's interest in this novel ER-like structure lining the ring canals. We have generated a detailed model of these structures within the stage 4 egg chamber (Fig. 5D,E). However, because we do not have data from a large number of egg chambers, we believe that performing statistics would not be appropriate. *

The addition of graphs showing the quantitative data with statistics in the figures would improve understanding of the results. This is particularly the case for the characterisation of RCs according to the stage of cyst development, as shown in Figure 3. This also applies to the characterisation of RCs within a cyst and the relationship between RC size and lineage, as shown in Figure 4, and to the characterisation (thickness) of the inner part of the RC.

*We have included graphs of ring canal diameter based on stage (Fig. 4B) or lineage (Fig. 4C); however, because we only have data from a few germline cysts, we have not performed any statistical analysis. *

The part on the structural analysis of the fusome is interesting but still secondary to the characterisation of the RCs. This part should be moved to the results and figures after the various parts concerning the RCs.

        *We have deemphasized the fusome structural analysis in the results section; however, we chose to leave these images in the figures, since there could be a connection between the novel ER-like structures and the fusome.  *

Minor comments The distribution of the fusome in Figure 2 is difficult to see with Hts labelling and does not really correspond to the schematic, especially in regions 2a and 2B.

*We have modified the images and the schematic. *

In panel C of Figure 2, it is a little disturbing that the legend is directly on the image of RC. It hides some information about the images and could be placed at the bottom of the panel. This also the case for the panel G.

We understand the possible confusion and have changed the layout in the figure.

With figure 3B, it would be good to highlight the position of cyst.

We have pseudocolored the portion that corresponds to the relevant cyst in the same color used for the reconstruction (which is now Fig. 3A).

Reviewer #1 (Significance (Required)): As mentioned above, this work can be divided into two parts. The part corresponding to the acquisition of images by volume electron microscopy and manual 3D reconstruction is new and a great source of valuable information. The part related to the spatial characterisation of the RC is important, but corresponds more to an extension and reinforcement of previously available information than to the contribution of significant new insights. I think it will be of great interest to an audience interested in Drosophila oogenesis.

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

This study presents a high-resolution volumetric analysis of germline ring canals (RCs) during Drosophila oogenesis. By combining two complementary electron microscopy techniques-Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and Array Tomography Scanning Electron Microscopy (AT-SEM)-the authors compare RC structural features at different developmental stages, ranging from the relatively small germarium to the significantly larger, later-stage egg chambers.

At early stages of oogenesis, FIB-SEM analysis confirms that the average RC size increases progressively with cyst development, in agreement with previous studies. The authors further show that lineage reliably predicts RC size (an observation previously reported, but here identified at an earlier stage in region 2a) and, importantly, that the thickness of the actin rim can also be predicted by lineage (reported here for the first time, at stage 1). FIB-SEM analysis also enables a clear delineation of the fusome, allowing for detailed characterization of its assembly and disassembly. Notably, the authors report, for the first time, structural evidence of ER-like membranes capping the inner rim of actin RCs.

At later developmental stages, AT-SEM analysis reveals that the microvilli observed by FIB-SEM evolve into extensive interdigitations extending beyond the outer rim in mid-stage egg chambers, a structural feature detected earlier than previously reported. Moreover, by analyzing a sample in which tissue organization was disrupted during preparation, the authors demonstrate that these interdigitations preferentially occur in proximity to the RC. In addition to RC analysis at later stages, the authors use AT-SEM to readily identify small cell populations, such as the germline stem cell niche and border cells, and provide high-resolution volumetric EM data for these structures.

MAJOR COMMENT My main comment is that we don't learn much new about the biology of these ring canals. The results primarily confirm findings from previous studies using conventional electron microscopy.

Although TEM data has been used to perform foundational studies in the field, there are limitations to this approach. Due to the size of the ring canals, it is challenging to locate them within the large volume of the egg chamber (especially at later stages). Even if ring canals can be located, they are typically not oriented the same way, so a single section is not sufficient. *Although some of the results shown by our complementary vEM approaches do confirm results that have been previously reported by TEM or fluorescence microscopy, our approach provides important additional insight into structures that have been studied for many decades that would not be possible using other approaches. Further, this approach has identified a novel membrane structure lining the ring canals, and it has provided structural details of the membrane interdigitations that would not be possible with conventional electron microscopy. Further, this complementary set of vEM approaches would be applicable to the study of many other structures within other tissue types. *

• *

One particularly interesting biological question, which is briefly mentioned in the text, is whether the oocyte is the cell that inherits the majority of the fusome. Since the authors are able to reconstruct the fusome using their data, they could measure the fusome volume in each cell (especially in the two pro-oocytes) and investigate whether the cell with the larger fusome ultimately becomes the oocyte. This question has been discussed for some time, and recent studies have proposed opposing models based on fusome volume to explain how the oocyte is selected among the 16 sister cells (Nashchekin et al., Science, 2021; Barr et al., Genetics, 2024).

        *We appreciate the reviewer's interest in the fusome, and we agree that our approach has provided significant insight into its three dimensional structure. The rendering of the fusome was performed using a large number of small isosurface volumes, and it is therefore difficult to accurately determine the fusome volume, since additional (non-fusome) material could be included in the model. Further, the fusomes that were rendered were within the germline clusters from region 2b, where the fusome has already started to break down, so these would not provide an accurate quantification of the full fusome volume. Because the focus of the manuscript is on the germline ring canals and associated structures such as the interdigitations (which we have tried to further streamline in this revised version), we believe that additional analysis of the fusome is outside of the scope of this work. *

MINOR COMMENT • The fluorescent markers used in the fly stocks are neither described in the Materials and Methods section nor depicted in the figures.

*We apologize if this was not clear in the original manuscript. Based on the comment from Reviewer #3 (see below), we have repeated the Hts staining using flies that do not have CheerioYFP in the background. We have also clarified the materials and methods section to indicate the panels that correspond with each strain used. *

• The authors should quote (Nashchekin et al., Science, 2021) when mentioning unequal partionning of the fusome (p4) and oocyte determination (p12). *We have added the reference to these parts of the manuscript. *

• P11-12, when mentioning electron dense regions reflecting strong cell-cell adhesion, the authors could refer to (Fichelson et al. Development, 2010), where AJ have been described around ring canals. *We have added the reference to this part of the manuscript. *

• Figure 2A: The schematic diagram (4th line) is not explained in the figure legend. *We have updated the figure legend to describe this schematic. *

• Figure 2D: Please clarify whether the RC stage shown corresponds to stage 1 or stage 10, as indicated in panel 2E. Alternatively, are these examples representing the minimum and maximum RC sizes observed across the entire dataset?. *These were not meant to be examples of the minimum and maximum ring canal sizes observed across the dataset. Instead, they were used to demonstrate the significant expansion that occurs during oogenesis. In the updated version of this figure, this panel has been removed. *

• Figure 5D: Please specify which panel in 5B this corresponds to. • Figure 5E: Please specify which panels in 5B this corresponds to. The two green boxes are not defined. Why is there a grey background under the ovariole assembly? • Figures 5G, 5H: Does panel 5G correspond to the left green box in 5E, and 5H to the right green box in 5E? Please clarify. *We have modified Figure 5 and merged it with the figure 6. In this updated format, panels 5B and 5E have been removed. *

• Figure 6: The figure title is not on the same page as the figure itself.

• We have made this change. *

• Figure 6A: The black box marking the germarium is not defined. *In this revised version, we have modified Fig. 6, and this panel has been removed. *

• Figure 6B-E: The arrows point to long interdigitations. However, arrowheads (which are not mentioned in the legend) appear to indicate the RC outer rim. Please specify this clearly in the figure legend. In the updated version of Fig. 6, these arrowheads have been removed.

Reviewer #2 (Significance (Required)):

I am not an expert in electron microscopy, so I cannot comment in detail on these techniques, but they appear to bridge the gap between conventional EM and optical microscopy in terms of resolution, user-friendliness, and other aspects. This is technically interesting, although these EM approaches have been previously described and applied. The images and movies are beautiful and clearly presented. My main comment is that we don't learn much new about the biology of these ring canals. The results primarily confirm findings from previous studies using conventional electron microscopy.

One particularly interesting biological question, which is briefly mentioned in the text, is whether the oocyte is the cell that inherits the majority of the fusome. Since the authors are able to reconstruct the fusome using their data, they could measure the fusome volume in each cell (especially in the two pro-oocytes) and investigate whether the cell with the larger fusome ultimately becomes the oocyte. This question has been discussed for some time, and recent studies have proposed opposing models based on fusome volume to explain how the oocyte is selected among the 16 sister cells (Nashchekin et al., Science, 2021; Barr et al., Genetics, 2024).

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

Kolotuev et al. used two volume-based electron microscopy based approaches to identify, segment, and document the changes in intercellular bridges, or ring canals, in early egg chambers of the fruit fly, Drosophila melanogaster. Using array tomography and focused ion beam scanning electron microscopy, Kolotuev et al., provide a high resolution and content rich lineage analysis of ring canal size, shape and orientation among early and late egg chambers. Their analysis included parameters such as the presence and shape of the fusome, the recruitment of actin to the inner ring, and development of membrane fingers that presumably spatially stabilize such structures. Last, Kolotuev and co-authors highlight additional aspects of their dataset including a reconstruction of the border cell cluster in stage 9 egg chambers. The data presented are a treasure trove of the ultrastructural features of the developing dipteran germline and subsequent ovarian follicle development. The data presented represent the highest resolution 3D dataset available and thus are a valuable worthwhile contribution to the field. My overall impression is that this paper sits intellectually between a valuable method and a loose experimental manuscript. This critique is not requesting additional experimental evidence because the data are unique and are the foundation for a new experimental paradigm. But there is not sufficient detail presented to be a full method, nor any hypothesis testing to be considered experimental. I suggest the authors consider amplifying their methods in detail and then note that using these methods provide a foundation for additional future investigations (as mentioned in the discussion). Problems with data interpretation and presentation should be addressed before publication. Below are the major and minor concerns that I believe need to be considered.

Major comments: In general images in figures are thought provoking, however changes to figure layout and design should be considered to better highlight the results. For instance, I don't know how to follow figure 1a. The arrow leads from a whole ovary to an ovulated egg with an ovariole strand connecting the two. What is the purpose of the arrow? Is it to represent time? And why is the mature egg in the figure when no data regarding this stage is presented. The authors should consider removing the mature egg and helping the reader understand that the ovariole is a subset of the whole ovary. They might do this by putting a box around a single ovarile in the whole ovary to indicate their ovariole illustration. Several other figures have similar problems. Throughout the authors used black and white arrows on black and white EM data and these arrows were lost. Color should be considered to effectively point out what they want the reader to see.

We have modified the layout of Fig. 1 and added additional explanation to the introduction and figure legend to guide readers through the introduction to the system. We have also added color to some of the arrows throughout the manuscript.

Can the authors provide additional information for the genotypes used? For instance the Cherrio-YFP (which might affect actin). When what this used and can the authors provide information on how this affected the data between when it was used and when it was not used. Additionally, why was analysis done in transgenic flies over fully wild-type?

*We have repeated the Hts staining in Fig. 2A in flies that do not express Cheerio-YFP and have made the appropriate changes to the methods section. For the AT-SEM experiment, we chose to use this genetic background since it would align with that of the negative controls that we often use in RNAi or over-expression experiments. FIB-SEM datasets were collected while imaging other tissues of the fly, so the choice of that genotype was not intentional. However, these datasets provided us with the opportunity to do this proof-of-concept work without such a large financial investment in the acquisition of new image stacks. In the future, we hope to expand this work to generate additional datasets from flies of different genotypes. *

Figure 1 seeks to lay out the ovary system and narrow the reader into the stages that will be analyzed in subsequent figures. Figure 1B is meant to show the types and kinds of electron microscopy, however lacks a full detailed description and legend for each of the colored arrows. And to that fact, so does figure S1. The authors need to provide additional information so the reader can glean what the authors point they are trying to convey. In addition, the authors might add pros and cons to each. I know this was attempted in S1, but did not fully come across.

We appreciate this feedback, and we have modified the layout of Figure 1 and updated Figures S1 to better highlight the technical challenge of EM in general and benefits of vEM in particular.

Figure 1 and 2 seek to set up both the biological and technical system to be understood. The authors might consider combining the two figures and eliminate elements that don't represent a result of any kind (Figure 1B, 2B, 3D and 3F). Or more fully explain the result and point they are trying to make with these illustrations. I fully understand and appreciate what they are trying to get across, but it does not come across clearly. For example, I don't know how figure 2B effectively gets across the point that rotation of the image has an effect on how it is sliced and segmented in EM data. Not sure it is necessary. Furthermore, what is the bottom panel with a green ring canal supposed to allow us to interpret or conclude? The same for 3D and F. The result in 3E is far more interesting and should be two panels that emphasize the growth characteristics between young and old rings or those of M1 and M4.

        *We greatly appreciate these suggestions, and we have modified and reorganized several figures to make the flow of scientific ideas easier to follow.* *We have moved panel 1B to the supplementary figure and gave additional indications in the text as to the differences between the EM methods. We have moved panel 2B to the supplementary material. We have moved Fig. 3D to Fig. S5A,B. Fig. 5 now provides more extensive rendering of membrane interdigitations from the stage 4 egg chamber. We have chosen to leave Fig. 3F to allow readers to compare the novel ER-like structures within the ring canals to the fusome that is present within younger germline clusters. *

The HTS and actin stain in figure 2A overlap significantly and obscure the fusome staining. Can the authors confirm that there is no bleed through in their staining and imaging procedure?

*We have repeated this staining and can confirm that there was no bleed through between the two channels. *

The data in Figure 2C are critical to showing the z-resolution enhancement of sectioned EM. However, the use of green psuedocolor only in one panel is confusing. Can the authors duplicate the whole panel and provide one without and one with psuedocolor? This would be ideal for fully orienting the reader to the sectioning and setting them up to understand the rest of the figures.

*In the revised version of Figure 2, we have split the sections into two rows of panels; we have added the pseudocolor to every other section (in the bottom row of panels). *

• *

The results section for figure 2 does outline the results presented. For example, the germarium contains syncytia of differing stages and ring canals with intervening fusomes... It does more to talk about the pros and cons of different technical aspects and their difficulty This should be saved for the rationale or the discussion. Rather the section should outline the results presented.

*We have modified the layout of figure 2 in order to describe the system in a more straightforward manner with a smoother transition from Figure 1 while further explaining technical points. *

I appreciate the color coding of the differentially segment cysts in Figure 3. The color coding helped orient me to which cysts were being evaluated. However I found the lack of detail bothersome. For instance, which ring canals are in the two panels of D? Are they M1 or M4?

*With the additional analysis of the interdigitations in the stage 4 cluster, we have moved panel D to Fig. S5. We did not have enough coverage of the region 2a cluster (red) to determine lineage, but we have added a statement to the legend to indicate that the ring canal shown in Fig. S5B is an M1 ring canal. *

Also, the presentation of ring canal size and distribution should be presented in a graph. Statistics are not necessary, but a dot-plot would go a long way to presenting the result. Two plots can add value, one in which the ring canals for each phase is shown, and the other is the distribution of sizes for each cyst.

*We have added these graphs in Fig. 4B, C. *

Lastly, the results section for figure 3 interprets the membrane bound vesicles in the ring canal as "ER-like". This should be removed since they neither look ER-like to me, nor have been shown to be ER in the data.

*We appreciate this suggestion, and although we cannot be absolutely certain of the identity of these structures without further study, with our additional analysis of the stage 4 egg chamber, we are further convinced of the similar appearance of these novel structures and the ER in other regions of the nurse cell (Fig. 5). We have clarified this point in the text. *

Figure 4A is not called out specifically in the results and thus should be interpreted or removed from the figure.

In this revised version, we have removed panel 4A.

Figure 5 was confusing. I understand the authors wanted to show the wafer and the ribbons, however, this is not a result and does not offer any interpretation of a result and is thus confusing on why it is in the figure. If this were a method paper, I would understand its presence.

*We have removed this panel from the figure. *

Can the authors comment on the shape of the nuclei in older egg chambers? They are not round at all. I am interested in whether this is a fixation artifact or the real ultrastructure of the nuclei. Of the border cell nuclei for instance. If it is an artifact, this should be added to the discussion.

*Some of the nuclei appear to have a peculiar shape in the cross-section. We cannot entirely exclude the role of the fixation in the shape irregularities. However, since not all the nuclei are subject to this phenomenon, we are inclined to attribute it to the intrinsic qualities of the late-stage nuclei. In numerous cases, different tissue and cell stages determine the shape of the nucleus, which frequently deviates from a spherical shape. *

Although data from "imperfect" samples is interesting, consider relegating Figure 6 to the supplement section, as it takes away from the pre-existing narrative flow established in the paper.

• In this draft, we have combined parts of figures 5 and 6, and much of the data from the imperfect sample has been removed. *

Interpretation of the data throughout the results should be left to the discussion section. For instance, interpretation of Figure 4 results on page 14 beginning with "these data demonstrate the importance...". The importance is not related to the result, but rather discussion of past and future studies.

We have removed this sentence from the results.

In another example, Figure 5I is introduced and discussed in the results section on page 15, second whole paragraph with an overall introduction/discussion on junctions, which convolutes the actual result. Discussion of future studies or how structures like the novel membrane fingers should be viewed in a larger biological context, should not be in the results.

We have made this change.

Minor comments: Remove words such as "pseudo-timelapse", they invoke precision on a point that is imprecise.

*This has been removed. *

Re-consider the acronyms for ring canal and egg chamber.

*We have removed these acronyms. *

Consider finding another way to call out each supplemental movie other than with another acronym.

*We have added small icons to indicate that a supplemental movie is associated with a given figure or panel. *

Reviewer #3 (Significance (Required)): The present manuscript is a technical advance in the field. The use of serial EM imaging with two separate modalities, on what is considered to be a challenging problem in the field, represents a useful technical advance. Light microscopy has thus far limited the resolution to which we can understand the spatial organization and the cellular features there in that regulate germline development. This manuscript brings to bear two serial EM methods to begin approaching this problem. The audience for this work are those working at the forefront of understanding germline architecture and development. I make these statements as an expert in live and super resolution of fruit fly egg chamber development, in addition to having performed 3D SEM in past works.

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Referee #3

Evidence, reproducibility and clarity

Kolotuev et al. used two volume-based electron microscopy based approaches to identify, segment, and document the changes in intercellular bridges, or ring canals, in early egg chambers of the fruit fly, Drosophila melanogaster. Using array tomography and focused ion beam scanning electron microscopy, Kolotuev et al., provide a high resolution and content rich lineage analysis of ring canal size, shape and orientation among early and late egg chambers. Their analysis included parameters such as the presence and shape of the fusome, the recruitment of actin to the inner ring, and development of membrane fingers that presumably spatially stabilize such structures. Last, Kolotuev and co-authors highlight additional aspects of their dataset including a reconstruction of the border cell cluster in stage 9 egg chambers. The data presented are a treasure trove of the ultrastructural features of the developing dipteran germline and subsequent ovarian follicle development. The data presented represent the highest resolution 3D dataset available and thus are a valuable worthwhile contribution to the field. My overall impression is that this paper sits intellectually between a valuable method and a loose experimental manuscript. This critique is not requesting additional experimental evidence because the data are unique and are the foundation for a new experimental paradigm. But there is not sufficient detail presented to be a full method, nor any hypothesis testing to be considered experimental. I suggest the authors consider amplifying their methods in detail and then note that using these methods provide a foundation for additional future investigations (as mentioned in the discussion). Problems with data interpretation and presentation should be addressed before publication. Below are the major and minor concerns that I believe need to be considered.

Major comments:

• In general images in figures are thought provoking, however changes to figure layout and design should be considered to better highlight the results. For instance, I don't know how to follow figure 1a. The arrow leads from a whole ovary to an ovulated egg with an ovariole strand connecting the two. What is the purpose of the arrow? Is it to represent time? And why is the mature egg in the figure when no data regarding this stage is presented. The authors should consider removing the mature egg and helping the reader understand that the ovariole is a subset of the whole ovary. They might do this by putting a box around a single ovarile in the whole ovary to indicate their ovariole illustration. Several other figures have similar problems. Throughout the authors used black and white arrows on black and white EM data and these arrows were lost. Color should be considered to effectively point out what they want the reader to see.

• Can the authors provide additional information for the genotypes used? For instance the Cherrio-YFP (which might affect actin). When what this used and can the authors provide information on how this affected the data between when it was used and when it was not used. Additionally, why was analysis done in transgenic flies over fully wild-type? Figure 1 seeks to lay out the ovary system and narrow the reader into the stages that will be analyzed in subsequent figures. Figure 1B is meant to show the types and kinds of electron microscopy, however lacks a full detailed description and legend for each of the colored arrows. And to that fact, so does figure S1. The authors need to provide additional information so the reader can glean what the authors point they are trying to convey. In addition, the authors might add pros and cons to each. I know this was attempted in S1, but did not fully come across. Figure 1 and 2 seek to set up both the biological and technical system to be understood. The authors might consider combining the two figures and eliminate elements that don't represent a result of any kind (Figure 1B, 2B, 3D and 3F). Or more fully explain the result and point they are trying to make with these illustrations. I fully understand and appreciate what they are trying to get across, but it does not come across clearly. For example, I don't know how figure 2B effectively gets across the point that rotation of the image has an effect on how it is sliced and segmented in EM data. Not sure it is necessary. Furthermore, what is the bottom panel with a green ring canal supposed to allow us to interpret or conclude? The same for 3D and F. The result in 3E is far more interesting and should be two panels that emphasize the growth characteristics between young and old rings or those of M1 and M4.

• The HTS and actin stain in figure 2A overlap significantly and obscure the fusome staining. Can the authors confirm that there is no bleed through in their staining and imaging procedure?

• The data in Figure 2C are critical to showing the z-resolution enhancement of sectioned EM. However, the use of green psuedocolor only in one panel is confusing. Can the authors duplicate the whole panel and provide one without and one with psuedocolor? This would be ideal for fully orienting the reader to the sectioning and setting them up to understand the rest of the figures.

• The results section for figure 2 does outline the results presented. For example, the germarium contains syncytia of differing stages and ring canals with intervening fusomes... It does more to talk about the pros and cons of different technical aspects and their difficulty This should be saved for the rationale or the discussion. Rather the section should outline the results presented.

• I appreciate the color coding of the differentially segment cysts in Figure 3. The color coding helped orient me to which cysts were being evaluated. However I found the lack of detail bothersome. For instance, which ring canals are in the two panels of D? Are they M1 or M4? Also, the presentation of ring canal size and distribution should be presented in a graph. Statistics are not necessary, but a dot-plot would go a long way to presenting the result. Two plots can add value, one in which the ring canals for each phase is shown, and the other is the distribution of sizes for each cyst. Lastly, the results section for figure 3 interprets the membrane bound vesicles in the ring canal as "ER-like". This should be removed since they neither look ER-like to me, nor have been shown to be ER in the data.

• Figure 4A is not called out specifically in the results and thus should be interpreted or removed from the figure.

• Figure 5 was confusing. I understand the authors wanted to show the wafer and the ribbons, however, this is not a result and does not offer any interpretation of a result and is thus confusing on why it is in the figure. If this were a method paper, I would understand its presence.

• Can the authors comment on the shape of the nuclei in older egg chambers? They are not round at all. I am interested in whether this is a fixation artifact or the real ultrastructure of the nuclei. Of the border cell nuclei for instance. If it is an artifact, this should be added to the discussion.

• Although data from "imperfect" samples is interesting, consider relegating Figure 6 to the supplement section, as it takes away from the pre-existing narrative flow established in the paper. Interpretation of the data throughout the results should be left to the discussion section. For instance, interpretation of Figure 4 results on page 14 beginning with "these data demonstrate the importance...". The importance is not related to the result, but rather discussion of past and future studies. In another example, Figure 5I is introduced and discussed in the results section on page 15, second whole paragraph with an overall introduction/discussion on junctions, which convolutes the actual result. Discussion of future studies or how structures like the novel membrane fingers should be viewed in a larger biological context, should not be in the results.

Minor comments:

• Remove words such as "pseudo-timelapse", they invoke precision on a point that is imprecise.

• Re-consider the acronyms for ring canal and egg chamber.

• Consider finding another way to call out each supplemental movie other than with another acronym.

Significance

The present manuscript is a technical advance in the field. The use of serial EM imaging with two separate modalities, on what is considered to be a challenging problem in the field, represents a useful technical advance. Light microscopy has thus far limited the resolution to which we can understand the spatial organization and the cellular features there in that regulate germline development. This manuscript brings to bear two serial EM methods to begin approaching this problem. The audience for this work are those working at the forefront of understanding germline architecture and development. I make these statements as an expert in live and super resolution of fruit fly egg chamber development, in addition to having performed 3D SEM in past works.

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Referee #1

Evidence, reproducibility and clarity

Summary

The possibility of observing 3D cellular organisation in tissues at nanometre resolution is a hope for many cell biologists. Here, the authors have combined two volume electron microscopy approaches with scanning electron microscopy: Focused Ion Beam (FIB-SEM) and Array Tomography (AT-SEM) to study the evolution of the shape and organisation of cytoplasmic bridges, the 'ring canals' (RCs) in the Drosophila ovarian follicle that connect nurse cells and oocyte. This type of cytoplasmic link, found in insects and humans, is essential for oocyte development. RCs have mainly been studied using light microscopy with various markers that constitute them, but this approach does not fully capture an overall view of their organization. Due to their three-dimensional arrangement within the ovarian follicle, characterizing their organization using transmission electron microscopy (TEM) has been very limited until now. This v-EM study allows the authors to document the evolution of RC size and thickness during the development of germline cysts, from the germarium to stage 4, and potentially beyond. This study confirmed previous findings, namely that RC size correlates with lineage: the largest RC is formed after the first division, while the smallest is formed during the last division. Furthermore, this work allowed a better characterisation of the membrane interdigitation surrounding the RCs. In addition, the authors highlight the important potential of v-EM for further structural analysis of the fusome, migrating border cells and the stem cell niche.

Major comments

• The output of this work can be divided into two parts. First, this work presents a technical challenge, involving image acquisition by volume electron microscopy and manual 3D reconstruction of the contours of the membranes, nuclei, RCs, and fusome in different cysts at different stages. Secondly, this work is based on a structural study of the RCs and their associated membranes. This work is descriptive but important, although the results largely confirm previous findings, both for the structure of the RCs and their relationship to the division sequence of the cyst cells, and for the organisation of the membranes around the RCs.

• Very interestingly, the authors report the spatial characterisation of membrane structures associated with and close to CRs that have already been identified (Loyer et al.). However, their characterisation is somewhat incomplete, as it lacks quantified data - how many CRs were analysed? and, above all, the characteristics of these membranes, their length and orientation according to their position and their connection in the lineage - these data could be obtained from the VEM data already collected and would be an important addition to the RC structural analysis in this work. In line with this, the authors importantly report the presence of an ER-like membrane structure lining the RCs. First, it would be nice to have statistics to support the observation of how many RCs..? Secondly, does this ER membrane structure vary according to the position of the RC in the cyst, are they related to the RC lineage? The addition of graphs showing the quantitative data with statistics in the figures would improve understanding of the results. This is particularly the case for the characterisation of RCs according to the stage of cyst development, as shown in Figure 3. This also applies to the characterisation of RCs within a cyst and the relationship between RC size and lineage, as shown in Figure 4, and to the characterisation (thickness) of the inner part of the RC.

• The part on the structural analysis of the fusome is interesting but still secondary to the characterisation of the RCs. This part should be moved to the results and figures after the various parts concerning the RCs.

Minor comments

• The distribution of the fusome in Figure 2 is difficult to see with Hts labelling and does not really correspond to the schematic, especially in regions 2a and 2B.

• In panel C of Figure 2, it is a little disturbing that the legend is directly on the image of RC. It hides some information about the images and could be placed at the bottom of the panel. This also the case for the panel G.

• With figure 3B, it would be good to highlight the position of cyst.

Significance

As mentioned above, this work can be divided into two parts.

The part corresponding to the acquisition of images by volume electron microscopy and manual 3D reconstruction is new and a great source of valuable information. The part related to the spatial characterisation of the RC is important, but corresponds more to an extension and reinforcement of previously available information than to the contribution of significant new insights.

I think it will be of great interest to an audience interested in Drosophila oogenesis.

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Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript by Dufour et al. is a follow-up on the groups' previous publication that introduced the photo-inducible Cre recombinase, LiCre. In the present work, the authors further characterize the properties and kinetics of their optogenetic switch. Initially, the authors show that light affects only LiCre-mediated recombination itself and not DNA binding. Following these observations, they measure and mathematically model LiCre kinetics demonstrating high efficiency in vivo and a surprising temperature sensitivity. Finally, Dufour et al. evaluate several mutations that affect the LOV photo-cycle and provide recommendation for LiCre applications. The study thoroughly investigates various aspects of the function of LiCre, confirming some previously known characteristics (i.e. temperature-dependence of Cre activity and functionality of LOV-based optogenetic tools in yeast without co-factor supplementation), while providing new LiCre-specific insights (kinetics, light-independent DNA binding). Please note that the reviewer is no expert in mathematical modeling and cannot fully judge the methodological details of the models. While I have some concerns as listed below, I believe study should be well-suited for publication after a revision.

Major comments:

1. After completing the initial experiment, the authors discovered that their plasmids carry different numbers of V5 epitopes. I am wondering whether this was due to a recombination event happening during the experiment or whether the constructs were not sequence verified prior to use? In any case, an additional ChIP experiment using Cre and LiCre constructs with the identical number of tag-repeats will be necessary. The result, i.e. the strong reduction of DNA-binding of LiCre (which is close to the negative control), is quite remarkable given that LiCre is still considerably active and high DNA affinities were observed in SPR experiments. In light of these counterindications, identical experiment conditions for test and reference group become even more important.
2. The conclusion that DNA-binding of LiCre is completely light-independent is not entirely convincing to me. The differences between the light and dark conditions in Fig. 2d are indeed small, but the values for LiCre are almost on par with the vector control and therefore hard to interpret. Based on this experiment alone, one could even be inclined to argue that LiCre does not bind DNA at all (which is of course falsified by the later experiments), showing that the resolution of the corresponding dataset is too low to draw final conclusions. Light-independent DNA binding should either be confirmed by a more sensitive method or the conclusion statements on this matter should be revised accordingly.
3. If I understand the explanations correctly, replicates and plotted data points refer to multiple samples (different colonies), that were handled in a single experiment, i.e. by one researcher at the same time/same day. As already mentioned by the authors in the main text, this workflow explains the considerable differences between some of the results in the present manuscript and an identical experiment in a previous publication by the same authors. Providing truly independent experiments (performed on different days) that are therefore independent towards variables such as the fluctuation in incubation temperature (which was the issue in the described experiments) will be crucial, at least for the key datasets.

Minor comments:

1. At the end of the Introduction, the authors mention that the interaction of the Cre heptamers was weakened via point mutations in LiCre. A short sentence about the engineering rationale behind this weakened interaction would help readers, who are not familiar with the author's prior work.
2. Fig. 2a-b depicts images relating to the purification procedure. These could be moved to the supplements as they don't provide any insight apart from the fact that the proteins were successfully purified.
3. The kinetic characterization was only performed for LiCre. Especially for scientists, who have worked with wildtype Cre before, a side-by-side comparison with wt Cre would be valuable to judge the loss in reaction speed that has to be expected when switching from Cre to LiCre.
4. The difference between the ChIP results and the SPR results is striking but not mentioned in the discussion section. Also, the statement: "Finally, our results have practical implications on experimental protocols employing LiCre. First, given its high affinity for loxP (Fig. 5b), over-expressing LiCre at high levels will probably not increase its efficiency." (line 502) refers only to the affinity but seems to ignore the low DNA-occupancy of LiCre observed in Fig. 2d. Adapting the discussion section accordingly would improve the manuscript.

Significance

General assessment and advance:

The present study provides a large set of experiments and analyses characterizing the optogenetic LiCre recombinase. In general, the study is well conceived and executed. Although some of my concerns listed above affect key aspects of the study, they should be straightforward to address. The manuscript is a follow-up study providing a more detailed characterization of an optogenetic tool previously developed by the same authors. Its novelty is therefore somewhat limited. While the study provides a rich body of additional data, many of the findings merely confirmed aspects that were to be expected based on the two proteins LiCre is built of (temperature-dependent activity of Cre, optogenetics in yeast w/o the need of co-factor supplementation, weaker DNA-affinity of the Cre fusion protein as compared to wildtype Cre). New insights are provided by the facts that (i) light only controls recombination but not DNA binding and (ii) light activation of only some protomers within the LiCre heptamer is likely to be sufficient to activate recombination. The former aspect is, however, not entirely evident from the results as described above.

Audience:

The study will be of interest for researchers focusing on inducible DNA recombination and especially relevant to those who plan to work with LiCre and can now rely on a more detailed and extended characterization compared to the original LiCre publication.

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Reply to the reviewers

1. General Statements

Thank you for providing an assessment of our manuscript. We suggest here a revision plan to address the points raised by the reviewers regarding code documentation, benchmarking, and biological applications.

As part of the revisions implemented we have:

Clarified the management of dependencies of our package Fixed the data download run times of test data Clarified the parameters of the normalization and optimization functions We plan to:

Extend our manuscript to include a section on cross-condition analysis that builds on our tutorials, where we will illustrate how ParTIpy can quantify shifts in the distribution of fibroblasts across the functional space defined by archetypal analysis between healthy and failing hearts. Extend our benchmarks of scalability of coresets, by reporting wall-clock time and peak memory usage across distinct data sizes. Extend our benchmarks of stability of coresets, by reporting the similarity of the estimated archetypes based on the original versus the sampled data. Include the original enrichment analysis of ParTI to provide users with distinct options to work with the archetypes, and provide a larger discussion on the distinct strategies. We believe these revisions will strengthen our__ software manuscript__ and will help us to provide a robust and practical tool to analyze functional trade-offs from biological data.

2. Description of the planned revisions

Reviewer #1

Summary

The paper "ParTIpy: A Scalable Framework for Archetypal Analysis and Pareto Task Inference" presents ParTIpy, an open-source Python package that modernizes and scales the Pareto Task Inference (ParTI) framework for analyzing biological trade-offs and functional specialization. Unlike the earlier MATLAB implementation, which required a commercial license and was limited in scalability, ParTIpy leverages Python's open ecosystem and integration with tools such as scverse to make archetypal analysis more accessible, flexible, and compatible with modern biological data workflows. Through advanced optimization and coreset algorithms, it efficiently handles large scale single cell and spatial transcriptomics datasets. ParTIpy identifies "archetypes", or optimal phenotypic extremes, to reveal how cells balance competing functional programs. The paper demonstrates its application in modeling hepatocyte specialization across the liver lobule, highlighting spatial patterns of metabolic division of labor.

Overall, ParTIpy represents a modern, accessible, and scalable Python-based solution for exploring biological trade-offs and resource allocation in high-dimensional data. The paper is clearly written and addresses an important methodological gap. However, the enrichment analysis differs from the original ParTI framework and should be discussed more explicitly, and the documentation and tutorials, while helpful, could be refined to improve usability and reproducibility.

Major Comments

1. The archetype enrichment analysis used in this paper differs from the original enrichment analysis implemented in ParTI. This is acceptable, but: a) The authors should explicitly state and discuss the differences between the two approaches. b) The enrichment analysis should be made more systematic. For each tested feature (e.g. gene or pathway), the analysis should report a p-value for the hypothesis that the feature is enriched near an archetype - that is, its expression (or value) is high close to the archetype and decreases with distance. Appropriate multiple-hypothesis correction should also be applied.

We thank the reviewer for this valuable comment and agree that the differences between our enrichment analysis and the original ParTI implementation should be stated more explicitly. We will incorporate the original enrichment algorithm into ParTIpy, enabling users to select their preferred method. In the revised manuscript, we will note that two enrichment algorithms are available and describe both in greater detail in the supplementary methods section. We also note that the current enrichment analysis already reports p-values adjusted for multiple hypothesis testing.

Reviewer #2

Summary

This paper introduces the software ParTIpy, a scalable Python implementation of Pareto Task Inference (ParTI), designed to infer functional trade-offs in biological systems through archetypal analysis. The framework modernizes the previous toolbox with efficient optimization, memory-saving coreset construction, and integration with the scverse ecosystem for single-cell transcriptomic data.

Using hepatocytes scRNA-seq data as a test case, the authors identify archetypes corresponding to distinct gene expression patterns. These archetypes align with known liver domains in spatial transcriptomics data, validating both the method's interpretability and its biological relevance.

Major comments

(1) Conclusions

The core computational and biological claims are well supported. ParTIpy clearly scales better than earlier implementations and reproduces known biological structure. However, claims about "scalability to large datasets" should be further qualified (see below).

We will implement further performance benchmarks as discussed below.

(2) Claims

Archetypal analysis based on current matrix computation formulation is non-parametric, and new data require recomputation of archetypes. Therefore, the method cannot generalize to unseen data in the way deep learning approaches, which could be further acknowledged and clarified.

We thank the reviewer for this insightful comment. We agree that deep learning frameworks are typically amortized, allowing them to generalize to unseen data without retraining, and we will clarify this distinction in the discussion of the revised manuscript. However, we note that mapping new cells into an existing archetypal space is computationally inexpensive, as it only requires solving a single convex optimization problem.

(3) Additional suggested analyses or experiments

1） Absolute performance benchmarks : it's suggested to report wall-clock time and memory for a few dataset sizes (10k, 100k, 1M cells).

We thank the reviewer for this helpful suggestion. We will extend the coreset benchmark to quantify how coreset size affects both archetype positions and biological interpretation. Specifically, we will match archetypes across coreset sizes by solving the linear sum assignment problem, as we currently do when comparing bootstrap samples. We will then compare the distances between archetypes inferred from the full dataset and those obtained from different coreset sizes. In addition to measuring displacement, we will assess biological stability by comparing the gene expression vectors of corresponding archetypes as well as their enriched pathways (using metrics such as cosine similarity and Jaccard index).

**Referee cross-commenting**

I agree with the other reviewer's suggestion to check consistency and reproducibility with previous implementation, and enhance the tutorial of the software for users from a biological background. Combined with my comments to further improve the biological application showcase, the revised manuscript could be an impactful contribution to the field, if these comments could be properly addressed.

(1) Advance

This paper is primarily a technical contribution. It modernizes the Pareto Task Inference framework into a scalable and user-friendly Python implementation, which is valuable. However, to further improve its significance especially for the broader biological audience, more detailed analysis could be performed (see below)

(2) Biological scope and applications [optional]

The current biological validation in hepatocyte is technically fine but limited in breadth and impact. It demonstrates that ParTIpy works but falls in short of showing what new insights it can reveal. Several promising applications could be further explored:

1) Cross-condition comparisons: could ParTIpy quantify how the Pareto front shifts between conditions (e.g., normal vs. tumor, treated vs. control)?

We thank the reviewer for this valuable suggestion. We have shown ParTIpy's applicability to cross-condition settings in our online tutorials (https://partipy.readthedocs.io/en/latest/notebooks/cross_condition_lupus.html). However, we agree that a more explicit mention in the manuscript is needed. Thus, we will include a cross-condition analysis as a second application in the revised manuscript, focusing on fibroblasts from heart failure patients from Amrute, et. al. (2023) 1. This will illustrate how ParTIpy can quantify shifts in the distribution of cells across the functional space defined by archetypal analysis.

Because the manuscript does not explore these scenarios, the biological impact remains narrow, and the framework's broader interpretive power is somehow underrepresented.

We hope that the additional application included in the revised manuscript helps better illustrate the framework's strength. We would also like to note that the online tutorials provide a comprehensive overview of ParTIpy's functionality, as we expect these will serve as a primary entry point for many researchers interested in archetypal analysis and Pareto Task Inference.

(3) Audience and impact

The paper will interest computational biologists, systems biologists, and bioinformaticians focused on single-cell analysis, and its impact will grow substantially if the authors demonstrate more biological applications.

(4) Reviewer expertise

Computational biology, single-cell transcriptomics, machine learning, computational math

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

Reviewer #1

2. The package documentation on GitHub and ReadTheDocs is a major strength, but the tutorials can be improved for clarity and accessibility:

We thank the reviewer for this positive feedback. Indeed, providing comprehensive documentation to facilitate ease of adoption was a major motivation behind this project. In response to the reviewer's suggestions, we have revised the tutorials to further improve their clarity, structure, and accessibility, as detailed below.

a) The documentation should list external dependencies that need to be installed seperately, e.g. pybiomart.

We thank the reviewer for pointing this out. We had added all dependencies under the optional-dependencies.extra header, which allows users to run pip install partipy[extra] to be able to run all tutorial notebooks. However, we forgot to explain that in the tutorial or Readme page, which we corrected now. The Readme now reads:

Install the latest stable full release from PyPI with the extra dependencies (e.g., pybiomart, squidpy, liana) that are required to run every tutorial:

``` pip install partipy[extra]

```

Additionally we include clarifications in every tutorial notebook that uses additional dependencies: "To run this notebook, install ParTIpy with the tutorial extras: pip install partipy[extra]".

b) The dataset used in the Quickstart demo appears to be inaccessible or extremely slow to download (the function load_hepatocyte_data_2() did not complete even after 30 minutes, at least in my experience). The authors should verify data availability on Zenodo and consider providing a smaller or cached version to make the demo more reliable and reproducible.

We thank the reviewer for this helpful comment. We agree that the previous implementation of load_hepatocyte_data_2() was not reliable due to slow download speeds from Zenodo. To address this, we now host the required AnnData object on figshare (https://figshare.com/articles/dataset/scRNA-seq_hepatocyte_data_from_Ben-Moshe_et_al_2022_/30588713?file=59459459), ensuring faster and more stable access for the Quickstart tutorial via scanpy.read:

```

adata = sc.read("data/hepatocyte_processed.h5ad", backup_url="https://figshare.com/ndownloader/files/59459459")

adata

```

c) The tutorial order could be more intuitive - for instance, "archetype crosstalk network" appears before "archetypal analysis". Consider starting with the simulated dataset and presenting the full pipeline before moving to more complex real-world examples.

We thank the reviewer for this helpful suggestion and agree that the previous ordering was not intuitive. We have reordered the tutorials such that the notebook introducing archetypal analysis now appears first, followed by the Quickstart tutorial and the subsequent applied examples.

Minor comments

1. In the Python function, the parameter "optim" could use more descriptive option names - for example, renaming "projected_gradients" to "PCHA" would make it clearer and more consistent with terminology used in the paper.

We thank the reviewer for this helpful suggestion. We agree that the previous naming could be misleading. While PCHA does not precisely describe the underlying algorithm, it is the term most users are familiar with from the literature. We have therefore updated the function to accept both "PCHA" and "projected_gradients", which now map to the same underlying optimization routine.

In the Quickstart preprocessing, the authors use the following code:

sc.pp.normalize_total(adata)

sc.pp.log1p(adata)

However, they do not specify the target sum in the normalize_total function. The authors should ensure that the data values before the logarithmic transformation span several orders of magnitude (e.g., 0-10,000); if normalization is performed to a sum of 1, the log transformation becomes ineffective.

We thank the reviewer for this helpful comment. By default, sc.pp.normalize_total scales the counts in each cell to the median total counts across all cells, which preserves the typical range of expression values prior to logarithmic transformation. We therefore consider this default behavior appropriate for the Quickstart example. Nonetheless, we will clarify this explicitly in the tutorial to avoid confusion.

**Referee cross-commenting**

I agree with Reviewer #2 observation that the paper's contribution is primarily technical; however, I consider this technical advance to be an important and timely one that will enable many biologists to apply archetypal analysis more effectively in their own work.

We thank the reviewer for this positive and encouraging assessment.

Reviewer #1 (Significance (Required)):

This study presents ParTIpy, a Python-based implementation of Pareto Task Inference (ParTI) that makes archetypal analysis more accessible, scalable, and compatible with modern single-cell and spatial transcriptomics workflows. Its main strength lies in translating a conceptually powerful but technically limited MATLAB framework into an open-source, efficient Python package, enabling wider use in computational biology. The package is well-documented, which further enhances its accessibility and adoption potential, though documentation could be improved to enhance reproducibility and ease of use. It will be of interest to computational systems biologists, particularly those working with omics data, and those interested in studying functional trade-offs and resource allocation.

We appreciate the reviewer's positive evaluation and are encouraged by their recognition of ParTIpy's relevance and potential impact in computational biology.

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

Reviewer #2

The current biological validation in hepatocyte is technically fine but limited in breadth and impact. It demonstrates that ParTIpy works but falls in short of showing what new insights it can reveal. Several promising applications could be further explored:

2) Transient or plastic states: Cells with mixed archetype weights or high mixture entropy can be interpreted as transient, functionally flexible states. ParTIpy can quantify such transience geometrically, even in static data, which providing a competitive counterpart to models like CellRank or CellSimplex (https://doi.org/10.1093/bioinformatics/btaf119).

We thank the reviewer for this interesting suggestion. While we agree that quantifying transient or plastic states based on archetype mixtures is an intriguing idea, validating whether cells with mixed archetype weights ("generalists") truly represent transient states would require additional data modalities such as temporal or lineage-tracing measurements. Although we find this direction highly interesting, given that the manuscript is intended as a software paper, we prefer to focus on more directly supported applications of cross-condition data, where labeled data is available.

However, we will expand our discussion to relate ParTIpy with CellSimplex since we believe this is an interesting angle that future users could explore.

5. References

1. Amrute, J. M. et al. Defining cardiac functional recovery in end-stage heart failure at single-cell resolution. Nat. Cardiovasc. Res. 2, 399-416 (2023).

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

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Referee #2

Evidence, reproducibility and clarity

Summary

This paper introduces the software ParTIpy, a scalable Python implementation of Pareto Task Inference (ParTI), designed to infer functional trade-offs in biological systems through archetypal analysis. The framework modernizes the previous toolbox with efficient optimization, memory-saving coreset construction, and integration with the scverse ecosystem for single-cell transcriptomic data.

Using hepatocytes scRNA-seq data as a test case, the authors identify archetypes corresponding to distinct gene expression patterns. These archetypes align with known liver domains in spatial transcriptomics data, validating both the method's interpretability and its biological relevance.

Major comments

(1) Conclusions

The core computational and biological claims are well supported. ParTIpy clearly scales better than earlier implementations and reproduces known biological structure. However, claims about "scalability to large datasets" should be further qualified (see below).

(2) Claims

Archetypal analysis based on current matrix computation formulation is non-parametric, and new data require recomputation of archetypes. Therefore, the method cannot generalize to unseen data in the way deep learning approaches, which could be further acknowledged and clarified.

(3) Additional suggested analyses or experiments

1. Absolute performance benchmarks : it's suggested to report wall-clock time and memory for a few dataset sizes (10k, 100k, 1M cells).
2. Coreset sensitivity analysis: Could authors show how coreset size affects archetype positions and biological interpretation?

Referee cross-commenting

I agree with the other reviewer's suggestion to check consistency and reproducibility with previous implementation, and enhance the tutorial of the software for users from a biological background. Combined with my comments to further improve the biological application showcase, the revised manuscript could be an impactful contribution to the field, if these comments could be properly addressed.

Significance

(1) Advance

This paper is primarily a technical contribution. It modernizes the Pareto Task Inference framework into a scalable and user-friendly Python implementation, which is valuable. However, to further improve its significance especially for the broader biological audience, more detailed analysis could be performed (see below)

(2) Biological scope and applications [optional]

The current biological validation in hepatocyte is technically fine but limited in breadth and impact. It demonstrates that ParTIpy works but falls in short of showing what new insights it can reveal. Several promising applications could be further explored:

1) Cross-condition comparisons: could ParTIpy quantify how the Pareto front shifts between conditions (e.g., normal vs. tumor, treated vs. control)?

2) Transient or plastic states: Cells with mixed archetype weights or high mixture entropy can be interpreted as transient, functionally flexible states. ParTIpy can quantify such transience geometrically, even in static data, which providing a competitive counterpart to models like CellRank or CellSimplex (https://doi.org/10.1093/bioinformatics/btaf119).

Because the manuscript does not explore these scenarios, the biological impact remains narrow, and the framework's broader interpretive power is somehow underrepresented.

(3) Audience and impact

The paper will interest computational biologists, systems biologists, and bioinformaticians focused on single-cell analysis, and its impact will grow substantially if the authors demonstrate more biological applications.

(4) Reviewer expertise Computational biology, single-cell transcriptomics, machine learning, computational math

the advantages of Artificial Intelligence in Historical research

Document d'information : Analyse des mécanismes de sortie de conflit

Résumé analytique

Ce document synthétise les perspectives d'experts sur les mécanismes de résolution des conflits et de construction de la paix, basées sur des recherches en sciences comportementales et des expériences de médiation sur le terrain.

L'analyse part du constat d'une "spirale tragique" du conflit, où l'agression et la représaille s'auto-alimentent, nourries par des biais psychologiques comme la déshumanisation de l'ennemi.

Les points à retenir sont les suivants :

1. L'inversion de la spirale : Le cycle destructeur peut être inversé pour devenir un "cercle vertueux".

La clé de cette transformation est l'humanisation de l'autre, qui consiste à le percevoir comme un acteur avec qui collaborer, une partie ayant des intérêts légitimes ou un semblable.

2. Le rôle central des victimes : De manière contre-intuitive, les études, notamment en Colombie, montrent que les victimes de conflits violents sont souvent plus prosociales, plus enclines à la coopération et à la réconciliation que les non-victimes.

Cette attitude s'explique par une forte aversion à la perte — ayant tant perdu, elles sont déterminées à empêcher la violence de se répéter — et une capacité à reconnaître la souffrance partagée.

3. Transformer la violence, pas éliminer le conflit : Les experts s'accordent à dire que l'objectif n'est pas d'éliminer le conflit, qui est inhérent aux sociétés humaines, mais de le transformer d'une forme violente à une forme non-violente et constructive, gérée par des moyens politiques et institutionnels.

4. Recommandations stratégiques : Pour favoriser la paix, les recommandations clés incluent une communication qui reconnaît la souffrance de toutes les parties, l'utilisation du cadre de l'aversion à la perte pour motiver l'action collective, la promotion du contact direct entre les groupes pour humaniser l'autre, et le fait de s'attaquer aux causes profondes des conflits (ex: inégalités).

5. Le changement climatique comme analogie : Le défi climatique est présenté comme un exemple de conflit global non-violent qui exige une "collaboration radicale".

La solution ne réside pas dans la création de nouveaux mouvements, mais dans la capacité à capter et à renforcer les énergies positives et les initiatives déjà existantes au sein de la société.

--------------------------------------------------------------------------------

1. La "Spirale Tragique" du Conflit

L'analyse des conflits commence par le concept de "spirale tragique", un mécanisme d'escalade auto-entretenu. Ce cycle destructeur se déroule selon les étapes suivantes :

• Stress initial : Des tensions ou des difficultés génèrent un stress collectif.

• Attribution et accusation : En raison du "biais fondamental d'attribution", les humains ont tendance à attribuer la cause des problèmes à des personnes plutôt qu'à des situations. Cela mène à l'identification et à l'accusation d'un ennemi.

• Déshumanisation et agression : L'autre groupe est déshumanisé, ce qui lève les inhibitions et permet l'agression et la violence. Ces actes permettent de libérer la tension accumulée.

• Destruction et représailles : La violence entraîne la destruction, ce qui génère davantage de stress et de souffrance, alimentant un désir de représailles de la part de l'autre camp.

• Auto-alimentation : Chaque partie, se percevant comme répondant à une agression initiale, perpétue un cycle sans fin de violence et de souffrance croissante, renforçant la dichotomie "nous contre eux".

Ce modèle, alimenté par des propensions humaines universelles, explique comment les conflits s'enracinent et s'intensifient.

2. Inverser la Spirale : L'Humanisation comme Moteur du Changement

La même dynamique de boucle de rétroaction qui alimente la violence peut être inversée pour créer un "cercle vertueux" où "le mieux mène au mieux". La clé de cette inversion réside dans le processus d'humanisation.

Selon Adam Kahane, l'humanisation consiste à choisir de voir les autres non pas comme des objets ou des non-humains, mais à travers des perspectives constructives :

• Perspective technocratique : Voir l'autre comme un co-acteur dans la résolution d'un problème commun.

• Perspective politique : Voir l'autre comme une partie ayant des intérêts légitimes dans le cadre d'une négociation.

• Perspective relationnelle : Voir l'autre comme un semblable ou un parent, reconnaissant une humanité partagée.

Ce changement de perspective est souvent déclenché par une prise de conscience pragmatique : la reconnaissance qu'aucune partie ne peut l'emporter unilatéralement et que la collaboration, même avec des adversaires, est indispensable pour assurer son propre avenir.

3. Le Rôle Contre-Intuitif des Victimes dans la Réconciliation

Un des constats les plus frappants issus des recherches menées en Colombie est le rôle moteur des victimes dans les processus de paix. Contrairement à l'idée reçue, les personnes ayant directement souffert de la violence sont souvent plus enclines à la coopération et à la réconciliation que celles qui n'ont pas été directement affectées.

Les Mécanismes Comportementaux sous-jacents

Les recherches d'Enrique Fatas et Lina Restrepo mettent en lumière plusieurs explications comportementales à ce phénomène :

• Aversion à la perte : Conformément à la théorie des perspectives, les pertes sont ressenties plus intensément que les gains équivalents. Les victimes ont subi des pertes immenses (famille, biens, sécurité) et sont donc extrêmement motivées à éviter que cette souffrance ne se répète, ce qui les rend plus ouvertes à la concession pour garantir la paix.

• Prosocialité accrue : Il est documenté à travers l'Afrique, l'Asie et l'Amérique latine que l'exposition à un conflit violent augmente la prosocialité des victimes envers les membres de leur propre groupe (in-group) mais aussi envers d'autres groupes vulnérables qu'elles perçoivent comme similaires. Si les ex-combattants sont perçus comme un autre groupe vulnérable plutôt que comme des ennemis déshumanisés, cette prosocialité peut s'étendre à eux.

• "Victimisation inclusive" : Dans des contextes comme la Colombie, où le conflit a été long et irrégulier, la victimisation est si répandue qu'elle transcende les clivages. Il n'y a pas un "nous" et un "eux" clairement définis, ce qui favorise une identification partagée et réduit la pensée conflictuelle.

La Reconnaissance de la Souffrance Partagée

Adam Kahane corrobore cette observation en soulignant que les participants qui avaient le plus souffert dans les ateliers de paix en Colombie étaient les plus déterminés à trouver une solution non-violente. La reconnaissance de la souffrance partagée avec l'adversaire permet de le voir comme un être humain. Citant Carl Rogers, il affirme que "ce qui est le plus personnel est le plus universel".

Distinction entre Attitudes et Comportements

Une étude de Lina Restrepo sur le financement participatif pour des entrepreneurs (victimes vs. ex-combattants) a révélé une nuance importante.

• Comportement : Les participants ont donné des sommes d'argent similaires aux deux groupes, ne montrant aucune différence comportementale.

• Attitudes : Cependant, les attitudes exprimées (peur, anxiété) envers les ex-combattants restaient négatives.

Cette dissociation montre que même les personnes non directement affectées sont capables de surmonter leurs préjugés et leurs peurs pour s'engager dans des actions coopératives lorsqu'une solution pacifique est en jeu.

4. Recommandations Stratégiques pour la Construction de la Paix

Les experts proposent une série de recommandations pour sortir des conflits violents et faire prévaloir la paix.

Recommandation

Description

Expert(s)

Communication efficace

Communiquer sur les politiques de réconciliation de manière à légitimer l'aide aux victimes et aux ex-combattants, en reconnaissant explicitement la souffrance de l'autre pour éviter le "renversement du stigmate" (une réaction négative de la part de ceux qui ne bénéficient pas des politiques).

Enrique Fatas

Gestion de la mémoire

Ne pas utiliser la mémoire du conflit de manière partisane, car cela perpétue le conflit et peut nuire aux compétences cognitives et aux perspectives économiques des victimes, même des années plus tard.

Enrique Fatas

Cadre de l'aversion à la perte

Communiquer non pas sur les gains de la paix, mais sur ce que la société a à perdre si le conflit violent persiste. Ce cadre est plus puissant pour motiver l'action.

Lina Restrepo

Empathie et perspective

Intégrer activement le point de vue des victimes dans le discours public pour que les non-victimes développent une plus grande empathie envers une solution pacifique.

Lina Restrepo

Hypothèse du contact

Faciliter le contact direct entre les membres des groupes opposés. Apprendre à connaître l'autre en tant que personne (avec une famille, une histoire) est un puissant antidote à la déshumanisation.

Lina Restrepo

S'attaquer aux causes profondes

S'assurer que les raisons sous-jacentes qui ont déclenché le conflit en premier lieu (inégalités, manque de confiance dans les institutions) sont résolues pour éviter une résurgence de la violence.

Lina Restrepo

Canaliser les énergies existantes

Au lieu d'essayer de "pousser les gens à agir", il est plus efficace d'identifier, de soutenir et d'aider à coordonner les énergies, les mouvements sociaux et les initiatives positives qui existent déjà au sein de la société.

Adam Kahane

Transformer le conflit

Accepter que le but n'est pas d'éliminer le conflit mais de le transformer en un processus non-violent. Le conflit est inévitable ; la violence ne l'est pas.

Adam Kahane, Enrique Fatas

5. Le Changement Climatique : Une Analogie pour la "Collaboration Radicale"

Le changement climatique est utilisé comme une analogie puissante pour les conflits complexes du 21e siècle.

• C'est un problème non-unilatéral et non-local : aucune nation ou groupe ne peut le résoudre seul.

• Il représente un "conflit sans violence" où des intérêts divergents (agriculteurs, industries, gouvernements) s'affrontent.

• Il est caractérisé par une urgence temporelle ("ticking clock") qui rend l'inaction catastrophique.

Face à ce défi, Adam Kahane préconise une "collaboration radicale" qui intègre la vitesse, l'ampleur et la justice. Cependant, un risque majeur, souligné par Lina Restrepo, est la normalisation : à force d'entendre parler de la crise, les populations s'y habituent et l'urgence perçue diminue, ce qui paralyse l'action.

Conclusion : De l'Espoir à l'Action

La discussion se conclut sur une note pragmatique et pleine d'espoir.

La clé pour résoudre les conflits les plus complexes, qu'il s'agisse de guerres civiles ou de crises globales comme le changement climatique, ne réside pas dans la création de solutions ex nihilo.

Elle réside plutôt dans notre capacité à "capter les énergies qui circulent déjà".

Des mouvements positifs, des leaders et des initiatives existent toujours.

Le véritable défi est de les identifier, de les unir et de les amplifier pour transformer les dynamiques de conflit en collaboration constructive.

Justice Pénale et Transitionnelle : Sortir des Violences Collectives

Résumé Exécutif

Ce document de synthèse analyse les mécanismes juridiques et politiques conçus pour répondre aux violences de masse, en s'appuyant sur l'expertise de Sandrine Lefranc et Sharon Weill.

Il met en lumière l'inadéquation du droit pénal traditionnel, conçu pour la criminalité individuelle, face à des crimes d'État ou de grande ampleur.

En réponse, la justice transitionnelle a émergé comme une alternative politique privilégiant la vérité, la réparation et la réconciliation à la sanction pénale.

Cependant, cette approche, bien que vertueuse, impose souvent aux victimes un langage de la souffrance au détriment de la colère politique.

Parallèlement, la justice pénale s'est renouvelée à travers des mécanismes internationaux (Cour Pénale Internationale), nationaux (compétence universelle) et hybrides (tribunal pour Hissène Habré), chacun présentant ses propres limites en termes de politisation, de légitimité et d'efficacité.

Le modèle colombien post-accord de paix de 2016 représente une nouvelle voie holistique, intégrant la responsabilité pénale à des projets de réparation en collaboration avec les victimes.

Enfin, le procès des attentats du 13 novembre 2015 en France illustre une "hybridation" inédite où un cadre pénal classique a incorporé des éléments de justice transitionnelle, offrant une place centrale à la parole des victimes tout en révélant les tensions inhérentes à cette démarche et la quête, par les victimes elles-mêmes, d'une justice plus restaurative.

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1. L'Impuissance du Droit Pénal Traditionnel Face à la Violence de Masse

Le droit pénal classique se trouve fondamentalement dépassé et "réduit au mutisme" lorsqu'il est confronté à la violence de masse.

Sandrine Lefranc souligne que ce système est structuré pour juger des crimes individuels et non des dynamiques collectives impliquant des milliers de victimes et d'auteurs, ces derniers appartenant souvent à l'appareil d'État.

• Problème d'échelle : Le droit pénal est débordé par le grand nombre de victimes et d'auteurs, ainsi que par des pratiques répressives inventives pour lesquelles il n'a pas de catégories juridiques (par exemple, les "disparitions" en Amérique latine, difficiles à qualifier en assassinats sans corps).

• Conflit d'intérêts : Lorsque l'ennemi à juger est l'État lui-même et ses agents, le système judiciaire national, dont les magistrats ont souvent été nommés par l'ancien régime, est paralysé.

L'État est peu enclin à se considérer comme criminel.

• Principe d'individualisation : Le droit pénal se concentre sur la responsabilité individuelle, ce qui est inadapté pour traiter des dynamiques collectives et des crimes systémiques.

Face à cette impuissance, la sanction est souvent "rangée au placard" au profit de lois d'amnistie, ouvrant la voie à la recherche d'autres formes de justice.

2. La Justice Transitionnelle : Une Alternative Politique

En réponse aux limites du droit pénal, la "justice transitionnelle" a été développée non pas comme un droit, mais comme une "justice politique". Il s'agit d'un compromis politique visant à permettre une transition vers la paix ou la démocratie.

• Piliers Fondamentaux :

◦ Vérité : Établir un récit partageable des événements.  

◦ Réparation : Offrir des compensations aux victimes.  

◦ Réconciliation : Pacifier le conflit social.

• Mécanismes emblématiques : L'institution la plus connue est la Commission de Vérité et de Réconciliation, comme celle mise en place en Afrique du Sud.

Ces commissions visent à construire une histoire nouvelle et audible par tous, où ceux qui étaient qualifiés de "terroristes" peuvent être reconnus comme "victimes".

• Limites et Contraintes :

◦ Une justice de l'impuissance : Elle naît de l'incapacité à poursuivre pénalement et ne raconte souvent qu'une partie de l'histoire.

En Afrique du Sud, elle a mis en lumière les souffrances individuelles mais a peu abordé les injustices structurelles de l'apartheid.  

◦ Cadrage de la parole des victimes : Ces institutions, pour éviter de raviver le conflit, encadrent fortement l'expression des victimes.

On leur impose un "langage très doux et chaleureux", les encourageant à pleurer plutôt qu'à exprimer leur colère, leurs revendications politiques ou matérielles.

Les victimes sont amenées à parler en tant que mères ou veuves plutôt qu'en tant que militantes, utilisant un langage de la souffrance traumatique plutôt que celui de la politique.

3. Le Renouvellement des Mécanismes de Justice Pénale

Parallèlement à la justice transitionnelle, les mécanismes de droit pénal ont évolué pour tenter de juger les crimes de masse. Sharon Weill distingue trois grandes catégories de tribunaux.

Type de Mécanisme

Exemples Clés

Caractéristiques et Limites

Justice Internationale

Cour Pénale Internationale (CPI) à La Haye.

Objectif : Mettre fin à l'impunité ("no safe heaven").<br>Juridiction : Limitée aux 123 États signataires (ou aux crimes commis sur leur territoire).<br>

Limites : Production de cas très limitée, forte influence des agendas politiques des États (ex: mandat d'arrêt rapide contre Poutine, inaction sur les crimes contre les migrants), complexité due à la diversité des cultures juridiques.

Justice Nationale

• Procès Papon (France)<br>• Procès Eichmann (Israël)<br>• Procès rwandais (France)<br>• Tribunaux militaires (Guantanamo, Israël)

Types :<br>

1. Juger ses propres citoyens : Souvent trop peu nombreux et trop tardifs.<br>

2. Juridiction universelle : Un pays juge des crimes commis à l'étranger sans lien direct. Pose des problèmes de légitimité et de perception (un jury français jugeant des faits au Libéria).<br>

3. Juger son ennemi : Remet en question l'indépendance et l'impartialité des cours.

Justice Hybride (Mixte)

Procès de Hissène Habré (ex-dictateur du Tchad) au Sénégal.

Modèle : Combine des éléments nationaux et internationaux pour "prendre le meilleur des deux mondes".<br>

Avantages : Juridiction spécialement créée, financement international, juges nationaux et internationaux, et surtout, une localisation plus proche des victimes (Sénégal plutôt que La Haye), favorisant leur participation.

4. Vers des Modèles Holistiques : L'Exemple Colombien

Le processus de paix colombien de 2016 illustre une nouvelle approche qui tente de réintégrer la justice pénale dans une démarche plus holistique et restaurative.

• Fonctionnement : Une cour spéciale a été créée. Les accusés qui reconnaissent leur responsabilité, contribuent à la vérité et dialoguent avec les victimes peuvent éviter la prison.

• Sanctions alternatives : Au lieu de l'incarcération, les accusés s'engagent dans des "projets de réparation" conçus avec les victimes (reconstruire des écoles, créer des monuments).

• Approche "Macro" : La justice ne se concentre pas uniquement sur des cas individuels mais sur des "macro-cas", analysant des dynamiques de violence sur un territoire ou d'un type particulier (ex: les enlèvements).

• Principes clés : Participation massive des victimes, responsabilisation des auteurs et réparation collective.

5. Étude de Cas : Le Procès des Attentats du 13 Novembre 2015 (V13)

Le procès V13 en France est un exemple fascinant d' "hybridation", où un système de droit pénal classique et sévère a intégré des pratiques issues de la justice transitionnelle.

5.1 Une Place Inédite pour les Victimes

Dans un cadre judiciaire très solennel (Cour d'assises spéciale sans jury), le procès a consacré deux mois entiers aux témoignages des victimes.

Plus de 2400 parties civiles ont pu s'exprimer, une démarche exceptionnelle dans un procès pénal français.

Cet espace a permis de prendre la mesure de la souffrance et de reconnaître le statut des victimes, transformant un procès pénal en une scène de reconnaissance collective.

5.2 La Parole des Victimes : Entre Reconnaissance et Contrainte

Comme dans les commissions de vérité, la parole des victimes a été majoritairement celle du traumatisme.

Le langage médical ("hypervigilance", "peur panique") et l'expression de la souffrance ont dominé.

• Limites de la reconnaissance : Toutes les victimes n'ont pas eu la même place.

Les habitants de la rue du Corbillon, touchés par l'assaut policier du 18 novembre, ont longtemps été considérés comme victimes d'une opération policière et non du terrorisme, les reléguant à un statut secondaire.

• Canalisation de la colère : Les victimes en colère, notamment contre les défaillances de l'État (prévention, gestion des corps), ont vu leur discours tenu en lisière.

• Demande de compréhension : Certaines victimes, particulièrement des intellectuels, ont exprimé leur besoin de comprendre au-delà du crime individuel.

Elles ont réclamé une analyse des "dynamiques collectives" ayant mené des jeunes hommes à commettre ces actes, soulignant le manque d'une partie du "scénario".

5.3 Le Rôle Inattendu des Accusés et la Quête d'une Justice Restaurative

Malgré l'absence d'incitation à coopérer (contrairement au modèle colombien), plusieurs accusés ont choisi de parler.

Salah Abdeslam, silencieux pendant six ans, a parlé pendant trois heures dès le premier jour. Des échanges spontanés, parfois tendus, ont eu lieu entre accusés et victimes.

Une scène finale troublante a marqué les esprits : à l'issue du procès, de nombreuses victimes se sont approchées des trois accusés sous contrôle judiciaire sur les marches du palais de justice pour leur parler.

Cet acte spontané illustre une quête, par les victimes elles-mêmes, d'une forme de justice restaurative allant au-delà de la sanction pénale. Cela démontre que pour elles, la sanction seule ne suffit pas.

6. Conclusion : Vers un Nouveau Paradigme Juridique ?

Les expériences de la justice transitionnelle et des procès comme le V13 bousculent profondément le droit pénal traditionnel, qui produit une "vérité judiciaire" et non une vérité sociale ou historique.

On observe une évolution d'un droit purement répressif vers un droit plus restauratif.

• Influence des critiques : Des approches critiques, notamment féministes, remettent en question les finalités du droit pénal.

• Convergence des luttes : Sandrine Lefranc établit un parallèle entre la réponse aux violences politiques de masse et celle aux violences sexuelles, une autre forme de violence de masse.

Dans les deux cas, le droit pénal est jugé insuffisant et des alternatives (comme la justice restaurative) sont explorées pour permettre aux victimes de trouver autre chose que la seule sanction.

• Rôle des sciences sociales : Ces nouveaux espaces judiciaires ou para-judiciaires offrent une place inédite aux sciences sociales pour contribuer à la compréhension des événements collectifs.

Reviewer #1 (Public review):

This work by Antonnen et al. was triggered by claims of auditory-mediated effects on altricial avian embryos, which were published without any direct evidence that the relevant parental vocalizations were actually heard. I agree with Anttonen et al. that, based on the available evidence about avian auditory development, those claims are highly speculative and therefore necessitate more direct experimental verification.

Attonen et al. have embarked on a comprehensive series of experiments to:

(1) Better characterize acoustically the relevant parental vocalizations (heat whistles; in a separate preprint, not reviewed here)

(2) Characterize the auditory sensitivity of zebra finches at various stages of their posthatching development. Despite the long-standing importance of the zebra finch as a songbird model in neuroethology of learned vocalizations, the auditory development of the species has not been studied so far.

(3) Explore an alternative hypothesis of how the parental vocalizations might be perceived.

The principal method used here is the non-invasive recording of ABR (auditory brainstem response), a standard neurophysiological method in auditory research. The click-evoked ABR provides a quick and objective assessment of basic hearing sensitivity that does not require animal training. Weaknesses of the technique include its limited frequency specificity and low signal-to-noise ratio. The authors are experienced with ABR measurements and well aware of those issues. ABR responses in zebra finches are shown to gradually appear during the first week posthatching and to mature in subsequent weeks, consistent with the auditory development in other altricial bird species studied previously. When matching the acoustic properties of parental heat whistles and auditory sensitivities, hearing of the parental heat whistles by zebra finch hatchlings was convincingly excluded. Although not directly measured, this also convincingly extrapolates to zebra finch embryos. Finally, the authors tested the hypothesis that parental heat whistles could induce perceptible vibrations of the egg and thus stimulate the embryo via a different modality. The method used here was laser doppler vibrometry, an appropriate, state-of-the-art technique that the authors also have proven experience with. The induced vibrations were shown to be several orders of magnitude below known vibrotactile sensitivities in mammals and birds. Thus, although zebra finch vibrotactile thresholds were not obtained directly, the hypothesis of vibrotactile perception of parental heat whistles by zebra finch embryos could also be rejected convincingly.

In summary, even when considering some weaknesses of the techniques (which the authors are aware of), the conclusions of the paper are well supported: Auditory and/or vibration perception of parental heat whistles can be excluded as an explanation for previous reports of developmental programming for high ambient temperatures. As a constructive suggestion towards resolving the apparent paradox, the authors recommend repeating some of the crucial, previous playback experiments at lower sound levels that better match the natural parental vocalizations.

Reviewer #2 (Public review):

This study by Anttonen, Christensen-Dalsgaard, and Elemans describes the development of hearing thresholds in an altricial songbird species, the zebra finch. The results are very clear and along what might have been expected for altricial birds: at hatch (2 days post-hatch), the chicks are functionally deaf. Auditory evoked activity in the form of auditory brainstem responses (ABR) can start to be detected at 4 days post-hatch, but only at very loud sound levels. The study also shows that ABR response matures rapidly and reaches adult-like properties around 25 days post-hatch. The functional development of the auditory system is also frequency dependent, with a low-to-high frequency time course. All experiments are very well performed. The careful study throughout development and with the use of multiple time-points early in development is important to further ensure that the negative results found right after hatching are not the result of the experimental manipulation. The results themselves could be classified as somewhat descriptive, but, as the authors point out, they are particularly relevant and timely. Since 2016, there have been a series of studies published in high-profile journals that have presumably shown the importance of prenatal acoustic communication in altricial birds, mostly in zebra finches. This early acoustic communication would serve various adaptive functions. Although acoustic communication between embryos in the egg and parents has been shown in precocial birds (and crocodiles), finding an important function for prenatal communication in altricial birds came as a surprise. Unfortunately, none of those studies performed a careful assessment of the chicks' hearing abilities. This is done here, and the results are clear: zebra finches at 2 and 6 days post-hatch are functionally deaf. Since it is highly improbable that the hearing in the egg is more developed than at birth, one can only conclude that zebra finches in the egg (or at birth) cannot hear the heat whistles. The paper also ruled out the detection on egg vibrations as an alternative path. The prior literature will have to be corrected, or further studies conducted to solve the discrepancies. For this purpose, the "companion" paper on bioRxiv that studies the bioacoustical properties of heat calls from the same group will be particularly useful. Researchers from different groups will be able to precisely compare their stimuli.

Beyond the quality of the experiments, I also found that the paper was very well written. The introduction was particularly clear and complete (yet concise).

Weaknesses:

My only minor criticism is that the authors do not discuss potential differences between behavioral audiograms and ABRs. Optimally, one would need to repeat the work of Okanoya and Dooling with your setup and using the same calibration. The ~20dB difference might be real, or it might be due to SPL measured with different instruments, at different distances, etc. Either way, you could add a sentence in the discussion that states that even with the 20 dB difference in audiogram heat whistles would not be detected during the early days post-hatch. But adding a (novel) behavioral assay in young birds could further resolve the issue.

More Minor Points:

(1) As mentioned in the main text, the duration of pips (from pips to bursts) affects the effective bandwidth of the stimulus. I believe that the authors could give an estimate of this effective bandwidth, given what is known from bird auditory filters. I think that this estimate could be useful to compare to the effective bandwidth of the heat-call, which can now also be estimated.

(2) Figure 5b. Label the green and pink areas as song and heat-call spectrum. Also note that in the legend the authors say: "Green and red areas display the frequency windows related to the best hearing sensitivity of zebra finches and to heat calls, respectively". I don't think this is what they meant. I agree that 1-4 kHz is the best frequency sensitivity of zebra finches, but they probably meant green == "song frequency spectrum" and pink == "heat call spectrum". In either case, the figure and the legend need clarification.

(3) Figure 5c. Here also, I would change the song and heat-call labels to "song spectrum", "heat call spectrum". The authors would not want readers to think that they used song and heat calls in these experiments (maybe next time?). For the same reason, maybe in 5a you could add a cartoon of the oscillogram of a frequency sweep next to your speaker.

(4) Methods. In the description of the stimulus, the authors describe "5ms long tone bursts", but these are the tone pips in the main part of the manuscript. Use the same terms.

Reviewer #3 (Public review):

Summary

Following recent findings that exposure to natural sounds and anthropogenic noise before hatching affects development and fitness in an altricial songbird, this study attempts to estimate the hearing capacities of zebra finch nestlings and the perception of high frequencies in that species. It also tries to estimate whether airborne sound can make zebra finch eggs vibrate, although this is not relevant to the question.

Strength

That prenatal sounds can affect the development of altricial birds clearly challenges the long-held assumption that altricial avian embryos cannot hear. However, there is currently no data to support that expectation. Investigating the development of hearing in songbirds is therefore important, even though technically challenging. More broadly, there is accumulating evidence that some bird species use sounds beyond their known hearing range (especially towards high frequencies), which also calls for a reassessment of avian auditory perception.

Weaknesses

Rather than following validated protocols, the study presents many experimental flaws and two major methodological mistakes (see below), which invalidate all results on responses to frequency-specific tones in nestlings and those on vibration transmission to eggs, as well as largely underestimating hearing sensitivity. Accordingly, the study fails to detect a response in the majority of individuals tested with tones, including adults, and the results are overall inconsistent with previous studies in songbirds. The text throughout the preprint is also highly inaccurate, often presenting only part of the evidence or misrepresenting previous findings (both qualitatively and quantitatively; some examples are given below), which alters the conclusions.

Conclusion and impact

The conclusion from this study is not supported by the evidence. Even if the experiment had been performed correctly, there are well-recognised limitations and challenges of the method that likely explain the lack of response. The preprint fails to acknowledge that the method is well-known for largely underestimating hearing threshold (by 20-40dB in animals) and that it may not be suitable for a 1-gram hatchling. Unlike what is claimed throughout, including in the title, the failure to detect hearing sensitivity in this study does not invalidate all previous findings documenting the impacts of prenatal sound and noise on songbird development. The limitations of the approach and of this study are a much more parsimonious explanation. The incorrect results and interpretations, and the flawed representation of current knowledge, mean that this preprint regrettably creates more confusion than it advances the field.

Detailed assessment

For brevity, only some references are included below as examples, using, when possible, those cited in the preprint (DOI is provided otherwise). A full review of all the studies supporting the points below is beyond the scope of this assessment.

(A) Hearing experiment

The study uses the Auditory Brainstem Response (ABR), which measures minute electrical signals transmitted to the surface of the skull from the auditory nerve and nuclei in the brainstem. ABR is widely used, especially in humans, because it is non-invasive. However, ABR is also a lot less sensitive than other methods, and requires very specific experimental precautions to reliably detect a response, especially in extremely small animals and with high-frequency sounds, as here.

(1) Results on nestling frequency sensitivity are invalid, for failing to follow correct protocols:

The results on frequency testing in nestlings are invalid, since what might serve as a positive control did not work: in adults, no response was detected in a majority of individuals, at the core of their hearing range, with loud 95dB sounds (Figure S1), when testing frequency sensitivity with "tone burst".

This is mostly because the study used a stimulation duration 5 times larger than the norm. It used 25ms tone bursts, when all published avian studies (in altricial or precocial birds) used stimulation of 5ms or less (when using subdermal electrodes as here; e.g., cited: Brittan-Powell et al 2004; not cited: Brittan-Powell et al 2002 (doi: 10.1121/1.1494807), Henry & Lucas 2008 (doi: 10.1016/j.anbehav.2008.08.003)). Long stimulations do not make sense and are indeed known to interfere with the detection of an ABR response, especially at high frequencies, as, for example, explicitly tested and stated in Lauridsen et al 2021 (cited).

Adult response was then re-tested with a correct 5ms tone duration ("tone-pip"), which showed that, for the few individuals that responded to 25ms tones, thresholds were abnormally high (c.a. by 30dB; Figure 2C).<br /> Yet, no nestlings were retested with a correct protocol. There is therefore no valid data to support any conclusion on nestling frequency hearing. Under these circumstances, the fact that some nestlings showed a response to 25ms tones from day 8 would argue against them having very low sensitivity to sound.

(2) Responses to clicks underestimate hearing onset by several days:

Without any valid nestling responses to tones (see # 1), establishing the onset of hearing is not possible based on responses to clicks only, since responses to clicks occur at least 4 days after responses to tones during development (Saunders et al, 1973). Here, 60% of 4-day-old individuals responding to clicks means most would have responded to tones at and before 2 days post-hatch, had the experiment been done correctly.<br /> Responses to tones are indeed observed in other songbirds at 1day post-hatch (see #6).

In budgerigars, hearing onset occurs before 5 days post hatch, since responses to both clicks and tones were detectable at the first age tested at 5dph (Brittan-Powell et al, 2004).

(3) Experimental parameters chosen lower ABR detectability, specifically in younger birds:

Very fast stimulus repetition rate inhibits the ABR response, especially in young:

(a) The stimulus presentation rate (25 stim/ sec) is 6 times faster than zebra finch heat-calls, and 5 to 25 times faster than most previous studies in young birds (e.g., cited: Saunders et al 1973, 1974: 1 stim/sec or less; Katayama 1985: 3.3 clicks/sec; Brittan-Powell et al 2004: 4 stim/sec). Faster rates saturate the neurons and accordingly are known to decrease ABR amplitude and increase ABR latency, especially in younger animals with an immature nervous system. In birds, this occurs especially in the range from 5 to 30 stim/sec (e.g., cited: Saunder et al 1973, Brittan-Powell et al 2004). Values here with 25 rather than 1-4 stim/min are therefore underestimating true sensitivity.

(b) Averaging over only 400 measures is insufficient to reliably detect weak ABR signals:

The study uses 2 to 3 times fewer measures per stimulation type than the recommended value of 1,000 (e.g., Brittan-Powell et al 2002, 2024; Henry & Lucas 2008). This specifically affects the detection of weak signals, as in small hatchlings with tiny brains (adult zebra finches are 12-14g).

(c) Body temperature is not specified and strongly affects the ABR:

Controlling the body temperature of hatchlings of 1-4 grams (with a temperature probe under a 5mm-wide wing) would be very challenging. Low body temperature entirely eliminates the ABR, and even slight deviance from optimal temperature strongly increases wave latency and decreases wave amplitude (e.g., cited: Katayama 1985).

(d) Other essential information is missing on parameters known to affect the ABR:

This includes i) the weight of the animals, ii) whether and how the response signal was amplified and filtered, iii) how the automatised S/N>2 criteria compared to visual assessment for wave detection, and iv) what measures were taken to allow the correct placement of electrodes on hatchlings less than 5 grams.

(4) Results in adults largely underestimate sensitivity at high frequencies, and are not the correct reference point:

(a) Thresholds measured here at high frequencies for adults (using the correct stimulus duration, only done on adults) are 10-30dB higher than in all 3 other published ABR studies in adult zebra finches (cited: Zevin et al 2004; Amin et al 2007; not cited: Noirot et al 2011 (10.1121/1.3578452)), for both 4 and 6 kHz tone pips.

(b) The underlying assumption used throughout the preprint that hearing must be adult-like to be functional in nestlings does not make sense. Slower and smaller neural responses are characteristic of immature systems, but it does not mean signals are not being perceived.

(5) Failure to account for ABR underestimation leads to false conclusions:

(a) Whether the ABR method is suitable to assess hearing in very small hatchlings is unknown. No previous avian study has used ABR before 5 days post-hatch, and all have used larger bird species than the zebra finch.

(b) Even when performed correctly on large enough animals, the ABR systematically underestimates actual auditory sensitivity by 20-40 dB, especially at high frequencies, compared to behavioural responses (e.g., none cited: Brittan-Powell et al 2002, Henry & Lucas 2008, Noirot et al 2011). Against common practice, the preprint fails to account for this, leading to wrong interpretations. For example, in Figure 1G (comparing to heat call levels), actual hearing thresholds would be 30-40dB below those displayed. In addition, the "heat whistle" level displayed here (from the same authors) is 15dB lower than their second measure that they do not mention, and than measures obtained by others (unpublished data). When these two corrections are made - or even just the first one - the conclusion that heat-call sound levels are below the zebra finch hearing threshold does not hold.

(c) Rather than making appropriate corrections, the preprint uses a reference in humans (L180), where ABR is measured using a much more powerful method (multi-array EEG) than in animals, and from a larger brain. The shift of "10-20dB" obtained in humans is not applicable to animals.

(6) Results are inconsistent with previous findings in developing songbirds:

As expected from all of the above, results and conclusions in the preprint are inconsistent with findings in other songbirds, which, using other methods, show for example, auditory sensitivity in:

(a) zebra finch embryos, in response to song vs silence (not cited: Rivera et al 2018, doi: 10.1097/WNR.0000000000001187)

(b) flycatcher hatchlings at 2-3d post hatch (first age tested), across a wide range of frequencies (0.3 to 5kHz), at low to moderate sound levels (45-65dB) (cited: Aleksandrov and Dmitrieva 1992, not cited: Korneeva et al 2006 (10.1134/S0022093006060056)).

(c) songbird nestlings at 2-6d post hatch, which discriminate and behaviourally respond to relevant parental calls or even complex songs. This level of discrimination requires good hearing across frequencies (e.g., not cited: Korneeva et al 2006; Schroeder & Podos 2023 (doi: 10.1016/j.anbehav.2023.06.015)).

(d) zebra finch nestlings at 13d post-hatch, which show adult-like processing of songs in the auditory cortex (CNM) (Schroeder & Remage‐Healey 2021, doi: 10.1002/dneu.22802).

(e) zebra finch juveniles, which are able to perceive and learn song syllables at 5-7kHz (fundamental frequency) with very similar acoustic properties to heat calls, and also produced during inspiration (Goller & Daley 2001, doi: 10.1098/rspb.2001.1805).

NONE of these results - which contradict results and claims in the preprint - are mentioned. Instead, the preprint focuses on very slow-developing species (parrots and owls), which take 2-4 times longer than songbirds to fledge (cited: Brittan-Powell et al 2004; Köppl & Nickel 2007; Kraemer et al 2017).

(7) Results in figures are misreported in the text, and conclusions in the abstract and headers are not supported by the data:

For example:

(a) The data on Figure 1E shows that at 4 days old, 8 out of 13 nestlings (60%) responded to clicks, but the text says only 5/13 responded (L89). When 60% (4dph) and 90% (6dph) of individuals responded, the correct term would be that "most animals", rather than "some animals" responded (L89). Saying that ABR to loud sound appeared "in the majority only after one week" (L93) is also incorrect, given the data. It follows that the title of the paragraph is also erroneous.

(b) The hearing threshold is underestimated by 40dB at 6 and 8Kz on Fig 2C, not by "10-20dB" as reported in the text (L178).

(B) Egg vibration experiment

(8) Using airborne sound to vibrate eggs is biologically irrelevant:

The measurement of airborne sound levels to vibrate eggs misunderstands bone conduction hearing and is not biologically meaningful: zebra finch parents are in direct contact with the eggs when producing heat calls during incubation, not hovering in front of the nest. This misunderstanding affects all extrapolations from this study to findings in studies on prenatal communication.

(C) Misrepresentation of current knowledge

(9) Values from published papers are misreported, which reverses the conclusions:

Most critical examples:

(a) Preprint: "Zebra finch most sensitive hearing range of 1-to-4 kHz (Amin et al., 2007; Okanoya and Dooling, 1987; Yeh et al., 2023)" (L173).<br /> Actual values in the studies cited are:

1-to-7kHz, in Amin et al 2007 (threshold [=50dB with ABR] is the same at 7kHz and 1KHz).

1-to-6 kHz, in Okanoya and Dooling (the threshold [=30dB with behaviour] is actually lower at 6kHz than at 1KHz).

1-to-7kHz, in Yeh et al (threshold [=35-38dB with behaviour] is the same at 7kHz and 1KHz).

Note that zebra finch nestlings' begging calls peaking at 6kHz (Elie & Theunissen 2015, doi: 10.1007/s10071-015-0933-6), would fall 2kHz above the parents' best hearing range if it were only up to 4kHz.

(b) The preprint incorrectly states throughout (e.g., L139, L163, L248) that heat-calls are 7-10kHz, when the actual value is 6-10kHz in the paper cited (Katsis et al, 2018).

(c) Using the correct values from these studies, and heat-calls at 45 dB SLP (as measured by others (unpublished data), or as measured by the authors themselves, but which is not reported here (Anttonen et a,l 2025), the correct conclusion is that heat calls fall within the known zebra finch hearing range.

(10) Published evidence towards high-frequency hearing, including in early development, is systematically omitted:

(a) Other studies showing birds use high frequencies above the known avian hearing range are ignored. This includes oilbirds (7-23kHz; Brinklov et al 2017; by 1 of the preprint authors, doi: 10.1098/rsos.170255) and hummingbirds (10-20kHz; Duque et al 2020, doi: 10.1126/sciadv.abb9393), and in a lesser extreme, zebra finches' inspiratory song syllables at 5-7kHz (Goller & Dalley, 2001).

(b) The discussion of anatomical development (L228-241) completely omits the well-known fact that the avian basilar papilla develops from high to low frequencies (i.e., base to apex), which - as many have pointed out - is opposite to the low-to-high development of sensitivity (e.g., cited: Cohen & Fermin 1978; Caus Capdevila et al 2021).

(c) High frequency hearing in songbirds at hatching is several orders of magnitude better than in chickens and ducks at the same age, even though songbirds are altricial (e.g., at 4kHz, flycatcher: 47dB, chicken-duck: 90dB; at 5kHz, flycatcher: 65dB, chicken-duck: 115dB; Korneeva et al 2006, Saunders et al 1974). That is because Galliformes are low-frequency specialists, according to both anatomical and ecological evidence, with calls peaking at 0.8 to 1.2kHz rather than 2-6kHz in songbirds. It is incorrect to conclude that altricial embryos cannot perceive high frequencies because low-frequency specialist precocial birds do not (L250;261).

The references used to support the statement on a very high threshold for precocial birds above 6kHz are also wrong (L250). Katayama 1985 did not test embryos, nor frequency tones. Neither of these two references tested ducks.

(11) Incorrect statements do not reflect findings from the references cited

For example:

(a) "in altricial bird species hearing typically starts after hatching" (L12, in abstract), "with little to no functional hearing during embryonic stages (Woolley, 2017)." (L33).

There is no evidence, in any species, to support these statements. This is only a - commonly repeated - assumption, not actually based on any data. On the contrary, the extremely limited evidence to date shows the opposite, with zebra finch embryos showing ZENK activation in the auditory cortex in response to song playback (Rivera et al, 2018, not cited).

The book chapter cited (Woolley 2017) acknowledges this lack of evidence, and, in the context of song learning, provides as only references (prior to 2018), 2 studies showing that songbirds do not develop a normal song if the song tutor is removed before 10d post-hatch. That nestlings cannot memorise (to later reproduce) complex signals heard before d10 does not mean that they are deaf to any sound before day 10.

Studies showing hearing in young songbird nestlings (see point 6 above) also contradict these statements.

(b) "Zebra finch embryos supposedly are epigenetically guided to adapt to high temperatures by their parents high-frequency "heat calls" " (L36 and L135).

This is an extremely vague and meaningless description of these results, which cannot be assessed by readers, even though these results are presented as a major justification for the present study. Rather than giving an interpretation of what "supposedly" may occur, it would be appropriate to simply synthesize the empirical evidence provided in these papers. They showed that embryonic exposure to heat-calls, as opposed to control contact calls, alters a suite of physiological and behavioural traits in nestlings, including how growth and cellular physiology respond to high temperatures. This also leads to carry-over effects on song learning and reproductive fitness in adulthood.

(c) "The acoustic communication in precocial mallard ducks depends specifically on the low-frequency auditory sensitivity of the embryo (Gottlieb, 1975)" (L253)

The study cited (Gottlieb, 1975) demonstrates exactly the opposite of this statement: it shows that duckling embryos, not only perceive high frequency sounds (relative to the species frequency range), but also NEED this exposure to display normal audition and behaviour post-hatch. Specifically, it shows that duckling embryos deprived of exposure to their own high-frequency calls (at 2 kHz), failed to identify maternal calls post-hatch because of their abnormal insensitivity to higher frequencies, which was later confirmed by directly testing their auditory perception of tones (Dimitrieva & Gottlieb, 1994).

(12) Considering all of the mistakes and distortions highlighted above, it would be very premature to conclude, based on these results and statements, that altricial avian embryos are not sensitive to sound. This study provides no actual scientific ground to support this conclusion.

Author Response:

We thank all reviewers for their time and effort to carefully review our paper and for the constructive comments on our manuscript. Below we outline our planned revisions to the public reviews of the three reviewers.

In our revision, we will include more details regarding our ABR measurements (including temperature, animal metadata), analysis (including filter settings) and lay out a much more detailed motivation for our ABR signal design. Furthermore, we will provide a more detailed discussion on the caveats of the technique and the interpretation of ABR data in general and our data specifically. Furthermore, we will add more discussion on differences between ABR based audiograms and behavioural data. The authors have extensive experience with the ABR technique and are well aware of its limitations, but also its strengths for use in animals that cannot be trained on behavioural tasks such as the very young zebra finches in this study. These additions will strengthen our paper. We think our conclusions remain justified by our data.

Reviewer #1 and #2:

We thank both reviewers for their positive words and suggested improvements. The planned general improvements listed above will take care of all suggestions and comments in the public review.

Reviewer #3:

We thank the reviewer for the detailed critique of our manuscript and many suggestions for improvement. The planned general improvements listed above will take care of many of the suggestions and comments listed in the public review. Here we will highlight a few first responses that we will address in detail in our resubmission.

The reviewer’s major critiques can be condensed to the following four points.

(1) ABR cannot be done in such small animals.

This critique is unfounded. ABR measures the summed activity in the auditory pathway, and with smaller distance from brainstem to electrodes in small animals, the ABR signals are expected to have higher amplitude and consequently better SNR.  Thus, smaller animals should lead to higher amplitude ABR signals. We have successfully recorded ABR in animals smaller than 2 DPH zebra finches to support this claim (zebrafish (Jørgensen et al., 2012), 10 mm froglets (Goutte et al., 2017) and 5 mm salamanders (Capshaw et al., 2020). It is more surprising the technique still provides robust signals even in very large animals such as Minke whales (Houser et al., 2024).

(2) The ABR methods used does not follow protocol for other published work in birds. Particularly the 25 ms long duration tone bursts may have underestimated high frequency hearing.

There is no fixed protocol for ABR measurements, and several studies of bird ABR have used as long or even longer durations. Longer-duration signals were chosen deliberately and are necessary to have a sufficient number of cycles and avoid frequency splatter at our lowest frequencies used (see Lauridsen et al., 2021).

(3) Sensitivity data should be corrected from ABR to behavioural data.

We present the results of our measurements on hearing sensitivity using ABR, and ABR based thresholds are generally less sensitive than thresholds based on behavioural studies (presented in Fig 2c). Correcting for these measurements to behavioural thresholds is of course possible, but presenting only the corrected thresholds would be a misrepresentation of our sensitivity data. Even so it should be done only within species and age group and such data is currently not available. In our revision, we will include elaborate discussion on this topic.

(4) Results are inconsistent with papers in developing songbirds.

We agree that our results do not support and even question the claims in earlier work. These papers however do either 1) not measure hearing physiology or 2) do so in different species. To our best knowledge there is presently no data published on the auditory physiology development in songbird embryos. Our data are consistent with what is known about the physiology of auditory development in all birds studied so far. We will provide a detailed discussion on this topic in our revision.

References

Capshaw et al. (2020) J Exp Biol 223: jeb236489

Goutte et al. (2017) Sci Rep 7: 12121, doi 10.1038/s41598-017-12145-5

Houser et al. (2024) Science 386, 902-906. DOI:10.1126/science.ado7580).

Jørgensen et al. (2012) Adv Exp Med Biol 730: 117-119

Lauridsen et al (2021) J Exp Biol 224: jeb237313. https://doi.org/10.1242/jeb.237313

Synthèse : Comprendre le Logiciel de l'Esprit

Résumé Exécutif

Cette note de synthèse analyse les thèmes centraux de la présentation du professeur Uichol Kim, qui remet en question les paradigmes occidentaux dominants sur l'esprit humain et le succès.

L'argument principal est que le "logiciel" occidental de l'esprit, fondé sur des hypothèses d'individualisme, de compétition ("la survie du plus apte") et de déterminisme biologique, est fondamentalement erroné.

Le professeur Kim propose une vision alternative où la coopération, les relations et la co-création sont les véritables moteurs de l'évolution et du bien-être humains.

Il soutient que l'évolution humaine a été rendue possible non par la compétition, mais par des innovations sociales et technologiques comme la maîtrise du feu et le langage, qui ont favorisé la collaboration.

L'esprit humain n'est pas un système biologique fermé et prédéterminé, mais un système ouvert et socialement construit, façonné par les expériences et les relations interpersonnelles, un concept renforcé par les découvertes en épigénétique et en neurosciences.

Enfin, des études empiriques à grande échelle, notamment les travaux de Daniel Kahneman et l'étude longitudinale de Harvard sur le développement des adultes, convergent vers une conclusion univoque :

le véritable bonheur et une vie longue et saine ne découlent pas de la richesse ou du succès individuel, mais de la qualité des relations chaleureuses et du partage avec les autres.

La satisfaction dans la vie (liée au revenu) et le bonheur (lié aux expériences relationnelles) sont deux concepts distincts, souvent confondus au détriment du bien-être humain.

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1. Critique des Hypothèses Fondamentales du "Logiciel" Occidental

Le professeur Kim commence par souligner l'importance des "hypothèses de base sur la réalité" qui, selon Peter Drucker, forment le paradigme d'une culture et d'une science.

Ces hypothèses, souvent implicites et résistantes au changement, déterminent ce qui est considéré comme un fait. La pensée occidentale repose sur plusieurs hypothèses qui sont remises en question.

• L'Individu comme Unité de Base (Socrate) : L'injonction socratique "Connais-toi toi-même" a placé l'individu comme l'unité d'analyse fondamentale, considérée comme "indivisible".

• La Compétition comme Moteur de l'Évolution (Darwin) : La théorie de l'évolution de Charles Darwin, basée sur la compétition, la sélection naturelle et la "survie du plus apte", a été largement appliquée à la société humaine, aux entreprises et aux individus, créant une croyance fondamentale en la nécessité de la compétition.

• Le Déterminisme Biologique et Pathologique (Freud) : Sigmund Freud a adopté un modèle biologique, définissant le comportement humain en termes de pulsions sexuelles ou violentes.

Ses théories ont été généralisées à l'ensemble de la population à partir d'études de cas de patients "hystériques" et anormaux, ce qui constitue une extrapolation problématique.

• Le Comportementalisme Réductionniste (Skinner) : B.F. Skinner a étudié des pigeons et des rats pour comprendre les êtres humains, supposant que les comportements de base sont le fondement des comportements complexes, ignorant ainsi la spécificité humaine et le rôle du contexte social.

• Le Développement Cognitif sans Contexte (Piaget) : Le modèle de développement cognitif de Jean Piaget, bien qu'influent, est critiqué pour son omission quasi-totale du rôle des parents et des émotions, car Piaget observait principalement ses propres enfants de manière isolée.

2. Un Paradigme Alternatif : L'Agentivité et l'Auto-Efficacité

En opposition aux modèles déterministes, le professeur Kim met en avant le travail d'Albert Bandura sur le "soi en tant qu'agent proactif".

L'être humain n'est pas simplement déterminé par la biologie ou l'environnement, mais possède une agentivité qui lui permet de façonner son propre avenir.

• Concept d'Auto-Efficacité : Il s'agit de la "croyance en sa propre capacité à organiser et exécuter les actions requises pour gérer des situations futures".

Les personnes ayant une auto-efficacité élevée agissent, pensent et ressentent différemment, produisant leur propre avenir plutôt que de simplement le prévoir.

• Composantes Clés : L'intention, la connaissance, les objectifs, les croyances et les compétences sont essentiels.

• Influence Sociale : L'auto-efficacité n'est pas purement individuelle. Elle est modifiée et renforcée par :

◦ Le feedback : La pratique constante, comme le font les athlètes et les musiciens.    ◦ Le soutien social : Un élément crucial pour augmenter l'auto-efficacité d'une personne.

3. Réévaluation de l'Évolution Humaine : La Coopération Prime sur la Compétition

L'exposé conteste directement l'idée que la compétition est le principal moteur de l'évolution humaine en réexaminant notre héritage biologique et anthropologique.

• Deux Modèles de Chimpanzés : Il existe une distinction entre les chimpanzés communs (agressifs, violents, hiérarchiques) et les bonobos ou "chimpanzés pygmées" (dominés par les femelles, égalitaires, non-violents).

L'espèce la plus proche de l'ancêtre humain est le bonobo, suggérant que nos racines sont plus coopératives qu'agressives.

• Le Rôle du Milieu : Les Homo sapiens ont évolué dans la savane subsaharienne, un environnement ouvert, tandis que les chimpanzés vivent dans la jungle.

• Adaptations Clés pour la Coopération :

◦ La Bipédie : Marcher sur deux pieds a permis de réduire le stress thermique, mais a surtout provoqué une "descente du larynx", rendant possible la production de jusqu'à 20 000 sons différents, base essentielle du langage et de la communication complexe.   

◦ La Maîtrise du Feu : La plus grande transformation. Les humains ont appris à contrôler le feu, ce qui a permis de cuire les aliments. La cuisson a détruit les bactéries et permis de consommer cinq fois plus de calories que la viande crue.  

◦ Développement du Cerveau : Cet apport calorique supplémentaire est la cause principale de la taille du cerveau humain (quatre fois plus grand que celui du chimpanzé), en particulier du lobe frontal.

C'est en surmontant notre instinct (la peur du feu) que nous avons développé un plus grand cerveau, et non l'inverse.

4. L'Esprit Humain comme Système Ouvert et Socialement Construit

La présentation souligne une différence fondamentale entre les humains et les autres primates : la capacité de stocker et de transmettre l'information en dehors du corps.

• Le Corps comme Système Fermé, l'Esprit comme Système Ouvert : Alors que le corps est défini par la peau, l'esprit est un système ouvert.

Le cerveau humain, avec ses milliards de neurones et ses billions de connexions potentielles, intègre de nouvelles idées et se reconfigure en permanence par l'interaction avec les autres.

• L'Explosion de la Créativité : Il y a 30 000 à 40 000 ans, l'art rupestre est apparu comme la "première technologie de l'information", permettant de projeter des images et de combiner des concepts (ex: l'homme-lion).

• Stockage Externe de l'Information :

◦ Un chimpanzé comme Kanzi peut apprendre à communiquer avec des symboles, mais ne peut pas enseigner cette connaissance à sa progéniture.

À sa mort, tout son savoir disparaît.  

◦ Chez les humains, l'invention de l'écriture (cunéiforme), du papier et de l'imprimerie a permis un stockage et une transmission de l'information exponentiels, permettant aux générations futures de se connecter spirituellement et intellectuellement aux idées passées.

• Neurosciences et Épigénétique :

◦ Épigénétique : L'idée qu'un gène spécifique définit une expression unique est une simplification excessive. Les gènes peuvent être activés ou désactivés par des facteurs environnementaux (alimentation, exercice, stress, expériences). Nous naissons avec des gènes, mais leur expression dépend de l'expérience.  

◦ Le Cerveau comme Construction Sociale : Citant le neurobiologiste Gerald Hüther, le professeur Kim affirme que "le cerveau humain est une construction sociale".

Les connexions neuronales se forment et se renforcent par l'expérience sociale et la répétition (ex: faire du vélo, conduire).   

◦ L'Absence d'Objectivité Pure : Toute information sensorielle passe par le système limbique, où elle est connectée aux émotions.

Un même stimulus active un réseau cognitif et émotionnel.

5. Contrastes Culturels : Individualisme Occidental vs Relationalisme Oriental

Le "logiciel de l'esprit" varie considérablement selon les cultures.

• La Dualité Cartésienne : René Descartes, par son doute radical, a établi une dualité stricte entre le corps (soumis aux lois naturelles) et l'âme/esprit (capable de comprendre Dieu et la vérité).

Cela a conduit à une pensée dichotomique (noir/blanc, bien/mal).

• La Vision Relationnelle Est-Asiatique : En Asie de l'Est, le noir et le blanc (Yin et Yang) ne sont pas en opposition mais en relation.

Le caractère chinois pour "humain" (人間) signifie "entre les humains".

◦ La devise n'est pas "Je pense, donc je suis" mais pourrait être traduite par "Je suis entre, donc je suis" (I am between, therefore I am).

• Exemples Coréens :

◦ Culture du riz : La riziculture nécessite une coopération intense, favorisant une culture de l'harmonie.  

◦ Le concept de Cheong (情) : Une forme de connexion humaine profonde, de compassion et d'affection. Ne pas ressentir de compassion pour un enfant en train de se noyer signifie ne pas être humain.  

◦ Piété filiale : Le corps n'appartient pas à l'individu mais a été reçu des parents.

Le succès est donc un devoir envers eux. Les enfants représentent le futur et les parents le passé, créant une interdépendance où les parents ne peuvent être heureux que si leurs enfants le sont.

6. La Science du Bonheur : Les Relations Avant l'Argent et le Succès

Les recherches empiriques les plus récentes en psychologie et en économie convergent pour démanteler le mythe selon lequel l'argent et le succès individuel mènent au bonheur.

A. Les Travaux de Daniel Kahneman (Prix Nobel)

Kahneman fait une distinction cruciale entre la "satisfaction de vie" (liée au "soi qui se souvient") et le "bien-être émotionnel" ou bonheur (lié au "soi qui expérimente").

Caractéristique

Satisfaction de Vie

Bonheur (Bien-être Émotionnel)

Prédicteurs

Revenu, éducation, succès, atteinte d'objectifs

Santé, relations, absence de solitude, partage

Relation au Revenu

Augmente avec le revenu

Plafonne à un revenu médian (~75 000 $)

Concept du Soi

"Soi qui se souvient" (Remembering self)

"Soi qui expérimente" (Experiencing self)

Focalisation

Évaluation globale de la vie, réalisations

Expériences vécues dans le moment présent

Conclusion de Kahneman : Les gens poursuivent la satisfaction de vie (liée au statut social et à l'argent) en pensant qu'elle leur apportera le bonheur. Cependant, les personnes à hauts revenus sont souvent plus stressées et ne consacrent pas plus de temps à des activités agréables. C'est une "illusion de focalisation" où l'on surestime l'impact d'un seul facteur (l'argent) sur le bien-être global.

B. L'Étude Longitudinale de Harvard sur le Développement des Adultes

Cette étude, menée sur 85 ans auprès de deux groupes (hommes de Harvard et hommes de quartiers défavorisés de Boston), est l'une des plus longues jamais réalisées.

• Découverte Surprenante : Le facteur le plus puissant influençant la santé et la longévité n'est ni l'argent, ni le succès, ni le QI.

• Principaux Résultats :

◦ Les personnes les plus satisfaites de leurs relations à 50 ans étaient les plus en bonne santé à 80 ans.   

◦ Les relations chaleureuses sont un meilleur prédicteur d'une vie longue et heureuse que le statut social, le QI ou les gènes.  

◦ La solitude tue. Elle est associée à un décès plus précoce (jusqu'à 10 ans), au stress, à la dépression et à une mauvaise santé physique.   

◦ La qualité des relations avec la mère dans l'enfance prédisait l'efficacité au travail et des revenus plus élevés.   

◦ Des relations chaleureuses avec les parents étaient liées à moins d'anxiété et une plus grande satisfaction à l'âge adulte.

Conclusion de Robert Waldinger (directeur actuel de l'étude) : "La clé du vieillissement en bonne santé est : relation, relation, relation."

Les personnes les plus heureuses et en meilleure santé sont celles qui ont cultivé les "connexions les plus chaleureuses avec les autres".

7. Débat sur l'Analogie du "Logiciel"

Lors de la session de questions-réponses, l'analogie du "logiciel de l'esprit" est remise en question.

• La Critique : Un intervenant suggère que l'analogie est potentiellement trompeuse.

Un logiciel est un ensemble d'instructions spécifiques exécutées par un ordinateur standard.

Le cerveau ne fonctionne pas de cette manière ; il s'apparente davantage à un réseau neuronal artificiel complexe d'où émerge un comportement.

Des termes comme "culture", "récits" ou "habitudes" pourraient être plus appropriés et moins confus.

• La Réponse du Professeur Kim : Il reconnaît qu'il s'agit d'une analogie utilisée pour inciter les gens à penser différemment, en s'éloignant des vues déterministes (biologiques, cognitives-mécaniques) et en soulignant que le "logiciel" est invisible et que chacun fonctionne différemment.

L'analogie vise à introduire le concept d'agentivité et l'importance du soutien social.

Il admet ne pas avoir de meilleure analogie pour l'instant et souligne que les ordinateurs eux-mêmes sont des créations humaines qui imitent certaines de nos fonctions.

Document d'Information : Utilisation des Systèmes d'IA pour la Prise de Décision dans l'État Moderne

Synthèse Exécutive

Ce document synthétise les perspectives d'experts sur l'application des systèmes d'intelligence artificielle (IA) dans deux domaines sociétaux critiques : le droit en Europe et la santé en Afrique du Sud.

Dans le secteur juridique européen, l'IA est présentée comme une solution à la pression croissante entre l'augmentation des coûts du travail juridique et la nécessité de maintenir un état de droit de haute qualité face à une complexité réglementaire grandissante.

Les applications clés incluent l'optimisation de la recherche d'informations juridiques, la révision de contrats, la diligence raisonnable et l'analyse de cas complexes.

L'IA n'est pas considérée comme une menace pour l'emploi des juristes, mais plutôt comme un outil pour automatiser les tâches fastidieuses, leur permettant de se concentrer sur des activités à plus forte valeur ajoutée.

Cependant, des risques importants subsistent, notamment le manque d'explicabilité des décisions prises par l'IA (risque d'aliénation) et la multiplication des erreurs en cas de faille dans un système automatisé.

Dans le secteur de la santé sud-africain, confronté à des ressources limitées et à une forte prévalence de maladies transmissibles, l'IA offre un potentiel immense pour passer d'un modèle de santé curatif coûteux à un modèle préventif.

Les applications vont du diagnostic assisté par l'analyse d'images médicales à la prédiction de l'apparition de maladies grâce à des modèles d'apprentissage automatique.

Une vision d'avenir optimiste repose sur le déploiement de technologies à faible coût, comme les dispositifs portables (wearables), pour un suivi continu des individus.

Ces données pourraient créer des "jumeaux numériques" des citoyens et, à terme, des villes entières, permettant une surveillance, une simulation et des interventions proactives en matière de santé publique à une échelle sans précédent.

L'adaptation des technologies au contexte local à faibles ressources est une condition essentielle de succès.

Enfin, le document souligne l'importance cruciale de la collaboration interdisciplinaire pour développer des systèmes d'IA qui soient non seulement techniquement performants mais aussi socialement pertinents et responsables.

L'IA dans le Domaine Juridique : Relever les Défis en Europe

L'analyse du professeur Henrik Palmer Olsen de l'Université de Copenhague met en lumière les tensions et les opportunités liées à l'intégration de l'IA dans le système juridique européen.

Le Défi : La Pression entre le Coût et l'État de Droit

Le principal défi identifié est une "pression" économique et qualitative.

D'un côté, le travail juridique devient de plus en plus coûteux.

De l'autre, la demande pour ce travail augmente en raison de la complexification croissante de la réglementation, due au développement technologique, économique et social.

Les États européens sont donc confrontés au dilemme de maîtriser les dépenses tout en garantissant la haute qualité de l'état de droit, un principe fondamental de leur société.

Le Rôle de l'IA : Soutien et Optimisation du Travail Juridique

L'IA peut jouer un rôle de soutien essentiel pour résoudre cette tension de plusieurs manières :

• Recherche d'informations juridiques : L'IA peut analyser des milliers de pages de textes juridiques (lois, précédents judiciaires) de manière beaucoup plus rapide et fiable qu'un humain.

Cela réduit considérablement le temps consacré à la recherche de sources pertinentes pour la prise de décision.

• Révision de contrats : Pour les grandes entreprises gérant de nombreux contrats, l'IA peut automatiser la vérification de la conformité des contrats entrants avec les standards internes, en s'assurant que les clauses requises sont présentes.

• Diligence raisonnable (Due Diligence) : Lors de l'acquisition d'une entreprise, l'IA peut analyser rapidement le portefeuille de contrats pour évaluer leur valeur économique et identifier les obligations qui en découlent.

• Analyse de cas complexes : Dans des affaires longues et complexes (par ex. fraude fiscale, cas environnementaux) impliquant des milliers de documents sur plusieurs années, l'IA peut aider à construire et visualiser des chronologies et des séquences d'événements, offrant ainsi une meilleure vue d'ensemble aux humains.

Ces applications permettent d'accomplir un travail juridique de haute qualité à moindre coût.

L'Impact sur la Profession Juridique

Contrairement aux craintes courantes, l'IA ne devrait pas éliminer les emplois des juristes.

Au contraire, elle est susceptible d'améliorer leurs conditions de travail en prenant en charge les aspects les plus "fastidieux" et répétitifs du métier, qui ne requièrent pas une compétence juridique de haut niveau.

Les juristes pourront ainsi se consacrer aux tâches plus intéressantes et fondamentales, telles que la construction d'arguments, la défense des clients et la garantie de la justice.

Risques et Préoccupations Essentiels

L'utilisation de l'IA dans le domaine juridique n'est pas sans risques. Deux préoccupations majeures sont soulevées :

1. Le risque d'aliénation par manque d'explicabilité : L'IA fonctionne différemment de l'intelligence humaine.

Les décisions juridiques prises par certains algorithmes peuvent être difficiles, voire impossibles, à expliquer. Si les citoyens et même les professionnels ne peuvent pas comprendre comment une décision a été prise, cela peut entraîner une aliénation vis-à-vis des autorités de l'État.

2. Le risque de multiplication des erreurs : Une faille dans un processus juridique automatisé ne provoque pas une seule erreur isolée, mais une erreur multipliée sur potentiellement des milliers de cas.

Cela peut conduire à des violations massives des droits des citoyens si les systèmes ne fonctionnent pas correctement.

Ces risques ne sont pas une perspective lointaine ; il est jugé crucial de les prendre en compte dès maintenant, lors du développement des modèles d'IA, notamment en concevant des systèmes où les humains restent "dans la boucle" pour superviser et collaborer avec l'IA.

L'IA dans le Domaine de la Santé : Une Approche Préventive pour l'Afrique du Sud

Deshen Moodley, de l'Université du Cap, expose les défis uniques du système de santé sud-africain et le potentiel transformateur de l'IA.

Le Défi : Un Système de Santé sous Forte Pression

Le système de santé sud-africain est décrit comme "très tendu" en raison de plusieurs facteurs :

• Ressources limitées : En tant que pays en développement, les fonds alloués à la santé sont restreints.

• Fardeau élevé des maladies transmissibles : Le pays fait face à une forte prévalence du VIH et de la tuberculose, ce qui met une pression énorme sur le système.

• Pénurie de personnel qualifié : Il y a un manque critique de médecins et d'infirmières.

• Modèle de santé curatif : Le système est principalement réactif, traitant les patients une fois qu'ils sont malades, ce qui implique des traitements coûteux et une gestion de crise constante.

Le Rôle de l'IA : De la Détection à la Prévention

L'IA, bien qu'encore sous-explorée en Afrique du Sud, a un potentiel immense pour améliorer la détection et, surtout, la prévention.

• Détection et diagnostic : L'IA peut être utilisée pour analyser automatiquement des images médicales (radiographies, etc.) ou pour recommander des diagnostics et des interventions.

• Santé préventive : C'est le domaine le plus prometteur.

En utilisant des modèles d'apprentissage automatique et des techniques basées sur la connaissance, l'IA peut prédire l'apparition d'une maladie avant qu'elle ne se manifeste.

Cela permet des interventions proactives et un passage crucial vers un modèle de santé préventive, particulièrement pertinent pour les pays à faibles ressources.

Adapter l'IA aux Contextes à Faibles Ressources

Un simple transfert de technologie des pays développés n'est pas une solution viable. Il est impératif de prendre en compte le contexte local. L'approche privilégiée se concentre sur :

• Technologies à faible coût : Développer des solutions open source, avec des coûts de déploiement et de maintenance réduits et de faibles besoins en puissance de calcul.

• Interopérabilité : Un projet concret, le "Open Health Mediator", a été développé en partenariat avec une ONG africaine pour une fraction du coût des solutions équivalentes dans les pays développés.

• Dispositifs portables (Wearables) à faible coût : À l'instar des téléphones portables, le prix des wearables devrait chuter, permettant une adoption à grande échelle en Afrique pour un suivi continu de la santé des individus.

Vision d'Avenir : La Santé Préventive et les Jumeaux Numériques

La vision optimiste pour les 10 à 20 prochaines années est centrée sur la convergence de plusieurs technologies pour une santé préventive à grande échelle.

1. Suivi continu via les wearables : Une simple montre-bracelet mesurant la fréquence cardiaque ou l'ECG pourrait, grâce à l'IA, détecter l'humeur et l'état émotionnel d'une personne et prédire les états négatifs pouvant affecter sa santé.

2. Le Jumeau Numérique individuel : La collecte continue de données via ces dispositifs crée une "empreinte virtuelle" ou un jumeau numérique de l'individu, un miroir de sa personne dans le monde virtuel.

3. Le Jumeau Numérique d'une ville : En agrégeant les données des jumeaux numériques individuels, il devient possible de créer un jumeau numérique d'une ville entière.

Ce modèle permettrait de surveiller la santé et le bien-être à une échelle sans précédent, de simuler la propagation de maladies, d'apprendre des interactions entre les individus et leur environnement, et de mettre en place des interventions proactives.

Un tel système aurait été un "game-changer" lors de la pandémie de COVID-19.

Cette vision ambitieuse repose sur la convergence de l'IA, des systèmes cyber-physiques (jumeaux numériques) et de la réalité virtuelle.

L'Importance de la Collaboration Interdisciplinaire

Les deux experts soulignent la valeur de l'environnement de recherche interdisciplinaire de l'IEA de Paris.

Le fait d'être confronté à des spécialistes d'autres domaines (juristes, philosophes, technologues) a permis d'élargir leurs horizons, de générer de nouvelles approches à leurs propres problèmes de recherche et de repenser la manière de communiquer des idées complexes à un public non technique.

Cette expérience renforce l'idée que le développement futur de systèmes d'IA ayant un impact sociétal majeur doit impérativement adopter une approche interdisciplinaire pour être efficace et responsable.

Synthèse : Décomposition de la Discrimination

Résumé Exécutif

Cette étude, présentée par la Professeure Lina Restrepo-Plaza, propose une approche méthodologique innovante issue de l'économie expérimentale pour décomposer la discrimination en deux composantes distinctes :

• la discrimination fondée sur les préférences (ou les goûts) et
• la discrimination fondée sur les croyances (ou statistique).

En utilisant une version modifiée du "Jeu des Biens Publics" dans le contexte post-conflit en Colombie, l'expérience vise à isoler les motivations sous-jacentes des comportements discriminatoires.

Les résultats préliminaires révèlent des preuves claires de discrimination fondée sur les préférences.

Notamment, les participants non-victimes du conflit ont tendance à discriminer les victimes ainsi que les ex-combattants.

Un résultat majeur et contre-intuitif émerge : les victimes directes du conflit se montrent plus coopératives et moins discriminatoires envers les ex-combattants que ne le sont les non-victimes, suggérant une forme de résilience et une plus grande ouverture.

L'importance de cette décomposition réside dans ses implications pour les politiques publiques.

Une discrimination basée sur les croyances peut être corrigée par des campagnes d'information, tandis qu'une discrimination ancrée dans les préférences nécessite des interventions plus profondes, telles que la promotion du contact intergroupes pour réduire les préjugés.

L'étude ouvre ainsi des voies pour des interventions anti-discriminatoires plus ciblées et potentiellement plus efficaces.

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1. Contexte et Problématique de la Discrimination

La discrimination est un phénomène économique et social persistant et quantifiable à l'échelle mondiale. Des données récentes illustrent des disparités significatives :

• États-Unis (2022) : Les femmes gagnent 82 centimes pour chaque dollar gagné par un homme.

• États-Unis (2023) : Les Latinos gagnent 76 centimes pour chaque dollar gagné par un Américain blanc.

• Colombie : 75 % des Vénézuéliens gagnent moins que le salaire minimum, contre 43 % des Colombiens.

Du point de vue de la science économique, la discrimination est principalement conceptualisée selon deux axes :

1. Discrimination fondée sur les préférences ("Taste-based") : Un individu traite différemment une autre personne en raison d'une aversion ou d'un préjugé intrinsèque envers cette personne ou le groupe auquel elle appartient.

C'est un comportement motivé par une antipathie qui n'est pas nécessairement rationalisée.

2. Discrimination fondée sur les croyances ("Belief-based" ou statistique) : Un individu agit différemment en se basant sur des croyances ou des stéréotypes concernant les caractéristiques moyennes d'un groupe (par exemple, la productivité, la fiabilité).

Le comportement n'est pas motivé par une aversion personnelle, mais par une inférence statistique, même si celle-ci est erronée.

La principale difficulté méthodologique consiste à distinguer et mesurer l'influence respective de ces deux mécanismes, car les approches traditionnelles (comme la fourniture d'informations supplémentaires pour neutraliser les croyances) sont souvent "bruitées" et sensibles à des facteurs contextuels (voix, apparence, etc.).

2. Une Approche par l'Économie Expérimentale

Pour surmonter ces limites, la recherche utilise un protocole d'économie expérimentale basé sur le "Jeu des Biens Publics", un modèle canonique qui étudie la coopération et la confiance.

2.1 Le Jeu des Biens Publics

Le jeu se déroule entre deux participants anonymes. La mécanique est la suivante :

• Chaque joueur reçoit une dotation initiale (par exemple, 15 $).

• Chaque joueur peut décider de contribuer tout ou partie de cette somme à un "compte commun".

• L'équipe de recherche bonifie le compte commun en ajoutant 2 $ pour chaque 5 $ qui y sont déposés.

• La somme totale du compte commun (contributions + bonus) est ensuite divisée à parts égales entre les deux joueurs, quel que soit le montant de leur contribution individuelle.

Ce dispositif crée un dilemme social :

• Coopération maximale : Si les deux joueurs contribuent la totalité de leur dotation, le gain collectif est maximisé, et leur gain individuel final est supérieur à leur dotation de départ (21 $ chacun dans l'exemple).

• Incitation à la défection : Un joueur a un intérêt individuel à ne rien contribuer tout en bénéficiant de la contribution de l'autre, ce qui lui permet de conserver sa dotation initiale et de recevoir la moitié du pot commun (il termine avec 25,5 $ tandis que le coopérateur n'a que 10,5 $).

• Échec de la coopération : Si personne ne contribue, personne ne bénéficie du bonus.

La décision de contribuer est donc fortement influencée par les croyances qu'un joueur a sur le comportement de son partenaire.

2.2 Population et Contexte de l'Étude

L'expérience a été menée en Colombie auprès de 193 participants du SENA, un grand organisme public de formation professionnelle pour les populations vulnérables.

Après le processus de paix, le SENA a intégré des victimes du conflit, des non-victimes (issues de milieux économiques vulnérables similaires) et des ex-combattants.

Les participants savaient que leur partenaire anonyme pouvait appartenir à l'un de ces trois groupes :

• Victime du conflit

• Non-victime

• Ex-combattant

La présence d'ex-combattants dans le bassin de participants, bien que leur nombre soit faible (7), a rendu cette possibilité saillante et crédible pour tous.

3. Le Dispositif de Décomposition

L'étude utilise deux tâches successives pour isoler les composantes de la discrimination.

Tâche

Description

Mécanisme de Discrimination Capturé

1. Coopération Inconditionnelle

Les participants décident combien contribuer pour chaque type de partenaire possible (victime, non-victime, ex-combattant), sans connaître le montant que l'autre contribuera.

Préférences + Croyances. La décision est influencée à la fois par l'aversion potentielle pour un groupe et par les croyances sur la probabilité de coopération de ce groupe.

2. Coopération Conditionnelle

Les participants indiquent combien ils contribueraient pour chaque montant possible de contribution de l'autre (par ex. "si l'autre contribue 0, je contribue X ; s'il contribue 5, je contribue Y...").

Préférences uniquement. L'incertitude sur le comportement de l'autre est éliminée.

Si un participant contribue différemment face à une victime et une non-victime qui ont toutes deux contribué le même montant, cette différence ne peut être attribuée qu'à une préférence.

L'étude évite de demander directement aux participants leurs croyances afin de contourner les biais de désirabilité sociale et de dissonance cognitive, qui poussent les gens à rationaliser leurs réponses.

4. Résultats Préliminaires et Analyse

L'analyse des données, bien qu'encore en cours pour la partie "croyances", fournit déjà des conclusions claires sur la discrimination fondée sur les préférences.

4.1 Mise en Évidence de la Discrimination

• Discrimination envers les ex-combattants : Les victimes et les non-victimes discriminent toutes deux les ex-combattants.

Cependant, les non-victimes discriminent beaucoup plus fortement que les victimes.

• Relations entre victimes et non-victimes :

◦ Les non-victimes discriminent les victimes.   

◦ De manière surprenante, les victimes manifestent une discrimination positive envers les non-victimes, se comportant mieux avec elles qu'avec les membres de leur propre groupe.

4.2 Le Résultat Contre-intuitif : La Résilience des Victimes

Le résultat le plus marquant est que les personnes ayant été directement exposées au conflit (les victimes) se montrent plus coopératives et moins enclines à discriminer les ex-combattants que la population non directement affectée.

Ce constat suggère que l'exposition à des situations difficiles peut favoriser des comportements de résilience et une plus grande ouverture à la coopération.

Ce résultat est qualifié de "très surprenant" et "porteur d'espoir".

4.3 Données sur les Ex-Combattants

Avec seulement sept ex-combattants dans l'échantillon, les données sur leur propre comportement sont anecdotiques.

Cependant, l'observation initiale est qu'ils ne discriminent aucun groupe et se comportent de la même manière avec les autres qu'entre eux.

5. Implications et Perspectives

5.1 Implications pour les Politiques Publiques

La capacité à décomposer la discrimination est cruciale pour concevoir des interventions efficaces :

• Si la discrimination est principalement fondée sur les croyances, des campagnes d'information peuvent suffire à corriger les perceptions erronées et à mettre à jour les croyances des individus sur les autres groupes.

• Si elle est principalement fondée sur les préférences, des interventions plus profondes sont nécessaires.

Des stratégies basées sur le contact intergroupes, comme celles pratiquées au SENA où les différents groupes étudient ensemble, se sont avérées efficaces pour réduire les préjugés et les stéréotypes.

5.2 Pistes de Recherche Futures

La discussion a soulevé plusieurs axes pour de futures recherches :

• Adaptation à d'autres tâches : Appliquer cette méthode à d'autres jeux économiques (jeu de la confiance, jeu de l'ultimatum) pour tester la robustesse des résultats.

• Intégration de données qualitatives : Compléter l'approche quantitative en interrogeant les participants sur leurs représentations, même biaisées, pour comprendre les arguments qu'ils jugent "acceptables".

• Étude en jeux répétés : Analyser comment la discrimination évolue sur plusieurs tours d'interaction.

Une expérience positive répétée avec un membre d'un autre groupe est-elle suffisante pour modifier un préjugé, et si oui, à quelle vitesse ?

Cela permettrait de mesurer la "résilience du préjugé".

Document d'Information : Repenser la Collaboration avec l'Ennemi

Résumé Exécutif

Ce document synthétise les réflexions d'Adam Kahane, directeur de Reos Partners, sur la nature et les mécanismes de la collaboration dans des contextes de profonds désaccords.

L'analyse est issue de son travail de réécriture de son livre de 2017, Collaborating with the Enemy.

L'idée centrale de Kahane est que la collaboration est définie par une tension fondamentale : la nécessité de travailler avec des personnes avec qui l'on est en désaccord pour résoudre des problèmes complexes, et la crainte que ce faisant, on trahisse ses propres valeurs fondamentales.

Pour explorer cette dynamique, il propose un modèle de "cercles concentriques" qui classe les relations de la collaboration la plus proche à l'élimination de l'ennemi.

L'objectif principal est de trouver des moyens d'élargir le cercle de la collaboration.

Alors que la première édition de son livre se concentrait sur les approches individuelles, sa recherche actuelle vise à identifier et à comprendre les approches collectives qui favorisent une collaboration plus large et plus efficace.

Celles-ci incluent des cadres constitutionnels et juridiques, des systèmes politiques et réglementaires, des normes culturelles et des processus de réconciliation.

La discussion qui suit son exposé met en lumière des concepts clés tels que l'importance de trouver des objectifs communs, même minimes ; le rôle de la planification par scénarios non pas pour prédire mais pour façonner l'avenir ; et la prise de conscience que la collaboration peut également servir à créer des conflits en unissant un groupe contre un autre.

1. Contexte et Problématique Centrale

Adam Kahane est un praticien spécialisé dans la conception et la facilitation de dialogues multipartites sur des questions complexes depuis 1991.

Son travail l'a amené à intervenir dans divers contextes, notamment :

• Le processus de paix en Colombie, impliquant toutes les parties, y compris les factions armées.

• Les chaînes d'approvisionnement alimentaire durable, réunissant des communautés, des entreprises et des régulateurs.

• Les relations entre les États-Unis et la Chine, avec des acteurs de la sécurité et de la défense.

• Le travail avec des peuples autochtones et insulaires du détroit de Torrès en Australie.

Sa réflexion actuelle s'inscrit dans le cadre de la réécriture de son livre _Collaborating with the Enemy:

How to work with people you don't agree with or like or trust_.

La question fondamentale qui guide son travail peut être résumée par une formulation plus grandiose : "Comment diable pouvons-nous vivre ensemble ?"

Les Quatre Approches face à une Situation Problématique

Selon Kahane, face à une situation que nous jugeons problématique, quatre stratégies principales s'offrent à nous :

1. Forcer (Make) : Tenter d'imposer notre volonté, indépendamment de ce que les autres veulent.

2. S'adapter (Adapt) : Accepter la situation telle qu'elle est, car nous ne pouvons pas la changer.

3. Sortir (Exit) : Quitter la situation (émigrer, démissionner, divorcer).

4. Collaborer (Collaborate) : Travailler avec d'autres acteurs pour changer la situation.

Son travail se concentre sur cette quatrième option.

La Double Signification de la "Collaboration"

Kahane souligne une dualité sémantique cruciale dans le mot "collaboration", qui est au cœur des défis qu'il explore.

• Sens positif : Travailler ensemble avec d'autres. Les recherches Google pour "collaboration" montrent des images de coopération harmonieuse.

• Sens négatif : Collaborer de manière traîtresse avec l'ennemi. Il illustre ce point avec une photo de 1944 montrant deux collaboratrices françaises punies par la tonture de leurs cheveux.

Cette double signification révèle la tension inhérente à toute entreprise de collaboration :

"D'une part, nous pensons que nous pourrions avoir besoin de travailler avec ces autres personnes pour arriver là où nous essayons d'aller, et d'autre part, nous craignons que le faire nous obligerait à trahir ce que nous tenons pour précieux."

2. Un Modèle de Relations : Les Cercles Concentriques

Pour mieux comprendre les frontières de la collaboration, Kahane propose un modèle de cercles concentriques illustrant différents niveaux de volonté d'interaction avec autrui :

1. Collaboration : Le cercle intérieur, composé des personnes avec qui nous sommes prêts à travailler activement.

2. Cohabitation : Les personnes avec qui nous ne voulons pas collaborer, mais avec qui nous sommes prêts à partager un espace (un foyer, une ville, un pays).

3. Coexistence : Les personnes avec qui nous ne sommes pas prêts à cohabiter, mais dont nous tolérons l'existence à condition qu'elles restent séparées.

C'est le principe de l'apartheid ("apartness").

4. Élimination : Le cercle extérieur, composé de nos ennemis, des personnes que nous ne sommes même pas prêts à laisser coexister et que nous devons expulser ou éliminer.

L'objectif de sa recherche est de comprendre comment "déplacer la frontière entre les personnes avec qui nous sommes prêts à collaborer et celles que nous considérons comme nos ennemis".

3. Forces Motrices et Forces Restrictives

La décision de collaborer ou non est influencée par des forces contradictoires.

Forces Poussant à la Collaboration

Forces Freinant la Collaboration

Nécessité d'une action collective : Des défis qui exigent une réponse commune (ex: gestion des eaux usées dans la ville divisée de Nicosie, changement climatique).

Différences réelles : Désaccords, méfiance et conflits d'intérêts concrets et non imaginaires.

Peur du conflit violent : La crainte qu'une absence de collaboration ne mène à la guerre.

Fragmentation et polarisation : Tendance au tribalisme, à la partisanerie, aux bulles d'information, à la démagogie et à la diabolisation.

Sentiment d'interconnexion ("All My Relations") : Une conviction, notamment issue des traditions des Premières Nations, que nous sommes tous liés, que nous nous entendions bien ou non.

Identification exclusive à son groupe ("mon peuple") : Une vision qui empêche de s'ouvrir à la collaboration avec des "autres".

La diabolisation est un frein particulièrement puissant : "ces autres ne sont pas simplement nos adversaires ou nos ennemis, ce sont des démons, des diables. Et comment pourrions-nous collaborer avec le diable ? Nous ne le pouvons pas."

4. L'Enquête Actuelle : Des Approches Individuelles aux Approches Collectives

La question centrale qui anime la réécriture du livre de Kahane est de nature pratique : "Quelles approches permettent une collaboration plus nombreuse et de meilleure qualité ?".

Il s'agit d'identifier des méthodes pour élargir le cercle des acteurs avec lesquels nous sommes disposés et capables de travailler.

Le Passage de l'Individuel au Collectif

La première édition de son livre se concentrait sur les approches individuelles, destinées à aider les individus à mieux collaborer. Ces approches étaient :

• Accepter le conflit autant que la connexion.

• Expérimenter pour avancer.

• Reconnaître son propre rôle dans le système.

Pour la seconde édition, Kahane souhaite compléter cette perspective en explorant les approches collectives.

Il considère la relation entre le travail individuel et collectif comme une "bande de Möbius", où l'un ne va pas sans l'autre.

Exemples d'Approches Collectives à Explorer

Kahane a dressé une liste préliminaire d'approches collectives, qu'elles soient anciennes ou de pointe, qui permettent de collaborer au-delà des différences :

• Constitutions et accords : Cadres établis pour gérer les différences sans recourir à la violence.

• Organisation politique : Façons de s'organiser pour collaborer avec certains contre d'autres, ou contre un problème commun.

• Systèmes réglementaires : Mécanismes pour gérer les différences.

• Organisation des villes : Comment l'urbanisme peut faciliter la cohabitation et le travail en grande diversité.

• Politiques et "Nudges" : Interventions (comme celles d'Antanas Mockus à Bogota) conçues pour modifier les relations entre les gens, les faisant passer de la violence à la paix.

• Culture, valeurs et normes : Leur influence sur la capacité à collaborer.

• Réconciliation et guérison : Le rôle de la prise en charge des traumatismes collectifs et du rétablissement de la paix.

5. Perspectives Issues de la Discussion

Plusieurs intervenants ont enrichi la réflexion de Kahane avec des concepts et des exemples pertinents :

• Trouver un objectif commun, même minime : Même avec le pire ennemi, il est souvent possible de trouver un motif commun.

Commencer par ce petit objectif peut créer une expérience de collaboration positive qui change la dynamique de la relation.

• La finalité de la collaboration : Consensus ou Agonisme ? : La collaboration vise-t-elle à atteindre un consensus ou à gérer une tension permanente ("agonisme") ? Kahane adopte une posture pragmatique : l'objectif est de résoudre le problème en question.