Reviewer #1 (Public Review):

This is a study bearing on the longer term more trophic aspects of genetic modifications in RyR function. It welcomely provides physiological measures of the physiological outcomes of the experimental manipulations. It bears on the effects of such modifications on the important HDAC signaling pathway which modifies the extent of muscle hypertrophy and the extent to which pharmacological interventions in this pathway may rescue the resulting hypotrophic phenotype. The latter were assessed through grip strength and running distance, likely to evaluate overall muscle and isometric and dynamic function. These studies were then substantiated by measures of isometric twitch and tetanic tensions, evoked Ca2+ transients, and molecular and biochemical expression indicators. The methodology is clearly described. This is a broad and interesting paper.

Reviewer #2 (Public Review):

Ruiz et al have used their previously generated mouse model of congenital myopathy caused by recessive RYR1 mutations to investigate the therapeutic effects of inhibitors of DNA methylases and histone deacetylases. They target enzymes that they have previously shown to be upregulated (at the mRNA and protein level) in this myopathy model of multi-mini core disease. Remarkably there is improvement in the muscle function as tested by grip strength and wheel running activity. Further analysis revealed that the mechanical properties of muscles was improved but this improvement was restricted to slow oxidative soleus muscles and accompanied by an increase in RyR 1 protein expression. Calcium imaging experiments performed in a distinct set of muscles suggests that the treatment restores electrically evoked calcium transients. The findings are important and of interest to the scientific community.

Reviewer #3 (Public Review):

Mutations in ryanodine receptor are linked to many human diseases including muscle hypertrophy and weakness. Some of the mutations lead to reduction of the expression of the ryanodine receptor, which plays a pivotal role in control of calcium signaling during muscle contraction. Ruiz et al tested the hypothesis that epigenetic mutations in ryanodine receptor associated with muscle disease could be remedied through the use of pharmacological reagents that modulate DNA methylation and acetylation. Using a mouse model with knock-in of human mutations in RYR1, the authors demonstrated that combination of therapeutic drugs that inhibit DNA methylases and class II histone de-acetylases improve contractile function of the mutant mice. They further showed that the improved muscle function is associated with restoration of ryanodine receptor and other calcium signaling machinery, due to changes in methylation of the target DNAs. This study provides proof of concept for the pharmacological treatment of patients with congenital myopathies linked to recessive RYR1 mutations.

Reviewer #1 (Public Review):

The experiments and analyses presented here seem carefully designed executed for the most part. The manuscript is very well written and conveys a clear and interesting message. Dependence of the binding strength on mechanical force is an intuitive and elegant form of regulation for which the work presented here provides examples. The conclusions should be of broad interest. Some control experiments ruling out artifacts of the attachment strategy would strengthen the conclusions. Likewise, some of the analyses should be better described, and better measurement statistics would improve the precision of the extracted parameters that describe the MIDAS-UBL interaction. The model for how tension is exploited for regulating Mdn1 function is intriguing, but could be better described.

Reviewer #2 (Public Review):

The manuscript entitled "The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates" by Mickolajczyk reports on optical trap experiments of the Mdn1 MIDAS domain and the UBL domains of two of its known substrates, Rsa4 and Ytm1. The manuscript is clearly written and easy to follow. Using gel filtration shift and microscale thermophoresis studies, the authors demonstrate weak binding affinities between the purified MIDAS domain and the UBL domains of Rsa4 and Ytm1, respectively. This is followed by optical trap experiments that show an increase in bond life time when stronger forces are applied indicative of increased binding affinity. Further increasing the applied force leads to a decrease in bond life time, which reveals the catch-slip bond concept. The authors develop a "mechanical circuit model" in order to more precisely quantify the forces involved in the MIDAS-UBL catch-slip bond. All experiments are well designed, include the appropriate controls and the conclusions drawn are justified.

Early work by the Hurt group introduced the MIDAS-UBL catch bond concept based on the sequence homology with integrins (Ulbrich et al., 2009). However, until now no direct experimental proof of this concept has been published. The authors of the current study now demonstrate the catch-slip bond concept using a sophisticated optical trap arrangement. The MIDAS-UBL interaction is a key feature of Mdn1 mediated substrate removal from pre-ribosomal particles and therefore this study provides important insights into the Mdn1 mechanism. For the first time, the forces involved in the MIDAS-UBL interactions are quantified and the force dependency of the bond lifetime is revealed. This study is likely to make an important impact in the field.

Reviewer #3 (Public Review):

The manuscript by Mickolajczyk et al. reports on the development of a new optical tweezers-based unbinding-force assay to characterize the bond between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that while the affinity between the MIDAS domain and Rsa1 and Ytm1 is only weak (≥7 μM) in solution, it increases significantly under an applied load. Using the developed assay, the authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The authors suggest that the catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results are highly interesting for the ribosomal and single-molecule biophysics communities and the developed DNA-tether-based optical tweezers assay will be useful for characterizing other molecular bonds.

Reviewer #1 (Public Review):

Overall, the selectivity of Pn3a for CaV3.3 over CaV3.1 and CaV3.2 is convincingly demonstrated by the data presented. However, the lack of any experiments in native cells expressing a mixture of CaV3 channel isoforms is a significant omission in the study. Moreover, previous findings cited in the manuscript that Pn3a also inhibits Nav1.7 and high-voltage-activated (HVA) calcium channels further casts doubts as to how useful it would be in specifically dissecting effects of CaV3.3 in native tissues. Other weaknesses in the manuscript pertain to the lack of modeling to understand the mechanism of action of Pn3a on CaV3.3, and absence of experimental evidence to validate the computational docking model.

Reviewer #2 (Public Review):

The low voltage activated T-type calcium channels (CaV3.1-3.3) play a wide range of physiological roles, yet discerning their functions is challenging as options for selective pharmacology is limited. Although there are toxins such as ProTx-I and ProTx-II that target CaV3.1 and CaV3.2 with some specificity, there are no such tool available for CaV3.3. This study identifies the first subtype modulator of CaV3.3, which is interesting and will undoubtedly open new experimental avenues. The strength of the study is the extensive electrophysiological characterization of Pn3a modulation CaV3.1-3 channels, which is compelling. The weakness of the study is that the mechanisms that allow selective block of CaV3.3 is not fully clear.

Reviewer #3 (Public Review):

The authors show that the sodium channel blocking spider toxin Pn3a inhibits Cav3.3 channels selectively over Cav3.2 and Cav3.2. The inhibitory effect has the hallmarks of a classical gating modifier like agaIVA or grammotoxin, and by using chimeras with a K channel and molecular docking analysis, they identify the voltage sensing domain of DII as the target. Nice ephys overall, but some points should be addressed. My main concern relates to the use of a chimeric K channel for this work - I am not sure that I agree with the author's statement that a calcium channel chimera would not have worked. To what extent this work will lead to new tools for making a Cav3.3 channel selective inhibitor remains to be seen.

Reviewer #1 (Public Review):

White and colleagues have generated data that addresses the hypothesis that genes in linkage disequilibrium with a mutant allele in zebrafish could show allele-specific expression effects caused by high polymorphism rates in zebrafish (eQTLs). The experiments use a variety of mutant alleles with sophisticated analysis of RNA-seq data that is both allele-aware and chromosomal location-aware. They show a significant over-representation of gene expression changes from genes that are located close to the mutant allele on the same chromosome. They also performed global allele-specific expression analysis on the SAT line (a hybrid of Tü and AB strains) and showed significant correlations with gene level alterations when regions were one haplotype or the other.

The data are extensive, carefully analyzed and of sufficient depth and quality to support their claims convincingly. I do not have any significant critiques of the data or the conclusions drawn from them.

Reviewer #2 (Public Review):

In an extensive analysis of zebrafish wild-type vs. mutant RNA seq datasets, the authors find that differentially expressed genes are often enriched in the chromosomal region of the mutated gene.

An older paper by Miller et al. (2013, Genome Research 23: 679) also analyzed RNA seq data on wild-type and mutant zebrafish (in their case with the main goal of identifying the mutated gene), and they noted only a small number of differentially expressed genes near the mutated locus. White et al. could mention this paper and consider methodological differences that may explain these seemingly different conclusions.

By genotyping and performing RNA seq on individual animals from a large cross, the authors obtain convincing evidence that genomic polymorphisms cause many genes to be differentially expressed in different wild-type zebrafish strains. They show that most of the differentially expressed genes near mutant loci are likely to be caused allele-specific expression differences in linkage disequilibrium with the mutation rather than by any action of the mutated gene. The authors illustrate how one can determine which nearby genes may actually be regulated by the mutated gene, using polymorphisms among wild-type chromosomes and different mutant alleles of the muted gene. Another possible, complementary approach might be to rescue the mutants with a wild-type transgene, which should rescue the mutant phenotype and genes that are regulated by the mutated gene but not affect differentially expressed genes caused simply by allelic differences. In some cases, one could also use transgenic over expression of the gene of interest to compare loss of function and gain of function of the gene to assess possible inverse effects on target genes (e.g. a putative target gene may be reduced in the mutant and increased in transgenic over expression animals). As the authors note, these approaches would represent significant extra effort, and they reasonably suggest that a simpler alternative is for investigators to consider the chromosomal position differentially expressed genes when interpreting their RNA seq data from outbred strains.

The authors are likely correct that many investigators analyzing zebrafish RNA seq data may have overlooked this clustering of differentially expressed genes and its causes, but the paper does not contain examples where this has significantly affected pathway analysis or other follow-up experiments. Are there enough differentially expressed genes misattributed to an effect of the mutated gene instead allele-specific expression to lead to major errors in interpretation? For example, the authors show that lama1 mutants have 12 differentially expressed genes, of which 3 are near lama1. Are there major differences in the conclusions drawn from the sets of 9 and 12 differentially expressed genes? Evidence of this type might increase the impact of the paper. As it stands, the demonstration of allele-specific expression is convincing but not surprising in light of the outbred structure of zebrafish strains and much prior work in other species.

Reviewer #3 (Public Review):

The authors used transcriptome analyses by RNA-seq to identify differentially expressed (DE) genes in a series of previously identified forward genetic mutants emerging from outbred crosses as well as in clusters of wild type zebrafish embryos emerging from newly-generated cross of well-defined genotypes. The authors present experiments, which convincingly demonstrate physical linkage of DE genes to the mutated locus and their predominant localisation on the mutation-carrying chromosome. Next the authors demonstrate, that allelic variation of expression is common in a wild type hybrid cross of SAT double haploid strains and demonstrate haplotype-dependent allelic gene expression variation. Finally, White et al. offer an example approach for distinguishing gene expression change caused by a mutation from that caused by allelic variation of expression of genes in linkage disequilibrium with the mutation by analysing segregation of alleles and their expression dynamics.

The data from a series of mutants and well-defined wild type crosses convincingly demonstrates the impact of strain polymorphism and linkage disequilibrium on differential gene expression. The provided evidence suggests the generality of differential gene expression readouts arising independently from generated mutations in outcross experiments in zebrafish.<br> These observations are potentially important for the design of transcriptomic analyses of forward genetic screens and other experiments involving RNA-seq from outcrosses such as inter and transgenerational epigenetic inheritance studies.<br> Evidence on the actual impact of misinterpretation of gene expression differences on biological conclusions drawn from mutants generated in outbred crosses would strengthen the study.

The conclusions of this manuscript are well supported by the experimental data, some aspects would benefit from further clarification.

1.) Figure 4 demonstrates separation of differential expression due to sox10 mutations from those arising from allele-specific variation in LD with sox10 by providing an individual example for both. In this section a global demonstration of the distinct segregation-associated expression dynamics would strengthen the claim. It is recommended that the expression variation for the full set of genes quoted in the text (10 and 15 genes respectively) are shown.<br> 2.) Demonstration of the importance of the problem of appropriately drawing conclusions from RNA-seq data may be achieved by comparing the features of mutation-dependent and mutation-independent differentially expressed genes in relation to the biological or biochemical functions of the mutated gene.

Reviewer #1 (Public Review): 

Here, Tai-De Li et al. exploit an AFM system established by the same team in 2016 to measure precisely rates of actin filament elongation, nucleation and CP binding when branched actin networks grow under force. They observe that all rates decrease when growth stress increases, albeit to different extent. Rate of capping decreases faster, leading to a densification of actin networks as stress increases. A nice observation is that capping and elongation follow the same trend, suggesting a similar mechanism. A logical explanation is that insertion of both G-actin and CP are limited by the same Brownian ratchet mechanism, an idea which seems to be coherent with the fact that a larger CP has even more difficulty to insert between actin filament barbed ends and the surface. 

This is also a beautiful and very informative experimental setup. Data are convincing although their presentation should be improved for clarity. Few controls are missing (mainly whether bulky CP diffuses at the same rate than CP within branched networks and checking if the protein pool is the same at the time the AFM measurements are performed in experiments at different CP concentrations ), but the authors should be able to control these points by running some additional experiments.

Reviewer #2 (Public Review): 

This study proposes to explain why branched actin filament networks become denser when they grow against a mechanical load. This important property provides these networks with mechanical adaptability (nicely demonstrated by the authors in Bieling et al. Cell 2016), and has yet to be understood from a molecular perspective. 

Here, the authors use a suite of very powerful in vitro experiments, which they have developed over the past years, to study the impact of force on the main molecular reactions involved in the growth of branched network. They convincingly show that: (1) the incorporation of actin monomers and capping protein into the network is limited, when a mechanical load is applied, by a Brownian ratchet mechanism; while (2) the incorporation of Arp2/3, responsible for branching, is limited by the unavailability of its surface-anchored activator, as it interacts with the free barbed ends of filaments from the network. This 'barbed end interference' mechanism was recently identified by the authors (Bieling et al. EMBO J 2018, Funk et al. Nat Comm 2021) and they show here that it provides a negative feedback on branch nucleation, to control network density when a mechanical load is applied. 

These are new and important results. A clear demonstration of the Brownian ratchet mechanism for actin polymerization has been missing for decades, observing it on single filaments seems out of reach, and the present study provides the best evidence to date. The authors also show the relevance of the Brownian ratchet for capping, which had been mostly overlooked until now. 

However, I disagree with the the way the authors compare the impact of load on the capping and branching rates, and with their claim that it explains why networks become denser when a load is applied. 

The authors write that, upon the application of force, the drop in the rate of filament capping is larger than the drop in the rate of nucleation (Arp2/3-mediated branching) but this is because they are comparing the capping rate per filament to the nucleation rate per surface area. This presentation is misleading: the difference between the two rates simply reflects the change in filament density, by definition, and does not explain it. If the same definition of the rate is used (i.e. per filament, or per area) for both capping and branching, the authors find that they decrease the same way with increasing loads - as they should: this is imposed by their being at steady state, where the birth rate of growing barbed ends (branching) must match their death rate (capping). 

The new and interesting result is that the two identical drops in branching and capping rates have different molecular origins, but the authors do not explain how this leads to an increase in density. However, I think that this explanation is within reach, thanks to the experimental results provided by this study: the Brownian ratchet and the barbed end interference mechanisms depend on filament density in very different ways.

Reviewer #3 (Public Review): 

In this paper, the authors' goal was to identify the molecular mechanisms behind the results the obtained in a previous paper (Bieling, Li et al, 2016). The authors reused similar experimental setup and some previously acquired data and performed a deeper analysis of the data to determine the effect of load on different parameters related to actin subunits, filaments barbed ends, capping protein and the Arp2/3 complex. 

The experimental novelties in this paper are the clever use of an arrest and quenching strategy to determine the relative amount of actin monomers and barbed ends bound to the WH2 domain of the Arp2/3 complex activator. 

The quantitative analyses of this paper are quite interesting and nicely demonstrate the authors' points. The clever use of a larger capping protein also clearly demonstrates the effect of size on the on-rate constants under load. 

The only major point that remained unclear to us is whether the conclusions are universal for all Arp2/3 complex activators. What would happen to the results if another activator was used, if activators were prevented from binding to the barbed ends or monomers, or able to bind several actin monomers or barbed ends at the same time? The authors could also discuss more the effect of other proteins usually present in branched actin networks (e.g. crosslinkers, severing proteins), ideally with new experiments, or at least as speculations in the discussion. 

Overall the authors do a good job at convincing the reader.

Public Review: 

This manuscript from Pacheco-Moreno et al. compares the microbiome of potato fields with and without irrigation. Irrigation is known to control potato scab caused by Streptomyces scabies and the authors hypothesized that changes in the microbiome may contribute to disease suppression after irrigation. Using 16S rRNA sequencing, they identified a number of taxa, including Pseudomonas that are enriched after irrigation. They went on to isolate and sequence the genomes of many Pseudomonas strains. By correlating the ability of Pseudomonas to suppress Streptomyces growth in vitro with genomic data, the authors identified a novel group of cyclic lipopeptides (CLPs) that can inhibit Streptomyces in vitro and in planta. 

This work provides a substantial contribution that advances our understanding of disease suppressive soil mechanisms. It is novel in scope in that it focuses on suppression of a bacterial pathogen, while many prior studies focus on suppression of fungal pathogens. Additionally, the large-scaled comparative genomics is a useful resource, and the identification of CLPs that inhibit Streptomyces is novel. Importantly, the authors provide in planta data to show role a for CLPs in disease suppression in vivo. The manuscript is well written and the data are well presented. The analyses are quite thorough and I appreciate the extensive use of genetics and metabolomics to support the genomic predictions. The main weakness is a lack of data the conclusively links the change in microbiome function to disease suppression after irrigation in the field. However, I think the data they've presented, combined with those in the drought literature, might suggest that an increase in total Pseudomonas (and the corresponding disease-suppressive genes) in well-watered soil might contribute to suppression, rather than a change in function of Pseudomonas.

Reviewer #2 (Public Review):

Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to gene expression regulation. Our understanding of archaeal transcription termination mechanisms is still limited. Li et al present new evidences that support their model of aCPSF1-dependent transcription termination in Archaea (Yue et al NAR 2020). Importantly, they show that aCSPF1 recognize U-rich terminator signals with its KH-domain, using the methanogen Methanococcus maripaludis, as study model.

The reported results of the manuscript are the continuity of their work published in Nucleic Acid Research journal in 2020 (Yue et al NAR 2020). Previously, the authors demonstrated the importance of the absolutely conserved ribonuclease aCPSF1 to terminate transcription by cleaving the transcripts at their 3' ends. They proposed a model for transcription termination in Archaea in which they defined aCPSF1 as general transcription termination factor. In here, by reinvestigating Term-seq data and by using RNA-protein binding assays (EMSA & SPR) and genetics experiments, Li et al argue that (i) PolyU-tracts are signals for transcription termination in M. maripaludis, (ii) aCPSF1 binds to PolyU-tract signals in transcript 3'-ends through its KH domain, and (iii) transcription termination is more effective in presence of aCPSF1.

In general, the experiments and analyses that are shown are well-conducted. A major criticism is the lack (i) of quantification of RNA-protein binging assays which will allow going deeper in the understanding of how aCPSF1 specifically recognized PolyU-tract signals and (ii) of data on the oligomerisation status of aCPSF1. It is important to decipher if aCPSF1 is acting as a monomer or a dimer. Both will be helpful for proposing a more precise model for transcription termination mechanism in Archaea.

Reviewer #3 (Public Review):

In this manuscript by Li et al. examine U-rich motifs enriched for the transcript end sites in archaea M. marpaludis and analyze the role of aCPSF1 and its KH domains in binding these U-rich motifs. Their data indicate aCPSF1 binding to U-rich motifs is necessary for efficient 3' end definition of transcripts. Overall this work is well carried out, but there are also several key issues that should be addressed in order to more fully support the conclusions of the authors.

Major:

1. The conclusion that aCPSF1 functions as a back-up termination is not supported by their data. As far as I can tell, back-up termination should happen at non-primary sites, which have a lower frequency of U quadruplex. It is not clear how aCPSF1 functions at those sites.

2. The authors appear to indicate that aCPSF1 is the sole factor for 3' end cleavage of archaeal transcripts. But this is not supported by the data. Their data indicate both U-rich motif and aCPSF1 are necessary for cleavage. No data are shown to indicate that these two alone are sufficient for 3' end cleavage.

3. The U-rich motif (U quadruplex) was recently reported in their NAR paper (Yue et al.). There appears to be limited additional information on motifs in this work. It would be useful to readers to know if U-rich motifs are the only type for 3' end cleavage. The authors may want to examine motifs beyond single nucleotide models (which is what they are doing in this work). For example, are there any k-mer enrichments besides U quadruplex?

Minor:

1. Strictly speaking, they are measuring transcript ending sites, not polymerase termination sites. The Term-seq data do not map the polymerase termination site. The authors should make this distinction in their writing. Likewise, it is better to rename transcription termination efficiency (TTE) to transcript termination frequency, because they are measuring steady state RNAs which embody both 3' end cleavage efficiency and RNA stability. The efficiency implies termination kinetics, which is not what they are measuring.

2. Figure 3 needs better quantification. Binding curves showing binding constant are needed to make quantitative conclusions.

3. Figure 7. the eukaryotic model seems based on budding yeast. This should be noted. 3' end motifs in other eukaryotes are quite different than those in the budding yeast.

Reviewer #1 (Public Review):

The authors conducted an extensive analysis of transcription termination in Methanococcus maripaludis, which is an archael species. Using a combination of functional genomics, statistical analyses, in vitro/biochemical approaches, and reporters; the authors explore mechanistic aspects of termination. The authors determine that the archael CPSF protein binds an upstream uridine tract using a KH domain. The RNA binding activity of aCPSF is not present in eukaryotes and is shown to be important for termination in a uridine tract dependent manner.

Overall, the work is well-conceived and, in general, the conclusions by the authors are supported by the data. Investigation of archael species is not mainstream but still has significant potential to impact the field of transcription as the process of termination is still be unraveled. Moreover, several aspects of the methodology would benefit other researchers - notably, the development of Term-seq. The readers would benefit from consulting early structural studies of aCPSF to more fully gain a perspective on the interesting aspects of KH domain presence in this homology of CPSF.

Reviewer #1 (Public Review):

The foundational differentiation protocol up until day3 (formation of PSM) has been published previously in Diaz-Cuadros et al., 2020; Matsuda et al., 2020. The main difference between this manuscript and published protocol being the 2D (published) vs 3D differentiation. In this manuscript the authors were able to generate Pax3+ Somites (day4-5) from PSM. Both Diaz-Cuadros et al., 2020; Matsuda et al., 2020 were unable to generate Pax3+ somite in their 2D culture system but instead could only obtain a TCF15+ somatic mesoderm intermediate state. Moreover, the somites obtained in this manuscript could be further differentiated to sclerotome.

The experiments across the paper were validated using three repeats using appropriate quantitative microscopy. Imaging data are high quality and mostly presented in a clear manner. However, it is unclear exactly what the authors are scoring as a somite. Moreover, for each figure it is not clear whether technical or biological replicates are presented. Similarly, the heatmap block presented in Fig2C,D, 3C,D apparently represents just one organoid/replicate. Authors should comment on the efficiency of the protocol over the different cell lines used. Transcriptome data presented strongly support the reproducibility and accuracy of the 3D differentiation, although comparison with the in vivo situation is highly limited - in part due to lack of availability of human in vivo data at these developmental stages.

Reviewer #2 (Public Review):

This study is interesting in the sense that it brings us one step closer to the formation of complex structures (such as somites) from human iPSCs. Markers that are typical of somites are indeed present at the end of the (rather complex) culture protocol. There is also quite a lot of work involved and the illustrations are of good quality.

However, the spatial organization that is typical of somites is lacking in a number of important ways. Early somites in amniotes are epithelial (in fact it is a pseudo-stratified epithelium made of bottle shaped cells), apical facing the somitocoele (a cavity filled with a loose mesenchyme) and basal to the outside. Quite similar to the organization of the neural tube. This organization is initiated in the anterior last third of the PSM and amplified concomitant to segmentation. It would be important to show that somitoids display such structure. It does not seem that there is a central cavity. It is also unclear from the picture whether somitoid cells are bottle-shaped. Importantly, one sees (Figure 5a) that F-actin (which labels the apical side of epithelial cells) is facing the outside of the somitoid, and not the inside as it should. In this condition, the term "somitoid" seems quite inaccurate in comparison to other organoid systems that faithfully reproduce their in vivo counterparts, not only the "classical" intestinal crypts but also the more recently published neural tube organoids. Aggregates of somite-like cells may be more accurate.

Reviewer #1 (Public Review): 

This work significantly advances our understanding of the role of Strongyloides stercoralis nuclear receptor Ss-DAF-12 in determining parasite life cycle and infection outcomes. Strongyloidiasis is a facultative soil-transmitted nematode that infects hundreds of millions of humans. While infections are typically subclinical, 'hyperinfections' associated with host immunosuppression bring about severe morbidity and mortality that are not well-controlled by existing anthelmintics. DAF-12 signaling in free-living nematodes is known to promote growth and development, lifespan extension, and avoidance of the dauer dormancy state. Some of these findings have been shown to be conserved in parasitic nematodes, where infective stages are argued to be akin to dauer, motivating the study of the orthologous DAF-12 pathway as an antiparasitic substrate. 

Through a lipid fractionation strategy, the authors first show that Δ7-DA is a potent endogenous ligand for Ss-DAF-12 and that Δ7-DA abundance throughout the life cycle is correlated with states of parasite reproductive development. The authors show that S. stercoralis possess the machinery to synthesize Δ7-DA from dietary cholesterol and use an insect cell expression system to identify biosynthetic enzymes in this pathway: Ss-DAF-35, Ss-DHS-16, and the cytochrome P450 Ss-CYP-22a9. CRISPR/Cas9-mediated disruption of Ss-CYP-22a9 is shown to near-completely inhibit cGMP-mediated Δ7-DA production and activation of infective larvae as measured by feeding, which can be rescued by exogenous Δ7-DA. Finally, a gerbil infection model is used to show that Δ7-DA treatment drastically reduces fecal output of larvae in uncomplicated strongyloidiasis and that a combined Δ7-DA/ivermectin regimen can decrease the burden of autoinfective intestinal larvae and prevent death in hyperinfection induced by immune compromise. 

Overall, the conclusions are well-supported and this work provides important new insights on a pathway that is of broad relevance to the regulation of the complex and diverse life cycles of parasitic nematodes. The discovery that the combinatorial action of a DAF-12 agonist and ivermectin can synergistically control hyperinfective strongyloidiases is a major and impactful finding. This work will be of great interest to the larger parasitology and nematode biology community. My enthusiasm is only slightly tempered by the acknowledged caveats that currently limit the therapeutic outlook of this approach. The eventual development of therapeutics targeting this pathway could aid the treatment of uncontrolled strongyloides infections and be of potential value for the treatment and control of other parasitic worm infections. 

Strengths: 

- Experiments are generally well-designed and rigorous, clearly establishing that Δ7-DA is a primary ligand for Ss-DAF-12 and resolving the primary biosynthetic pathway for the production of Δ7-DA in S. stercoralis. While Δ7-DA is the known ligand of C. elegans DAF-12, significant difference in primary sequence justified caution and experimental validation. Similarly, while two of the Δ7-DA biosynthetic pathway enzymes have one-to-one C. elegans orthologs, the role of Ss-CYP-22a9 as the DAF-9 isoenzyme could not have been bioinformatically inferred. 

- CRISPR-based knockdowns are not trivial in this system and both the HDR and NHEJ pathways were elegantly leveraged to confirm the in vitro activity of Ss-CYP-22a9 and its essentiality to the life stage responsible for infection. 

- Animal studies convincingly reveal the synergistic effect of Δ7-DA and ivermectin in disseminated strongyloidiases. The burdens of intestinal larvae and adults in both uncomplicated and disseminated infection after treatment align with the standing model that Δ7-DA is acting against the intestinal larval stages (L3a) that are naturally deficient in the hormone. 

- While there is precedent for combinatorial drug therapies in antifilarial control, there is great novelty in combining drugs that are known to target different stages as opposed to just different molecular targets. This provides the first clear demonstration of this as far as parasitic nematodes are concerned. 

Weaknesses: Weaknesses are categorically minor. 

- As the authors recognize, the animal studies required daily Δ7-DA dosing over a two-week period. While some of this is explained by a short half-life and poor pharmacokinetics, drug was continually delivered directly to the gut at high (uM) concentrations. This is a major hurdle to surmount and it is entirely possible that even if drugs with equivalent potency and more favorable pharmacokinetics were discovered, they would have to be administered in multiple dosing regimens or at prohibitively high concentrations to achieve a curative effect. 

- While there is some evidence in other nematode clades that modulation of the DAF-12 pathway can affect developmental phenotypes, many of these parasites have huge phylogenetic separation from strongyloides and lack dormant stages requiring 'activation' or free-living stages. Given the independent evolution of parasitism across the phylum, it is just as likely that drugs acting on DAF-12 will have subtle (and not curative) effects in these other parasite systems.

Reviewer #2 (Public Review): 

Mangelsdorf, Kliewer and colleagues here identified the endogenous ligand in Strongyloides stercoralis that governs that parasitic nematode's capacity to autoinfect its mammalian hosts. The ligand, [Delta]7-DA, interacts with nuclear receptor Ss-DAF-12, just as it does with its C. elegans ortholog, Ce-DAF-12, governing transcription of genes essential for metabolism and reproductive growth. Specifically, [Delta]7-DA appears to mediate a switch in DAF-12 function: in unfavorable conditions, [Delta]7-DA is absent and unliganded DAF-12 is said to arrest growth in both species and produces developmentally quiescent infective S. stercoralis larvae; in favorable conditions, the ligand is synthesized and the liganded DAF-12 triggers infection by S. stercoralis and subsequent development and reproduction in both species. The authors determined the ligand's biosynthetic pathway and showed by mutating the rate-limiting enzyme in the pathway that [Delta]7-DA is essential for parasite reproduction, whereas its absence is required for infectious larval development. In an animal model, they demonstrated that administration of [Delta]7-DA suppresses autoinfection and host lethality. Given in combination with an existing drug targeted to actively developing stages, [Delta]7-DA virtually cures the disease. 

This work establishes a finding and an implication. The finding: gene circuits for growth and reproduction in nematode species with distinct life cycles -- parasitic vs free-living -- are regulated by a hormonal signal and cognate receptor that are structurally and functionally conserved. This is evolutionarily unremarkable, made mildly surprising because S. stercoralis lacks a cytochrome P450 with strong sequence identity to C. elegans DAF-9, which catalyzes the rate-limiting step in [Delta]7-DA synthesis in C. elegans; a screen of S. stercoralis P-450s demonstrated that Ss-CYP22a9 is the DAF-9 isozyme. The implication: targeting DAF-12 function (either agonism to block lethal hyperinfection or antagonism to prevent development of adult worms) or ligand synthesis may offer a therapeutic route to treating nematode parasitism. This is a valuable implication, identifying three therapeutic target approaches -- Ss-DAF-12 agonism or antagonism, and Ss-CYP22a9 inhibition -- that potentially might be be advanced from these pre-clinical observations. Overall, the manuscript makes a modest yet significant contribution. 

Typical of the work from Mangelsdorf and Kliewer, the research plan and experiments are rigorously designed, executed and interpreted. My only quibble is that the requirement for Ss-DAF-12 (unliganded) to produce infectious L3i larvae is claimed (lines 165-167) but not directly demonstrated here. Instead, the authors depend on, and eventually cite in the Discussion (line 340) their nice PNAS paper earlier this year, which makes this case. Because of its importance in rounding out the implication noted above, my preference would be for the authors to add an experiment to this work that documents that Ss-DAF-12 is essential both pre- and post-ligand production.

Reviewer #1 (Public Review): 

The work presented here describes the application of a tool (MorphographX 2.0) that opens up possibilities for new image analyses. MorphographX 1.0 is already a valuable tool in the field and the improvements and new functionalities, and approaches presented in this paper allow for the integration and analysis of more positional and temporal information. Specifically, adding positional annotation to analyze the distribution of cell properties across a plant organ will be of great use for the community. The case studies used to showcase MorphographX 2.0's applications highlight the diversity in questions that can be addressed using this tool. As a result, we expect to see MorphographX 2.0 applied in a variety of future plant biology stories. In addition, we believe this tool could also be useful to those outside the plant community. While probably less of use in tissues where there is extensive migration, it can be applied to any system with clearly visible cell membranes. 

The examples presented in this story highlight some great applications of the MorphographX 2.0 software. Analyses using more positional, temporal and 3D information will enable new findings across plant tissues and potentially across species. It is however important to be aware that for optimal use this software is designed to analyze high quality, high contrast stacks that can be difficult and time-intensive to acquire. MorphographX 2.0 also requires a powerful computer setup. The presence of both Linux and Windows versions that do not require a nVidia graphics card does open up possibilities. In addition, extensive documentation and the presence of a community forum allow use of the software without intensive training.

Reviewer #2 (Public Review): 

The manuscript is of wide interest to the plant development community. It gives an overview of recent extensions to MorphoGraphX, a tool to analyse confocal microscopy images of plant tissues to cell-level. In particular the advance concerns the annotation of cell morphometric and tissue-level topological properties (cell volume, cell shape, cell neighborhood relations, cell lineages) as a function of local coordinate systems in plant organs, such as distance to the root tip along the length of a (potentially curved) root. This allows the end-user to annotate static and time-lapse images in relation to a positional coordinate system close to those likely used by the developmental mechanism itself. 

The useful novel methodology to annotate 3D confocal images of plant organs is illustrated using data of roots, ovules, and leafs. Many of these analysis tools seem to have been developed in the context of research papers cited in the manuscript. The main novelty of the present manuscript seems the bundling and release of these new features in MorphoGraphX 2.0. The software is available from the authors' website allowing users to read it. 

The tools presented in the manuscript will attract the keen interest of plant biologists who (want to) carry out quantitative analyses of developing plant tissues, alongside animal developmental biologists working, e..g, with epithelial tissues that can be well segmented. However, with the manuscript in its present form, it would be very hard for a researcher new to the approach to get started with MorphoGraphX2.0. 

The datasets of the examples are available on the website of MorphoGraphX2.0. It would be very helpful if detailed instructions were given in the manuscript or in the supplements of how the analyses can be reproduced. This would allow the end-user to get started with their own data. Also it would be useful to give more detail on how the data must be generated to make it suitable for use in MorphoGraphX. 

Altogether, the annotation tools presented in the manuscript will be of interest to the community. However, as many of the tools seem to have been presented already elsewhere, the main contribution of the present manuscript would be (apart from 'advertising' MorphoGraphX2.0, which is useful in itself) in a set of instructions and/or directions to help get users started.

Reviewer #3 (Public Review): 

This manuscript reports the advancement in the image analysis software package MorphGraphX, which was developed about ten years ago. A comprehensive first report of this platform was published five years ago, and now the 2.0 version is being described. 

Included are useful tools to capture the developmental dynamics of growing tissues at cellular resolution. The analyses using this software can provide precise quantitative information of cellular behaviours, such as cell division and expansion, within the context of positional information in the growing organs. The tools developed and reported here are great assets to developmental biologists in general. 

The manuscript is well written in some places, while it reads confusingly in other parts. For example, figures are introduced in the main text in out of order and back-and-forth fashion. 

This article will become a go-to resource for future users of MorphGraphX to learn the power of the software package, with which they can quantify the spatiotemporal dynamics of the growth and development of living organisms.

Reviewer #1 (Public Review):

This is a nicely and well-performed study on the role of Rap1 and Rif2 in telomere homeostasis of the yeast Saccharomyces. Using a tlc1 mutant that generates telomere repeats with a lower Rap1 binding plus additional mutations, the authors show that Rap1 binding to telomeres is not necessary for cell viability and causes lengthening of telomeres. The authors show that the viability produced without Rap1 binding is not due to Tfb1 action, as it could have been predicted from previous studies. Analysis of the need for Rif1 or Rif2, which when bound to Rap1 inhibits telomerase, the authors find that it is Rif2 and not Rif1 the essential counterpart. The authors show indeed that indeed Rif1 and Sir4 binding to telomeres tlc1-tm is decreased but not that of Rif2 and Sir3. The authors focus the manuscript on the role of Rif2 which was known to bind dsDNA and MRX to show that Rif2 protects telomere ends in the absence of RAP1 by inhibiting MRX and promoting Rad51-dependent homologous recombination. Further analysis allows the authors to conclude that Rif2 and the Yku complex protects such telomeres. The study is well-executed with the appropriate molecular analysis of telomeres, protein binding by ChIP and senescence studies using a clever and well-explained rational combination of mutants, but the authors need to emphasize more the novelty of their conclusions, they should clarify and better support the conclusions about the function of Rif2 and provide more details on the role of Sir3 in tlc1-tm mutants.

Reviewer #2 (Public Review):

This is a very interesting study addressing how telomeres can be maintained when the main budding yeast shelterin protein (Rap1) is missing. The authors used a telomerase RNA template mutant to generate telomere repeats lacking high-affinity Rap1 binding sites. Nicely, the elongation of the mutant telomeres becomes length-independent, the expected hallmark of Rap1-depleted telomeres combined with a compromised telomerase. ChIP analyses also show that mutant telomeres are to a large extent Rap1-depleted. Among the Rap1-interacting factors tested, Rif1 and Sir4 are also missing or reduced but surprisingly, not Rif2 and Sir3. Furthermore, Rif2 is now essential for telomere stability and cell viability by antagonizing the uncontrolled action of the MRX complex (mostly end resection). The authors conclude that Rif2 recruitment to telomeres is in part Rap1-independent, highlighting the built-in redundancy among the pathways ensuring telomere protection.

Reviewer #3 (Public Review):

This manuscript reports a detailed description of telomere homeostasis in a strain expressing a previously described mutant allele of TLC1, the gene encoding the RNA component of the telomerase. The tlc1-tm RNA produces telomere repeats without GGG. It is shown here that tlc1-tm telomeres present a strong defect in Rap1 binding. Nevertheless, tlc1-tm cells are viable with a growth rate similar to wild-type cells. As expected the association of Rif1 and Sir4 is decreased but not that of Cdc13, Ku and more surprisingly Rif2 and Sir3. The tlc1-tm telomeres also show increased recruitment of the MRX complex. Rif2 binding is dependent on RAD50 specifically in tlc1-tm cells. Taken together, it seems that the stability of tlc1-tm telomeres combines the classical telomere protection functions provided by Cdc13 and KU with features of a double-strand break with increased recruitment of MRX and therefore of Rif2 that in turn restricts MRX action.

The manuscript is well written. Nevertheless, there are two major points that should be clarified:

1. The vocabulary used to describe the decreased binding of Rap1 to tlc1-tm telomeric repeats should be standardized taking into account that 1) Rap1 binding is not totally abolished 2) telomeres (except TELVII-L mut) retain RAP1 binding sites in their most proximal part as well as on interstitial telomeric repeats in Y' telomeres. It seems therefore more correct to speak of a decreased level of Rap1 rather than loss or lack.<br> 2. The role of recombination in the homeostasis of tlc1-tm telomeres should be discussed because stochastic recombination events or processing of intermediates could be at the origin of variations of the telomere profiles between isogenic tlc1-tm clones. In this line, the figure 5-fig Supp2 shows a clear difference in the telomere profiles of tlc1-tm and tlc1-tm rad52 cells but this is not commented in the text. Similarly, the large heterogeneity of X-telomeres is not mentioned though it could be due to de novo acquisition of Y' sequences at least in some cases. Finally, Rad52 is required for the growth of rif2 tlc1-tm cells.

Reviewer #1 (Public Review):

Essential functions of biomacromolecules such as enzyme catalysis, receptor activation or inhibition, etc. depend on the thermodynamics and kinetics of ligand binding. Recent NMR data support the model that conformational entropy of protein side chains directly impacts ligand binding affinity. Also, it has been shown that conformational entropy is responsible for modulating positive and negative cooperativity. However, NMR data on complex systems are still sparse, and it is unclear whether the conclusions derived from the analysis of NMR order parameters are generally applicable. Here, the authors use a multiconformer modeling of time- and space-averaged electron density to determine the conformational heterogeneity in matched pairs (holo and apo) of crystallographic datasets and evaluate protein responses to ligand binding. In their analysis, the authors include both anharmonic and harmonic (i.e., static and dynamic) disorder using the qFit software package as well as crystallographic order parameters. The most important observation is that ligand binding globally affects the structural dynamics of proteins. When the enthalpic contribution dominates in and near the ligand-binding sites (i.e., rigidification of the binding pocket due to protein-ligand interactions), the conformational dynamics are redistributed throughout the remainder of the protein, and distal sites become more disordered. Although the hypothesis on the redistribution of conformational free energy upon ligand binding has been previously contemplated, the direct experimental proof has been difficult to achieve using thermodynamic methods alone. The latest NMR techniques, however, enable one to estimate the conformational entropy from the calculation of site-specific order parameters for methyl-bearing side chains. Therefore, the distinct merit of this paper is to demonstrate the direct link between the disorder observed in the X-ray coordinates and the conformational entropy as measured by NMR spectroscopy. The connection between these orthogonal techniques leads to the conclusion that conformational entropy must modulate ligand binding phenomena.

Overall, the paper is exciting and offers new insights into ligand binding affinity and specificity mechanisms, suggesting that distal (allosteric) perturbations represent a possible avenue to modulate protein function. There are, however, a few weaknesses. The authors have stated some of them in the discussion section, i.e., the possibility of underestimating motions from frozen samples. Additionally, the authors offer a concise explanation for the redistribution of the conformational disorder. In fact, from the analysis of the holo/apo pairs, the authors deduced that packing optimization and rearrangements within the protein cores may be responsible for the redistribution of heterogeneity upon ligand binding. The current analysis of the data does not allow the reader to appreciate these conclusions entirely. Is there a way to look at the packing of the structures, both the apo and holo state? Additionally, the authors suggest that possible hydrogen bond rearrangements may also occur upon ligand binding. However, this aspect is poorly developed or discussed in the current version of the paper. Another shortcoming is the lack of consideration for other electrostatic interactions involving charged side chains that often dominate ligand binding affinity. Despite these shortcomings, the authors' work is solid.

Reviewer #2 (Public Review):

Overall, this is a comprehensive examination of the structural transitions associated with small molecule binding to proteins. A database of protein-small molecule complexes has been well curated from the PDB and the criteria explicitly stated. The resulting structures are carefully analyzed and compared and the observations very clearly presented. Since a continuum of response is seen, an excellent statistical framework is provided. Many new insights into the structural (energetic) and entropic contributions to ligand binding thermodynamics are discovered and synoptically put together. The main observation is the heterogeneity of response of proteins to small molecule ligand binding. One imagines that this will become a classic step forward in our understanding of the thermodynamics of molecular recognition by proteins and will be of great utility for biochemists, structural biologists and drug developers.

Reviewer #3 (Public Review):

Protein function emerges from a fine balance between enthalpic and entropic contributions of the various interactions between constituent atoms. While protein structures derived from X-ray diffraction data often yield rich information about enthalpic contributions, extraction of entropic information remains challenging. Here, Wankowicz et al. present a three step approach to quantitatively map changes in conformational heterogeneity for side chains across hundreds of different proteins in response to ligand binding. First, the authors create a well-curated, high resolution set of paired apo and holo structures from the PDB. The models were re-refined against the published data, and then multiconformer models were generated with qFit. The authors calculated crystallographic order parameters for most residues across all pairs. In cases where the binding site was found to rigidify upon ligand binding, a compensatory increase in disorder was observed at distant sites-an observation consistent with free energy considerations as well as with results of single protein studies conducted using other methods.

This work is interesting as it is the first systematic attempt to make such measurements from X-ray data at scale, spanning hundreds of different crystal structures bound to a variety of ligands. This approach leverages existing data, and could thus prove to be hypothesis generating in specific cases related to goals such as drug design and the identification of allosteric pathways/sites. Importantly, the development of this approach directly builds upon previous work (Fenwick et al. 2013) and represents a conceptual step towards the general goal of estimating of configurational entropy from X-ray crystallography data.

While the authors are candid about the limitations of the study and describe a plan to overcome some of the issues they describe, this work does have some weaknesses which should be addressed. From a technical perspective, definitions of different types of residues (binding site vs. distant) can seem arbitrary, so explaining the criteria thoroughly and showing that the results are robust to variation in these definitions is important. Conceptually, this work suffers somewhat from a lack of comparison with other studies of configurational entropy and, more generally, propagated structural change in proteins. Some quantitative comparison with data from the literature for specific cases is warranted.

Reviewer #1 (Public Review):

Molecular probes that respond to disease-specific activities to produce a diagnostic readout have had a major impact in the clinical management of cancer. The current study extends the teams previous work on the development of a molecular sensor for the cancer-associated, fibroblast activation protein (FAP). The molecular sensor is based on CGRP (Calcitonin gene-related peptide), a potent vasodilator of human arteries which mediates relaxation of arteries via activation of the CGRP(1)-type receptor. The sensor is fused to biotin with a linker sequence that contains FAP cleavage sites. In its intact form, the sensor fails to activate the CGRP-receptor, however, in the presence of FAP, proteolytic release of CGRP from inhibition, leads to CGRP-receptor engagement, which is then detected by changes in MRI contrast. Receptor activation on the vasculature, provides a diagnostic readout via local changes in hemodynamic image contrast for MRI. This is a technical report that provides a proof of principle evidence that a sensor for FAP proteolytic activity can be used in rodent models, with a robust signal to noise. However, the discussion and abstract overstate the clinical impact of the findings.

Reviewer #2 (Public Review):

In this manuscript, the authors create an imaging probe for magnetic resonance imaging (MRI) that is based on triggering vasodilation in a protease-dependent manner. The imaging probe is a steric blocking domain fused to the N-terminus of a vasoactive peptide connected by a linker that is sensitive to proteolytic cleavage by fibroblast activation protein (FAP). The linker design was optimized for FAP-mediated cleavage and led to a 34-fold increase in activity when there was FAP present. The imaging probe detected cells overexpressing FAP implanted into the rat striatum when infused directly at the transplantation site or into the cerebrospinal fluid. The authors also create a kinetic model to determine FAP catalysis rate, k, from temporal MRI signal. Lastly, the authors demonstrate in a proof-of-concept experiment that the vasoactive peptide is able to create imaging contrast in a nonhuman primate brain.

This group has previously described using peptide-mediated vasodilation as a method for image contrast in MRI. In this work, they advance this concept to make the peptide activity triggered by protease cleavage, thus creating an activity-based molecular imaging probe. The design presented in this work could likely be adapted to a wide range of proteases through substitution of the substrate that links the steric blocking domain and the vasoactive peptide allowing for the study of a wide range of protease activity in diseases that affect the brain. The creation of activity-based imaging probes is an important area of study for advancing precision medicine because the imaging signal may more accurately represent disease prognosis and stratification over conventional imaging probes.

A claim made in the abstract that is provided with limited support is the whether the probe allows for quantitative analysis of FAP activity. A useful measure for a diagnostic would be whether the imaging signal can quantitate the amount of enzyme activity. In this study, all in vivo experiments were conducted with the injection of a single concentration of cells overexpressing FAP transgene.

Reviewer #1 (Public Review):

In this study, Kim et al. perform comparative studies on the nuclear import dynamics of AR-FL versus a truncated, ligand-independent splice variant, AR-V7. Their main findings are as follows:

1) Under basal conditions without ligand, AR-FL remains largely in the cytoplasm, as does another splice variant of the AR, AR-v567. In contrast, AR-V7 exhibits fast nuclear import kinetics and 75% of AR-V7 accumulates in the nucleus by 90 minutes after microinjection.

2) In contrast to AR-FL, which is imported into the nucleus through a typical importin a/b import mechanism, AR-V7 is not dependent on either importin-b or microtubules for nuclear import.

3) When AR-V7 and AR-FL are co-expressed in the same cell, the nuclear localization of AR-FL increases two-fold over AR-FL expressed alone.

4) AR-V7 is transcriptionally active in the nucleus but exhibits very short chromatin residence time as assayed by fluorescence recovery after photobleaching as well as high intranuclear mobility.

AR-V7 was proposed as a biomarker of resistance to antiandrogen therapies several years ago but the functional significance of AR-V7 and other AR splice variants in driving resistance has remained unresolved, as have the precise mechanisms by which these variants contribute to AR signaling relative to AR-FL. The main strength of this manuscript is that it performs careful characterization of one aspect of AR biology - namely the nuclear import kinetics - in AR-Fl, AR-V7 and AR-v567. The data clearly show that AR-V7 exhibits distinct nuclear import kinetics from AR-FL in the experimental systems used. There remain some unanswered questions - namely: 1) what is the precise nuclear import machinery used by AR-V7? And 2) is the mechanism of AR/AR-V7 import in physiologic contexts similar to that observed in the ectopic expression systems used in this study?

Reviewer #2 (Public Review):

The data presented in this manuscript are both novel and seminal in our understanding of the molecular pathways for ARv7 driven progression of castration resistant prostate cancer. This data documents an entirely novel mechanism for this variant's nuclear uptake and transcriptional targeting. This novel information will be critical in developing therapeutic approaches to selectively inhibit ARv7 oncogenic abilities. As sole critique, it would be very helpful if the authors could include a discussion of how this new understanding of the molecular pathways of ARv7 can be used to develop new approaches for castration resistant prostate cancer.

Reviewer #3 (Public Review):

The emergence of AR-V7 and other AR splice variants in patient tumors has been shown to track with inferior response to AR targeted and chemotherapies in prostate cancer. Using a series of sophisticated imaging techniques the authors have determined that the AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR and yet AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. This study provides new insights into how AR-V7 may contribute to the pathology of CRPC.<br> Although it is well accepted that the AR-Vs serves as a strong biomarker for resistance to antiandrogen treatment, it is still being debated as to whether and how AR-Vs contribute to the pathology of castration resistance prostate cancer. In this regard, significant efforts have been invested to understand how these splice variants work and how to best target them. Several models of have been proposed, some of which suggested that the AR-Vs can dimerize with the full-length (FL) receptor and require FL-AR for activity, others indicated that AR-V has unique activities independent of FL-AR. Using a series of sophisticated imaging techniques, the authors have addressed some of these unresolved issues in the field, in particular determining that AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR; and yet, AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. Overall, this is a valuable study, and the authors are to be commended for the high-quality figures and illustrations. Specific strengths include the following points:

1. It has been observed that the AR-V7 predominantly resides in the nucleus; however, the mechanism(s) by which AR-V7 used for nuclear import is not clear. Using live imaging combined with pharmacological and genetic approaches the authors have determined that the importin / complex is required for FL-AR, but not AR-V7 nuclear import. However, nucleoporin complex and Ran-GTP activity are required for FL-AR import, as expected, and are at least partially required for AR-V7 nuclear accumulation. These series of studies have confirmed previously finding that AR-V7 uses a mechanism for nuclear import, distinct from the FL-AR and the AR-V567 variant.

2. Using mutagenesis of the D-box and DNA binding mutants, the authors have also contrasted the structural requirements within FL-AR vs AR-V7 for nuclear localization and transcription. It was determined that the dimerization surface is required for AR-V7 nuclear retention but is dispensable for FL-AR. However, disruption of DNA binding with a single point mutation has demonstrated that DNA binding is not an obligatory step for FL-AR and AR-V7 nulcear retention. This series of experiments have enhanced our understanding of the nuclear cytoplasmic dynamics of AR-V7 and how it differs from FL-AR and suggests that interfering with the dimerization interface may be a means by which AR-V7 can be targeted.

3. Using FRAP and photoconvertible fluorescent protein tag, the authors were able to track the intranuclear dynamics of AR and AR-V7 in real time, which is a strength of this study. They have determined that AR-V7 has higher sub-nuclear mobility compared to FL-AR and that DNA binding is required for even the short residence time of this mutant AR on the chromatin. Together these data suggested that AR-V7 and FL-AR may use different means to activate transcription.

There are some weaknesses that could be addressed to improve the work:

1. Most of the studies were done in PC3 AR negative cell line. It would be helpful to confirm some the key findings in AR positive cell line as the import mechanism may not be the same in AR negative vs AR-positive cell lines.

2. In Figure 2 and 3 where authors used mutagenesis to determine the structural requirements of FL-AR and AR-V7 for nuclear import/retention. These studies used nuclear:cytoplasmic ratios as readouts, not transport kinetics, and thus the observed changes in N/C ratios could be the results of changes in nuclear export and should be discussed appropriately.

3. The observation that co-expression of AR-V7 increased nulcear FL-AR in the absence of ligand is interesting. The fact that IPZ interferes with nuclear accumulation of FL-AR in the presence of AR-V7 indicated that FL-AR import still requires importin but does not rule out the possibility that FL-AR via its dimerization with AR-V7 within the nucleus could lead to increased retention of FL-AR within the nucleus, a possibility that the authors should consider.

4. The authors used ChIP assays to confirm the fast chromatin mobility of AR-V7 they have observed using FRAP, however ChIP efficiency could differ significantly using different antibodies and the results should be discussed with caution. Although the authors tried to confirm the same using chromatin bound fraction as another readout for transient chromatin binding of AR-V7, it was unclear why the authors didn't use endogenous AR-V7 in 22RV1 cells to look at chromatin bound fraction, as overexpressed protein may have different behavior compared to endogenous protein.

Reviewer #1 (Public Review):

In their manuscript, Urtatiz and colleagues propose that gain-of-function mutations affecting the G-alpha-q signaling pathway are not tolerated in melanocytes residing in the interfollicular epidermis because of paracrine signals from neighboring keratinocytes. This is an interesting and important hypothesis that would explain a mystery to the melanoma field - i.e. why are GNAQ/11 mutations common in uveal melanoma, among other rare subtypes, yet are exceedingly rare in cutaneous melanoma.

Specific comments on experimental work:<br> Previously, this group showed that forcible expression of oncogenic GNAQ during embryogenesis depletes melanocytes in the interfollicular epidermis. This paper offers an advance because TYR-cre mouse is inducible at later points in life, which also permits in vitro explant cultures to perform more in-depth studies. This is a major strength of the manuscript.

Major concerns:<br> The most significant issue with the experimental results is that in most of the explant cultures, the melanocytes are not proliferating. Instead, the authors are observing which melanocytes are dying slower than others. This seems a bit strange because there are countless examples of laboratories who have established healthy murine melanocytes in tissue culture, and it raises the question that there is something off with the culture conditions.

In addition, when sorting the GNAQ-mutant melanocytes, there is a selection for a subpopulation that did not die. This introduces a bias and seems, at minimum, worthy of discussion. One potential experiment to remove these doubts would be to isolate the GNAQ-mutant melanocytes prior to tamoxifen treatment and then induce the mutation formation in vitro.

Specific comments on bioinformatic work:<br> Major issues:<br> The evidence that there is selection against PLCB4 in cutaneous melanoma is weak. It is true that mutations frequently affect PLCB4, but this is equally true for a great number of genes in melanoma because of the high mutation burden in this cancer type. Following their lines of reasoning, the authors could make an equally compelling case that TTN, the gene encoding the muscle fiber titin which is the largest gene in the genome, is under selection. Unfortunately, the ratio of nonsynonymous to synonymous mutations is not supportive of the authors' argument that PLCB4 is under selection. It is somewhat bizarre that the authors' entirely disregarded synonymous mutations, but this reviewer looked them up in the TCGA study, and they are abundant. To identify genes under selection, there are much more sophisticated strategies that take into account the trinucleotide context of mutations, the ratio of nonsynonymous to synonymous mutations, and/or the ratio of exonic to intronic mutations. To be sure, the authors correctly point out these sophisticated algorithms have missed driver mutations in the past, but the missed mutations tend to be exceedingly rare, hotspot mutations. If the authors are going to make the case that loss-of-function PLCB4 mutations are under selection in melanoma, then the onus is on them to explain why the much more sophisticated strategies, previously invoked, have missed this finding, and they should employ even better methods to make their point. Unfortunately, the strategies that the authors do employ fall for the same traps that many older papers in the field stumbled upon. For example, they make the case that PLCB4 mutation are more frequent in melanomas with high mutation burdens. While this seems to denote a biological signal, it is exactly what one would expect to observe if a gene only harbored passenger mutations.

The Semaphorin mechanism is interesting but remains too speculative to warrant so much attention and space. In addition, there was too much speculation regarding signaling mechanisms in the apoptosis section of the manuscript. Generally speaking, RNA-sequencing is a powerful tool, but when there are >1000 differentially expressed genes, it is too easy to construct "in silico" mechanistic stories centered around a handful of genes. These would need be backed up with biological data, but in this case, additional mechanistic studies would go beyond the scope of the manuscript. Instead, this reviewer suggests removing these points.

Reviewer #2 (Public Review):

The authors aimed at addressing the question of why constitutively activating somatic mutations in GNAQ and GNA11 that occur in almost 90% of uveal melanomas, and are common in blue nevi and melanomas of the central nervous system, are very rare in cutaneous melanoma tumors. They hypothesized that the restriction of GNAQ and GNA11 mutations to non-epithelial melanomas can be due to i) exposure of melanocytes in different anatomical locations to different mutagens that affect specific genes, ii) difference in the potential for melanomageneiss of melanocytes arising from different anterior-posterior positions during embryogenesis, and/or iii) differences in the microenvironment of melanocytes in different anatomical locations, that affect their regulation by paracrine factors. The authors arrived at the conclusion that epidermal factors convert the impact of GNAQQ209L mutation from being oncogenic to being inhibitory to melanocyte survival and proliferation, and reduce the maintenance, rather than the establishment of interfollicular epithelial (IFE) melanocytes.

The strengths of the methods lie in the:<br> 1) In vivo comparison of the impact of GNAQQ209L mutation on melanocyte establishment and maintenance, by expressing the mutation in melanocytes, and comparing their number in the interfollicular epidermis (IFE) of the tail of wild type versus mutant mice.<br> 2) in vitro validation of the differences between wild type and GNAQQ209L mutant IFE melanocytes by sorting them from the tail IFE by FACS, and comparing their proliferation on fibronectin and their morphology. By removing the mutant melanocytes from their microenvironment (the tail IFE), they proliferated better than wild type melanocytes on a fibronectin matrix, demonstrating that the reduction in their number in vivo is due to paracrine regulation by their epithelial microenvironment.<br> 3) Demonstrating the impact of the microenvironment of GNAQQ209L mutant IFE melanocytes, melanocytes were co-cultured with IFE cells or tested in a transwell assay, with IFE cells in the bottom well, and the melanocytes plated on top of a permeable membrane. In both cases, IFE cells resulted in inhibition of proliferation and survival, and significant increase in the size and morphology of melanocytes.<br> 4) RNA sequencing to determine any change in gene expression that might account for the reduced survival, proliferation, and altered morphology of IFE melanocytes expressing GNAQQ209L , as compared to wild type melanocytes, using melanocytes immediately after sorting from the tails of mice. Bioinformatics analysis revealed altered expression of genes involved in pigmentation, cell adhesion, focal adhesion, extracellular matrix receptor interactions and structural constituents, axon guidance, nervous system development, stress and apoptosis, which account for the differences observed in the survival, proliferation, and morphology between mutant and wild type melanocytes.<br> 5) Analysis of TCGA-SKCM dataset to re-examine the data related to PLCB4, which encodes a protein that bins to Gαq and Gα11, and is the primary effector of signaling to downstream components of 1 class heterotrimeric G proteins, to confirm the previous report that PLCB4 is targeted by loss of function gene fusion events in some cutaneous melanomas. Demonstrating expression of loss of function of PLCB4 supports the authors' findings that activation of the q class heterotrimeric G proteins inhibits, while in contrast, inhibiting their signaling pathway is permissive to, epithelial melanoma tumor formation.<br> The data presented are solid, and the results support the conclusions derived by the authors. The data were validated using the different experimental methods listed above. The results clarify the differences in the mutations observed in non-epithelial versus epithelial melanomas, and should improve the understanding of the various pathways that drive melanomagenesis under the influences of different microenvironments in different anatomical locations.<br> The only weakness is in the detailed discussion of the various other mutations (pages 14-17). The data included could be summarized, since the detailed analysis detracts from the main take-home message of the manuscript that the epidermal microenvironment impairs the survival of GNAQQ209L mutant melanocytes, and deficiency of PLCB4 promotes cutaneous melanoma formation.

Reviewer #1 (Public Review):

Reichardt M. et al investigated cardiac tissue of Covid-19 samples compared to other infections (influenza and coxsackie) and control. Using X-ray phase-contrast techniques, they provide interesting results on the microstructure description. For instance, the orientation of the cardiomyocytes, their degrees of anisotropy, their shapes (obtained via structure tensor analysis). They also present interesting findings thanks to the segmentation of the vascular network analysis (via deep learning method).

This paper is using state of the art techniques. The experiments use the latest development of X-ray phase-contrast techniques (at laboratory and synchrotron). Analysis are using machine learning approach. This paper will therefore serve as a reference for future analysis on cardiac tissue. Furthermore most of the tools used are publicly available.

The results presented show that X-ray imaging is providing more information than standard histology by accessing 3D information. This is illustrated for example by the 3D vasculature tree to assess intussusceptive angiogenesis.

In overall the paper is well written and giving clear background and explanation that people outside the fields can follow. The findings and conclusions of this paper are mostly well supported by data and analysis, but some aspects in the image acquisition, sample choice and data analysis need to be clarified.

1/ In the abstract, the authors don't mention the laboratory setup which is a big part of their results and actually the technique that could pave the way to a clinical translation of the technique. A sentence mentioning it is necessary.

2/ The authors present their results as "fully quantified". It would be nice to see how the results presented in this paper are comparable to the gold standard analysis used so far (i.e. conventional histology analysis). This technique seems to provide more or different information not accessible by 2D slices analysis as done in histology.

3/ A major drawback of the technique presented here is that it is necessary to make a biopsy punch on the initial paraffin block. It means that the original sample is destroyed (which also goes again a bit the "non-destructive" claim of the method). For the high resolution acquisition done with the Wave-Guide setup, a second biopsy punch is even done. Several questions can then be raised: How those biopsy punches have been selected, how is this representative compare to the entire samples, etc.

4/ The statistics obtained are based on sparse data with large error bars. Only 26 samples have been used. For instance, the parameters obtained for the shape of the cardiac tissue represented in a ternary diagram in Figure 5, present tendencies but it would need more statistics to clearly affirm that there is a clear difference between the groups.

5/ Concerning the sample selections, several samples have been taken for control (2 patients and 6 samples) and coming from 2 young patients while for the diseased samples only one sample per patient have been collected, on older patients. Furthermore, the majority of the patients are men. How the authors are sure for instance that the control patients didn't have another disease that could have affected the cardiac structure? Could they see any differences between gender, as it has been shown in other COVID-19 studies? Could the difference in age also have an impact?

Reviewer #2 (Public Review):

The works aim to characterize cardiac tissues from patients which have succumbed to Covid-19.<br> The authors studied pathological and normal tissues using microtomography scans performed at different resolution scales. Starting on the reconstructed volumes, special automatic analytical procedures were developed to extract some quantitative structural parameters about the samples themselves. This characterization method was used previously in the study of murine heart models. The main outcome of the research is that there are some well defined characteristics found in Covid tissues that are not revealed in other pathological and normal samples.<br> The authors achieved the proposed aims and their conclusions are supported by the obtained data. The samples statistics should be further improved but it is already enough significant to validate the outcomes.

Reviewer #1 (Public Review): 

This paper uses intracellular patch-clamp recordings of hippocampal CA1 pyramidal cells in awake mice running on a cued treadmill to investigate whether dendritic plateau potentials, which can induce place fields in silent cells through Behavioral Time Scale Plasticity (BTSP), could also modify the spatial modulation of existing place cells. They report that plateau potentials can lead to the formation of a secondary place field by synaptic potentiation while reducing the primary place field by synaptic depression. As for place fields induced in silent cells, the spatial extend of this bi-directional plasticity depends on the speed of the animal during induction suggesting a fixed time course. Further analysis revealed that the sign and magnitude of Vm changes varied in a distance/time -dependent manner from the location/time of plateau induction such that Vm tended to increase at plateau location and to decrease away from the plateau in both directions adding a bidirectional property to previously described BTSP. The sign and magnitude of the plasticity also depends on the value of Vm at the time/location of plateau induction such that if Vm is more hyperpolarized than -55 mV the plasticity induces a depolarization at the plateau location and less hyperpolarization away from that location as observed in silent cells while if Vm is more depolarized than -55 mV the plasticity induces a smaller depolarization and more hyperpolarization further away. The authors then used Vm manipulations and computational modeling to show that the critical factor was not the absolute Vm level but instead the level of potentiation of activated synapses. Finally, the authors used a network model of CA1 to show that BTSP can account for over-representation at reward location within a familiar environment. Altogether, this work represents a nice combination of cutting-edge experimental work and modeling that shed new lights on cellular mechanisms allowing experience -dependent modifications of spatial maps in familiar environments. 

Major comments: 

1) The procedure for BTSP induction is described without much details in the text and method. According to the text from 1 to 8 laps/ stimulation were used to induce the secondary place field. Why is there such variability? I guess that sometimes one stimulation is not enough to induce the place field but this should be clearly stated in the introduction. Also how does one know if a new place field is induced? Sometimes the new and old place field strongly overlap (e.g. Fig. 1C, blue trace) and it must be difficult "by eye" to decide if a new place field was induced. For readers to get a better idea of the all process could you report the average number of laps/stimulations used to induce the secondary place field? Could you mention how many laps/stimulations were used for the example traces shown in Figure 1C. Was the success rate 100%? How many laps were recorded after induction and used to compute the average traces shown in Figure 1C? Could you please report those numbers in the text and the figure legend? Also, in case a primary place field was induced by BTSP how many laps/stimulations were used? Was it more difficult to induce a secondary place field compared to a primary one? 

2) Along the same line the behavior of the mice is not extensively described. However, behavior and notably running speed seems to have a major impact on the spatial extend of the plasticity. Could you plot the speed profile of mice below voltage traces for example in Figure 1B, S1I and so on. Also could you show lap by lap speed profiles superimposed for one recording session to have an idea of the variability and overall stereotypy of behavior. It appears that the place fields can span the border between laps (i.e. start before and end after the reward zone). How is it possible if animal stops at the end of a lap to get reward? Usually these stops induce a state change with reduced theta oscillations, which is less favorable to place cell coding. Is it the case that some animals do not stop at reward location? Could you give more details here? 

3) The rationale behind the analysis of delta Vm against time from plateau induction shown in Figure 2E, 3 and 4 and associated supplementary figures is not clear from the text and method sections. If I understood well this analysis uses the difference between average Vm of several laps after the induction laps minus the average Vm of several laps before the induction laps but then uses the speed of the animal during the induction laps to convert this delta Vm trace in the temporal domain. But this assumes a relatively constant behavior of the animal during induction. If induction is performed over 1 or 2 laps the chance of a constant speed are probably higher than if it is performed over say 7-8 laps. If the animal slows down consistently or even stops during induction laps 6-8 but runs fast during induction laps 1-5 how does one interpret the DeltaVm over time representation? Authors should report the number of laps used for induction for the traces illustrated in Fig. 2 and the time against position traces for all individual induction laps superimposed on top of the average in Fig. 2C and delta Vm against time traces for all individual induction laps superimposed on top of the average in Fig. 2E. 

4) In Figure 3D it is unclear where the PCs within-field data comes from. The n = 26 suggests that this data includes all stimulation but in most cases induction was performed by stimulating outside place field location (as shown in Figure 1D) and induction is done by stimulating always in the same position (except for 2/24 cells were there was a third induction). Could you please specify? 

5) In the modeling experiment (Figure 6 A) it is unclear why the dV/dt trace show no change for synapses activated before the plateau (unlike what is illustrated in Figure 5C). In my understanding the eligibility trace of these synapses shown in green allow them to be potentiated by a certain amount that depends on the overlap of their eligibility trace with the instructive signal. Maybe to facilitate understanding authors could show the post-synaptic potentials before and after plateau induction in Figure 5A.

Reviewer #2 (Public Review): 

What causes the synapses to give way in long-term potentiation (LTP) or long term depression (LTD)? Much work in the past 30 years has focused on the relative timing of pre- and post-synaptic spikes (STDP), post-synaptic voltage and the presence of neuromodulators as the most salient factors. But theory has not been able to lead to fast and accurate learning using only these factors, nor has experiment been able to show their causal role in producing representations. The discovery that relative timing between pre-synaptic activity and plateau potentials CA1 pyramidal cells regulates a fast and strong expression of LTD, which causes new representations in behaving animals, has offered a new hope. 

The original studies on behavioural time-scale plasticity (BTSP) left many crucial questions unanswered. Foremost is the absence of LTD. Are plateaus not engaging LTD at all or is LTD regulated by other factors altogether? Milstein et al. provide an answer to this question. LTD is engaged by plateaus, but only for synapses that have been potentiated, or synapses we expect have a strong weight. According to this point of view, previous attempt did not observed LTD because the protocol triggered only synapses with weak synaptic weights. 

Surprisingly, the dependence of synaptic plasticity on pre-post timing remains approximately symmetric (as in Bittner et al.); with LTD being engaged at the same time whether the post-synaptic plateau or the pre-synaptic firing came before one another. This enhances the paradigm shift offered by BTSP. First because it suggests a convergence of synaptic mechanisms: the effect of pre-synaptic firing on the trace left by the post-synaptic plateau is the same as the effect of post-synaptic plateau on the trace left by pre-synaptic firing. And second, because this asymmetry is firmly against Hebb's postulate. Pre-synaptic firing does not have to 'repeatedly or persistently take part in firing' the post synaptic neuron, a plateau generated by another pathway before the occurence of pre-synaptic firing is sufficient to induce both LTP and LTD. 

The work of Milstein et al. is also important because it contains a computational model, a hypothesis consistent with their experimental data and the molecular pathways involved in synaptic plasticity. They show that when the two opposing forces for LTP and LTD have distinct parameters (LTD being triggered by lower temporal coincidence but with a higher sensitivity and saturating earlier than LTP), they can recapitulate their experimental observations in vivo. 

I believe this is likely to become a seminal article in the field. The only weak point I see is I think the results from the modelling section could come out more clearly, particularly the crucial dependence of the model on having distinct functions for LTP and LTD.

Reviewer #1 (Public Review): 

Given that lipid metabolism plays an important role in viral infections, that lipid lowering drugs especially statins (HMGCoA reductase inhibitors) are widely used and that observational studies investigating the association between statin use and COVID-19 outcomes have given mixed results it's attractive to resort to mendelian randomization to minimize confounding and avoid reverse causation to address the potential benefits of lipid lowering drugs in this context. 

The authors use selected target genes for three lipid lowering drug classes - HMGCoA reductase inhibitors (statins), PCSK9 inhibitors and the NPC1L1 inhibitor ( ezetimibe) ; large well characterized COVID GWAS case and control data sets; summary -data-based MR (SMR) and inverse variance weighted MR ( IVW-MR) and sensitivity analysis. 

They demonstrate with SMR that a higher expression of HMGCR was associated with a higher risk of COVID-19 hospitalization. With IVW-MR they observed a positive association between HMGCR -mediated LDL cholesterol and COVID-19 hospitalization. For the other two drug classes no clear associations were shown. 

This suggests but does not prove that HMGCR drugs ie statins might improve outcomes. For the other two classes its unclear if there would be sufficient users of these drugs in the data sets to provide enough power to adequately address the issue. 

Overall this is a well conducted study providing important further insights but the final sentence of the abstract as well as the related discussion section including the limitations should be more expressed more conservatively.

Reviewer #2 (Public Review): 

Lipid metabolism plays an important role in viral infection. The study aimed to assess a putative causal effect of lipid-lowering drugs including HMGCR inhibitors, PCSK9 inhibitors and NPC1L1 inhibitors on COVID-19 outcomes. 

Two kinds of genetic instruments to proxy the exposure of lipid-lowering drugs, including eQTLs of drugs target genes, and genetic variants within or nearby drugs target genes associated with LDL cholesterol from GWAS were used. Mendelian randomisation analysis was performed. A higher expression of HMGCR was associated with a higher risk of COVID-19 hospitalisation (OR=1.38, 95%CI=1.06-1.81) at suggestive significance. Similarly, a positive association between HMGCR-mediated LDL cholesterol and COVID-19 hospitalisation (OR=1.32, 95%CI=1.00-1.74) was also observed. 

The authors conclude a potential causal evidence that HMGCR inhibitors could reduce the risk of COVID-19 hospitalisation, providing insights into the prevention and management of COVID19 infection.

Reviewer #2 (Public Review):

Luo et. al., have proposed a mechanism of action for a plant derived pentacyclic triterpene called celastrol. This drug has been previously shown to reduce obesity in mouse models by increasing leptin sensitivity and decreasing ER stress (PMC4768733) but a detailed mechanism of action has not been fully elucidated. The authors have identified GRP78, an ER resident chaperone, as a molecular target of celastrol. The evidence that authors provide in favor of their hypothesis shows that celastrol treatment is effective in reducing the deleterious effects of high fat diet on tissue hypertrophy in mouse liver and adipose. They further show that celastrol mitigates the high fat diet induced changes in lipid composition in mouse liver but not in adipose tissue. They also provide data to show that the expression of lipolytic and lipogenic genes are regulated by celastrol in both mouse liver and adipose tissue. These results support and add to the previous literature that celastrol decreases lipid accumulation and affects the regulation of important lipogenic and lipolytic genes in mouse liver and adipose tissue. But these results lack important information about the status of ER stress response in their particular experimental set up and how celastrol is affecting this stress pathway.

Authors solely rely on GRP78 immunofluorescence staining to show that its expression is reduced only in resident macrophage cells after celastrol treatment in mouse liver and adipose tissue. Another approach would have been to isolate macrophages from mouse liver which then can be used for protein or mRNA analysis and further broaden the findings.

In figure 4, authors have done a transcriptome profiling after nano-celastrol treatment. This provides valuable high throughput data which shows major cellular pathways that are affected by celstrol in-vivo. But it is also interesting to see that the proposed target molecule for celastrol i.e GRP78 as well as other canonical markers of ER stress are not among the list of differentially expressed genes. This raises the question that the effects of celastrol might not be limited to the modulation of ER stress.

The authors have done several experiments which greatly increase the confidence in their findings that celastrol binds to GRP78. Their discovery that quinone methide is essential for the beneficial effects of celastrol in-vivo is remarkable and of great clinical significance. But their data does not rule out the possibility that celastrol has molecular targets other than GRP78 and that the physiological effects of celastrol might not be limited to inhibition of GRP78 only.

Reviewer #3 (Public Review):

The article focuses on the anti-obesity effects of celastrol on HFD-induced mouse model. The impact of the article provided evidences that celastrol contains anti-obese effects in animal model. By using RAW264.7 marcrophages cell model and biotinylation method, they revealed that celastrol covalently bonded to the residue Cys41 and inhibit the ER stress sensor GRP78. The findings provide insights that covalent inhibition on GRP78 may serve as target against diet induced obesity. A synthesized celastrol analogue lacking quinone function group in ring A provided further evidence that the triterpene celastrol was covalently bound to GRP78 due to its containing the specific quinone substructure.

Reviewer #1 (Public Review):

This manuscript integrates conditional mouse models for TRAP, PAPERCLIP and FMRP-CLIP together with compartment specific profiling of mRNA in hippocampal CA1 neurons. Previously, similar approaches have been used to interrogate mRNA localization, differential regulation of 3'UTR isoforms, their local translation, and FMRP-dependent mRNA regulation. This study builds on these previous findings by combining all three approaches, together with analysis of mRNA dysregulation in Fmr1 KO neuron model of FXS. The strengths of the paper are the rich data sets and innovative integration of methods that will provide a valuable technical resource for the field. The weakness of the paper is the limited conceptual advance as well as lack of deeper mechanistic insights on FMRP biology over previous studies, although the present study validates and integrates past studies, adding some new information on 3'UTR isoforms.

Despite the fact that FMRP targets are overrepresented in the dendritic transcriptome, it does not appear from this study that FMRP plays an active role in the mechanism of dendritic mRNA localization, at least under steady state conditions. One goal of the manuscript is to address a major question in the mRNA localization field, which is how FMRP may differentially modulate "localization" of functional classes of mRNAs such as those encoding transcriptional regulators and synaptic plasticity genes (Line 78-90). The data here indicate that FMRP directly interacts with functional classes of mRNAs in different cellular compartments, which has previously been shown in the field. However, no evidence is provided that mechanistically reveal a role for FMRP to promote subcellular localization of different functional classes of mRNAs. The correlative evidence presented in this manner does not add mechanistic insight.

Further related to a role of FMRP in mRNA localization, a recent paper in eLife reports that FMRP RGG box promotes mRNA localization of a set of FMRP targets through G-quadruplexes (Goering et al 2020). This relevant paper needs to be cited and discussed. This relates to earlier work from the Darnell lab that identified a substantial pool of FMRP targets mRNAs having G-quadruplexes (Darnell et al., Cell 2001). It will be interesting if G-quadruplexes are enriched in their dendritic transcriptome datasets.

Some additional discussion and evaluation of relevant literature would be helpful to explain what aspects fit or do not fit with the proposed model. FMRP biology is more complex and this study should tie together and integrate different mechanisms on translational control (both negative and positive regulation) and mRNA stability.

Reviewer #2 (Public Review):

The authors performed transcriptomic analyses from compartment-specific, micro-dissected hippocampal CA1 region tissue from transgenic mice. One feature that distinguishes this work from previous studies is the use of conditional knock-in of tags (GFP or HA) and tissue specific expression of the Cre recombinase to target a very specific population of pyramidal neurons in the CA1 region--as well as the combined use of TRAPseq, PAPERCLIP and FMRP-CLIP. Also, central to this work are the analysis pipelines that look at large populations of mRNA with the goal of finding features shared by those mRNA that bind FMRP.

First, they established the identity of mRNAs that are dendritically enriched or/and alternatively polyadenylated (APA) by sequencing; followed by validation of a few candidates using smFISH. Next, the APA data was filtered through the rMATS statistical program to identify alternatively spliced (AS) mRNA variants within the APA population. The authors concluded that the majority of splicing events were of the exon-skipping type with NOVA2 as the likely culprit leading to this differential localization of AS isoforms. The authors then proceeded to perform FMRP-CLIP which was analyzed against the TRAP dataset. The (413) mRNAs that were shared by the two experiments (TRAP and FMRP-CLIP) exhibited two notable features: dendrite-enrichment and longer average transcript length. More importantly, They demonstrated that FMRP can preferentially bind to an AS isoform that is enriched in dendrites. Further analyses of FMRP CLIP targets showed that they shared a significant level of genes designated by gene set enrichment analysis (GSEA) as involved in ion transport and receptor signaling and similarly for ASD-related candidate genes.

Strengths:<br> -The combined use of tissue-specific Cre and conditional tags for RPL22, PABPC1 and FMRP help make these pull-downs highly specific and robust.<br> -RNA sequencing approach allows for identification and comparison of populations of ribosome-, PABPC1- and FMRP-associated mRNAs.<br> -Preferential binding of FMRP to AS or APA isoforms in dendrites is an impactful and significant finding.

Weaknesses:<br> -A caution in interpreting comparative or differential RNA-sequencing results as some are correlative.<br> -Validation of FMRP interaction with AS or APA isoforms or ASD candidates by smFISH-IF is lacking.<br> -Although hippocampal CA1 region is an excellent site to study FMRP-RNA interactome, are there other projection systems where altered FMRP-RNA interaction may lead to greater dysfunction?

Taken together, the authors were largely successful in demonstrating that FMRP can preferentially associate with its target mRNAs. Previous observations of a large number of FMRP targets suggested that perhaps FMRP could promiscuously bind to many mRNAs. The results presented in this manuscript argues strongly against this idea and show that within a subset of FMRP targets, FMRP can selectively bind in an isoform-specific manner. This work will be of broad interest to the community and serve as a resource and a template for those studying RNA-binding protein(s) and its mRNA targets.

Reviewer #1 (Public Review):

Musardo and colleagues explore the neurobiological impact of social isolation on the adolescent versus adult male mouse brain. The authors suggest that isolation alters subsequent social behaviors by causing increased excitability of Oxytocin neurons in the PVN and, subsequently, increased insertion of GluA2-lacking AMPARs in DA neurons of the VTA that project to PFC. 

Strengths:

• The authors examine the acute effects of social isolation during 2 periods of adolescence and during a period of young adulthood. They show that acute social isolation in adolescent mice from P28-P35 (but not P21-P28) increases subsequent social interaction time while decreasing habituation to social novelty. In contrast, isolation during adulthood has the opposite effect in that it reduces social interactions.

• When adolescently-isolated mice are regrouped and tested as adults, the isolation-induced increase in social interaction persists, but not the reduction in habituation to social novelty, suggesting some effects of adolescent social isolation are long-lasting.

• The authors show acute adolescent social isolation increases activity of PVN neurons projecting to the VTA concomitant with an increased density of PVN oxytocin neurons. They also show that regrouping mice eliminates the increased activity of PVN and VTA neurons but results in a long-lasting increased rectification index for VTA neurons projecting to the PFC.

• The authors then go on to establish a causal role for oxytocin signaling in isolation-induced changes in social behavior by showing that inhibiting the activity of all oxytocin neurons using a chemogenetic approach prevents the effects of social isolation on 1) subsequent social behavior and 2) the electrophysiology of VTA neurons (although, not specifically PVN-VTA neurons). They further show that globally inhibiting GluA2-lacking AMPARs in the VTA rescues the long-lasting effects of isolation on social interaction behavior in the regrouped mice.

Weaknesses:

• It does not appear that inhibition of oxytocin neurons was specific to the PVN, leaving room for effects to be ascribed to other nuclei (e.g., supraoptic nucleus). The inhibition of GluA2-lacking AMPARs in the VTA was not specific to DA neurons nor to neurons projecting to the PFC. Nor was the VTA inhibition of these AMPARs performed in the acute social isolation condition, which exhibited more wide ranging social behavioral effects. Social isolation may affect oxytocin neurons elsewhere in the brain, and it may be inhibition there that rescues the behavior and VTA electrophysiological phenotypes. Even if it was increased activity of oxytocin neurons specifically in the PVN that was responsible, that increased excitability may well have other downstream effects beyond increasing excitability of VTA neurons. These limitations are not acknowledged in the manuscript.

• The paper describes significant differences between rodents and humans in terms of how social isolation affects the circuity of interest, yet there is no discussion of this in terms of limited applicability of the present findings to humans. This is particularly troubling when only male mice are tested.

• The social behavioral changes triggered by social isolation are interpreted with a relatively narrow and negative light. We urge caution in interpreting the present phenotypes as absolute negatives, particularly when the main phenotype associated with adolescent social isolation is a subsequent increase in social interaction. Perhaps these are better characterized as changes in preference for social interaction as opposed to social deficits-or they could be interpreted as changes in social recognition memory in some cases. Note that recent work in other mouse models has revealed that social approach behavior can significantly shift depending on whether or not the neurobiology of the subject and the stimulus mouse match. Very different patterns of social interactions might be observed if group vs. isolated mice were tested using an isolated stimulus mice as opposed to a group-housed stimulus mouse (see a cautionary tale: Smith et al., 2021 Molecular Psychiatry, https://www.nature.com/articles/s41380-021-01237-4?proof=t). Use of the term "social craving" so definitively throughout the discussion also seems unwarranted based on the type of data presented (e.g., no tests of motivation to engage in social interactions were presented). Craving implies having experienced something that is very rewarding and wanting more of that thing; however, these mice never experienced any social interactions with non-cage mates, so how can they crave it? To this end, it might be informative to test these behaviors using cage mates as stimulus mice.

• Many of the statistical analyses employed/reported were not appropriate (e.g., use of 2-Way ANOVAs when repeated measure ANOVAs should have been used; reporting main effects when a significant interaction between main effects was necessary to support the post hoc tests used and the conclusions made).

Reviewer #2 (Public Review):

Musardo et al have evaluated the lasting effects of acute social isolation on future social interactions in juvenile mice, revealing a compelling oxytocin-mediated mechanism. The authors laid out clear hypothesis within a well-contextualized framework, and evaluated them using appropriate behavioral paradigms, chemogenetic, and pharmacological tools. The work sheds light on Oxytocin as a key regulator that modulates DA neurons in VTA projecting to the mPFC imparting long-lasting effects on social interaction. Conceptually, this work provides important information on neural signatures of social craving.

1) Neurobehavioral disorders that impact sociality, such as Autism spectrum disorder (ASD) are sexually dimorphic in occurrence. While the behavioral experiments in this study are well controlled, only males are used as subjects, limiting the potential impact of this work. 

2) The study used unknown sex-matched conspecific juveniles in direct free interaction and three-chamber tasks (one week younger than the experimental animal; Page 9 line 18,31). There is no explanation for choosing this specific age and age difference. Does the exposure to same-age juvenile or adult sex-matched conspecifics elicit a different behavioral response? 

3) Line 29-31 page 3 states that social isolation-dependent behavioral deficits are age-dependent and isolated adult mice show less social interaction compared to isolated juveniles. The authors should further elaborate on this interesting point and consider developmental differences in oxytocin system that could relate to it. 

4) cFOS+ and Oxt+ coexpression images are needed to relate the increase in the activity of PVN (via the IEG proxy) and the density of Oxt+ cells in PVN (figure 3 and page 4, line 11-17). Also, it is unclear what is happening here-are neurons increasing Oxt production to become more efficiently labeled or changing identity of their synthesized neuropeptides? 

5) The expression of inhibitory (Oxt-hM4Di) DREADD in the PVN should be illustrated with immunohistochemical evidence to demonstrate expression and selectivity. Some interpretation related to whether SON Oxt neurons, labeled in the cross, could be involved in the observed effects would be useful. 

6) Page 11, lines 30-32: The authors identified putative DA neurons based on position, morphology, and capacitance, while providing sparse details in the methods. 

7) The authors have exclusively shown scaled-up excitatory responses by pharmacologically blocking GABAA receptors, determining synaptic scaling after social isolation without considering potential effects on inhibitory post synaptic responses. Can any predictions be made?

Reviewer #3 (Public Review):

This study investigated how one week of adolescent social isolation (p28-p35) in mice impact VTA dopamine neurons and social behavior acutely at p35 and later in the adulthood at molecular and circuit level. Authors found that isolation led to increase in social interaction in free social interaction test, but no change in sociability during 3 chamber test. At the neural level, they observed excitability of VTA DA neurons are increased at p35 but not in adulthood. However, in the adult isolated mice, they found long-lasting expression of GluA2-lacking AMPARs at excitatory inputs onto DA neurons projecting to frontal cortex. Importantly, they found that the excitability of oxytocin expressing PVN neurons projecting to VTA is enhanced by isolation, and this change seems to have a causal impact to isolation induced DA neuron and behavioral changes as chemogenetic suppression of oxytocin neuron activity during isolation period prevented these changes. 

Overall, these findings support a key role of oxytocin expressing PVN neurons in regulating the adolescent isolation induced changes in VTA dopamine neuron excitability in adolescent mice, and synaptic function onto VTA neurons projecting to NAc through CP-AMPA receptor in adulthood. These findings are significant to the field as it provides the molecular and circuit mechanism mediating the effect of adolescent social isolation on social behavior. While the questions and findings are overall interesting and significant, there are some concerns with the interpretations of social behavior data and synaptic plasticity results that will benefit from more careful considerations and addition of control experiments. 

Regarding the interpretation of social behavior data, as isolated mice show increased social interaction during direct social interaction, but not during 3 chamber sociability assay, it is informative to more thoroughly explore why isolated mice show different outcomes between these two tests. To better understand the cause of increased direct social interaction (social craving vs aggression), it would be informative to conduct additional behavioral testings from isolated mice (e.g. social CPP, aggression assay). In addition, more discussion on possible factors that could influence the long-lasting effects of adolescent social isolation on adult social behavior is helpful as previous studies found juvenile social isolation rather leads to reduced sociability, and reduced active approach in free direct interaction. 

Regarding other concerns, the current manuscript did not test whether the deprivation induced synaptic scaling changes reported in Fig 5 only emerges slowly in the adult, or it is already acutely happening at the end of isolation at p35. This information would help to interpret whether the synaptic changes are due to chronic adaptation uniquely happening slowly or not. It is also unclear how oxytocin neuron activity contributes to synaptic plasticity onto VTA-NAc projection. One possibility is that oxytocin expressing PVN neurons projecting to VTA themselves release glutamate and contribute to synaptic plasticity. It would be informative to optogenetically examine the synaptic connectivity between PVN->VTA projections and VTA->NAc projection neurons in isolated mice. Finally, there are some concerns of the selection of statistical testings, and data presentation. 

Overall, this study revealed novel contributions of oxytocin PNV neurons in regulating adolescent isolation induced changes in VTA dopamine neurons to impact social behavior. More thorough interpretation of social behavior data and control experiments related to synaptic plasticity in VTA would help better understand the interesting findings made in this study.

Reviewer #1 (Public Review):

This study set to test the hypothesis that alleles of paired NLRs Pik-1 and Pik2 have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors. They show that co-expression of Pikm-1 and Pikp-2 allowed weaker HR in response to Avr proteins compared to co-expression of Pikm-1 and Pikm-2, and this is attributed to a single amino acid residue at 230 in Pik-2. Most interestingly, they found that co-expression of Pikp-1 and Pikm-2 led to Avr-independent HR and this is also determined by polymorphism at residue 230. This HR requires Pikm-2 P-loop and MHD domains, which are known to be required for Pik-2 function. The authors reconstructed phylogenetic tree to trace the evolutionary history of the polymorphism of residue 230. The data showed that Gly230 is the ancestral residue and Pik-2 carrying this residue is functional in working with Pik-1 for Avr-D recognition, whereas Asp230 (Pikp-2) arose from Gly230. Glu230 (Pikm-2) likely resulted from a further mutation from Asp230. The authors further provided evidence that, while both matching and mismatching alleles of Pik-1 and Pik-2 can generally interact, there seems to be a preference for matching pairs, supporting the possibility for the co-evolution of Pik-1 and Pik-2. Other interesting results include greater accumulation of Pik-2 protein associated with autoimmunity. Overall, the study provides insight into the co-evolution of paired NLRs which has important implications in hybrid necrosis. However, the work could be further strengthened if the author could address the following issues:

1. The study relies solely on transient expression in Nb plants. It will be more convincing if the authors could show whether combination of Pikp-1 and Pikm-2 in rice by either crossing or transgenics leads to autoimmunity.

2. The observation that autoimmunity caused by Pikp-1 and Pikm-2 can be strengthened by extending to additional alleles. How general is this phenomenon? Do other combinations of mismatched pairs also show autoimmunity?

3. Figure 2A, Pikp-2D230E showed stronger HR compared to Pikm-2 when co-expressed with Pikm-1 and Avrs. What about co-exressing Pikp-2D230E and Pikm-1 without Avr? Does it show autoimmunity? Why is it stronger than Pikm-2?

4. Figure S5A, why Pikp-2T434S showed weaker HR compared to Pikp-2 in Figure 1A? It is necessary to compare Pikp-2 and Pikp-2T434S side-by-side.

5. Is the autoactivation associated with oligomerization? A blue-native gel assay would do.

6. It needs to be cautious to draw conclusion from the competition experiments where different ODs are compared, as these may not guarantee correlation with protein concentrations. For example, in Figure 9C, a OD of 0.1 gave stronger Pikp-2 band in co-IP compared to higher ODs.

Reviewer #2 (Public Review):

De la Concepcion and colleagues present the work investigating the mode of co-evolution in the rice NLR immune receptor pair, Pik-1 and Pik-2 using two different matching allelic series of Pikm and Pikp. The Pik NLR pair system has been extensively studied with the integrated domain (ID) HMA in the sensor Pik-1; its effector binding properties through the ID have been rigorously studied with structure determination as well as functional reconstitution of evolutionary changes in recent years by the same group of authors. The body of work on the Pik NLR pair clearly suggests that the sensor-helper pair must have handled the diversifying selection pressure mostly imposed on HMA while ensuring the operating system to fine-tune immune responses within the pair system. The authors tackled this issue of "allelic specialization within the pair" using the cell death assay system in the heterologous N. benthamiana with extensive efforts to quantify the cell death readout as a proxy of immunity signaling. The main finding on the mismatched NLR pair triggering autoimmunity in the N. ben system highlights that the helper NLR Pik-2 has evolved the fine-tuning switch at a single amino acid along with diversifying HMA domain in the sensor. Using a series of biochemical assays and competition assays, the authors nicely demonstrated molecular details in the Pik-autoimmune signaling complex, revealing preferential association between the co-evolved partners and contribution of P-loop/MHD motifs in immune signaling. They also emphasized the fact that the NLR pairs (different allelic combinations) exist in an oligomeric status in the plant cell regardless of signaling status, which is consistent with previous findings on certain NLRs. With current structure determination of resistosomes, the NLR research field tends to simplify the NLR activation model from a single molecule representing a pre-activation state to the final signaling complex as a fully activated status. In that sense, this work readdressing pre-active NLR assembly adds a value to the field to elaborate NLR activation model. The clever use of an autoimmune NLR heterocomplex is expected to further advance our understanding on successive processes of NLR immune complex activation.

This work is inspiring in many ways. First, the work confirms the notion that autoimmunity is a general phenomenon when an evolutionarily elaborated NLR network is disturbed. From hybrid necrosis cases, we learned that a mismatch of independently evolved NLRs can result in autoimmunity. Here, they nicely demonstrated that the same outcome occurs when the most elaborately and forcefully (by tight linkage) co-evolved NLR pair are juxtaposed as mismatched. Second, this bidirectional confirmation of autoimmunity has a huge implication in our breeding practices and how we can further engineer disease resistance. Many groups are already trying out to engineer ID and paired NLRs to enhance resistance traits, however, without understanding exact mechanisms of NLR pair activation, presumably hetero-NLR complex with distinct stoichiometry, the engineering would not be within a reach. For example, merely replacing an ID without considering the context of sensor-helper co-evolved modules may not result in a fruitful outcome. This work nicely addressed this issue with detailed biochemical analyses and hinted at the importance of the helper competence, suggesting variations on a "resistosome" theme, on which the field might have been too simplifying. Future work on structural determination of such autoimmune complexes will be an interesting avenue to follow.

Reviewer #3 (Public Review):

The molecular basis of co-adaptation and specialization mechanism of paired plant NLRs remains unclear. The authors examined allelic diversification of the rice NLR pair called Pik and found a single amino acid polymorphism in the helper Pik NLR that plays a role in the Pik pair function.

It is intriguing that the single amino acid polymorphism at the residue 230 has a striking impact on the Pik pair function despite their similar biochemical properties (Asp vs Glu). The results of the cell death assay and Co-IP experiments in N. benthamiana well supports the importance of the residue 230 of helper Pik. However, it is unclear how the polymorphic residue regulates the mode of action of Pik pair upon AVR perception. Also, I'm wondering how this finding is applicable to understand mode of actions of other NLR pairs.

Reviewer #1 (Public Review):

The authors of this manuscript present data suggesting that octopamine signals from neurons arising in the mesometathoracic ganglion, through cognate receptors on flight muscles that they innervate, drive thermogenesis in honey bees. They employ temperature measurements on castes of honey bees where octopamine function is pharmacologically inhibited or stimulated in with the appropriate controls in addition to showing the presence of Octopamine and its receptors in relevant tissue. They make a connection of these manipulations to possible downstream metabolic events through measured alterations in flight muscle cAMP levels. In all, these findings are very interesting and noteworthy from the standpoint of physiology.

The data presented in this study suggest that Octopamine signals to flight muscles contribute to thermogenesis. The downstream metabolic pathways that might underly this correlation is a "hypothetical cascade" albeit with support from studies in other species of insects. Results from testing these biochemical hypotheses may greatly substantiate this study.

Reviewer #2 (Public Review):

The authors have tested the hypothesis that the biogenic amine octopamine is of major importance for shivering thermogenesis in bees. This hypothesis is based on previous studies in which octopamine was correlated on the one hand with the control of muscle activity in insects and on the other hand with the known functions of noradrenaline in the context of thermogenesis in mammals. They could show that octopamine is indeed found in the relevant flight muscles responsible for shivering thermogenesis and that the amount of this hormone increases with age. Anatomical studies could directly show that octopaminergic neurons indeed innervate these muscle groups. With the help of pharmacological studies, they finally succeeded in demonstrating the importance of octopamine and selected octopamine receptors for thermogenesis.

I consider the investigations of Kaya-Zeeb and colleagues to be very interesting and relevant in principle, as they were able to elucidate an essential aspect of bee life in this way. In most aspects, the manuscript is well written and the experiments are comprehensible. The experiments are mostly adequately and competently performed.

Reviewer #1 (Public Review): 

This study addresses how spontaneous activity in the developing cochlea impacts the development of auditory response in the MTNB of the brain stem. To this end the authors make a targeted KO mouse in which the calcium activated chloride channel is knocked out in the developing cochlea. They showed that the targeted KO greatly reduces spontaneous activity, recapitulating what was show from Wang et al in the whole animal KO of the same channel. They show this reduction of activity is observed in the ex vivo cochlea leads to a change in the bursting activity in the MNTB in vivo, though units recorded exhibited many different firing patterns. They conclude that the change in the organization of the bursting activity leads to a reduction in refinement of spiral ganglion neurons to their target in the MTNB based on two measurements, 1) a reduction in frequency selectivity of MNTB neurons and 2) an expansion In the number and spatial distribution of glutamatergic inputs from MNTB to LSO neurons. 

The manuscript has many strengths - it is addressing an extremely interesting question as to how disrupting activity in the cochlea prior to hearing impacts the developing auditory circuits in the brain stem. Moreover, the targeted knockout approach removed difficulty associated with expression of this channel throughout the brain. However, the approaches used by authors are limited in their interpretation and therefore it is difficult to assign the changes they observed to a lack of refinement.

Reviewer #2 (Public Review): 

In this study, Maul and colleagues examined the impact of the spontaneous activity within the developing cochlea on the refinement of the medial nucleus of the trapezoid body (MNTB) and lateral superior olive (LSO) connectivity. Using the TMEM16A knock-out mouse, in which the spontaneous activity leading to the excitement of the cochlear sensory cells is strongly diminished, the authors demonstrate the frequency-coding within the medial nucleus of the trapezoid body (MNTB) is degraded in the TMEM16A KO mouse. Then, they show an increase in the MNTB- LSO projections. Interestingly, they find-out that both high- and low-frequency regions in the MNTB project onto LSO neurons suggesting a failure in the tonotopic refinement. Thus, the authors conclude that the spontaneous activity within the immature cochlea is essential for a proper maturation within the higher auditory centers. 

Although it was known that the deletion of TMEM16A led to a reduced activity of the cochlear epithelium before the onset of hearing, the authors took care to repeat part of these experiments. Next, the analysis of the receptive field is pretty straightforward to appreciate the frequency-selectivity coding of the MNTB neurons. Finally, the functional mapping of the MNTB-LSO projections is very elegant. In addition, the authors examined the functional state of the cochlea of the TMEM16A KO mouse as well as the TMEM16A expression along the ascending auditory pathway to link the central auditory neurons maturation to the sole defect in the developing cochlea. 

Thus, this study is an important contribution to the field on the input from periphery in the neural circuitry establishment and function.

Reviewer #3 (Public Review): 

Strengths of the paper include the use of complementary techniques to characterize both peripheral and central defects in TMEM16A knockout mice. They use calcium imaging in excised cochlear preparations to demonstrate the altered patterns of calcium waves in TMEM16A knockout mice, which are far smaller and faster than in control animals, and less frequent. Tests of auditory function (ABRs) in normal animals demonstrate overall normal hearing thresholds and auditory nerve function. They use in vivo recordings from medial nucleus of the trapezoid body (MNTB) neurons identified by their characteristic pre-spike to show altered patterns of spiking in developing MNTB neurons, with a smaller coefficient of variation that indicates less bursting activity. In hearing MNTB neurons, TMEM16A mutants have a higher sound threshold, and slightly wider tuning curves. They then use in vitro recordings from neurons of the lateral superior olive (LSO) which receive tonotopically refined inhibitory synaptic inputs from MNTB neurons to compute horizontal sound localization paired with glutamate uncaging activation of pre-synaptic MNTB neurons to demonstrate that single LSO neurons receive inputs from a larger region of the MNTB in TMEM16A mutants compared to control animals. This further indicates aberrant refinement of tonotopically distributed neurons in the ascending auditory system. 

There are a few weaknesses in interpretation of the data. There is an unsupported claim that TMEM16A is upstream of ATP release from inner supporting cells, the opposite of which has been proposed by other groups. The authors did not adequately discuss whether TMEM16A may influence other parts of the sound localization circuitry besides cochlear supporting cells. The alterations in frequency selectivity of MNTB neurons in TMEM16A mutants seems too subtle to account for the broader deficits in frequency-specific inputs to LSO neurons, which could be discussed. Finally, the authors did not adequately discuss how their present work fits in with previous work using extremely similar techniques in a different knockout animal that also implicated patterns of spontaneous activity in the cochlea in tonotopic refinement of ascending auditory projections (Clause et al 2014). The authors should compare the patterns of aberrant bursting measured in MNTB neurons in TMEM16A mutants to those measured in alpha9 AChR knockout animals used in the Clause et al 2014 paper. Then, they should clearly state what their work adds to the existing literature, namely that while other papers have linked the bursting patterns of the ascending auditory system with tonotopic refinement of MNTB projections to the LSO, this work more clearly links an earlier step in the process, the spreading calcium waves in developing supporting cells, with refinement of ascending circuits.

Reviewer #1 (Public Review): 

The major strength of the work is that the procedures are enabling, robust, well-characterized, and described in detail. The expansion and staining procedures are therefore likely to be readily adopted by the community. Unlike advances in microscopy hardware that require purchasing new equipment or having specialized expertise for building instrumentation, improvements in sample preparation can be performed for low cost with relatively easily learned skills. So, although the fine tuning of a hydrogel recipe may appear at first as a simple optimization, the end result is a substantial breakthrough that will be useful to the community. 

The simple but practical gel deformation methodology for assessing hydrogel sturdiness was executed well. I agree with the authors that although more advanced methodology could be appropriate in a materials science or polymer physics context, the tilted gel methodology was sufficient to achieve the authors' aims and is simple enough that other researchers can easily adopt it when troubleshooting TREx or possibly when adapting the TREx for new applications. 

It is much appreciated that the authors included validation of their new gel recipes using both pre-expansion / post-expansion distortion analysis as well as distance measurements of nuclear pore complexes(NPC) that have a well known size (Fig. 3). The distortion analysis in Figure 3 shows very strong performance with ~3% distortion over a range of length scales. The comparison of NPC diameter to prior published results is good but leaves some room for future improvement and suggests that the local / nanoscale expansion may be smaller for tight complexes than for the overall gel. 

The imaging demonstrations in cell and tissue labeling were convincing. The experiments with amine-reactive dyes and membrane-labeling stains were good to see in combination with TREx and help illustrate the potential of the method.

Reviewer #2 (Public Review): 

In this study, the authors developed a new expansion microscopy (ExM) method called Ten-fold Robust Expansion Microscopy (TREx). This method emphasizes one-round sample expansion of cells by systematically optimizing the monomer recipe. Compared to existing ExM methods which expand samples to similar scale (~ 10 folds), TREx aims for a robust procedure that can be handled more easily. The reviewer experimentally tested the TREx protocol, and validated the TREx 10x gel can be made robustly by researchers who have experience with standard ExM. 

Specific comments: 

1) The authors claimed in the abstract that "TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes". The authors only demonstrated one NHS ester dye, BODIPY-FL NHS dye (lined 151-159) without justification why this dye was selected. Does BODIPY-FL NHS dye work better than other off-the-shelf NHS dyes? The reviewer recommends the authors to validate a few more widely used dyes with TREx, e.g. Cy3/Cy5, Alexa 488, Alexa 568, to guide the readers to choose the appropriate dyes. 

2) Page 8: The reviewer is happy to see the discussion on the heterogeneous local expansion factors in cells. It is critical for evaluating the expansion isotropy and avoid pitfalls in the applications of TREx. Based on this work and previous work (e.g. U-ExM), organelles with higher protein density may have smaller local expansion factors than the macroscopic expansion factor. The authors discussed the local expansion factor of organelles with different protein density, including centrioles, NPCs, and microtubules. To evaluate the local expansion factors comprehensively, the reviewer asks the authors to add a figure or plot to compare the local expansion factors of different organelles, ideally including centrioles, NPCs, microtubules, clathrin-coated pits, mitochondria, ER, and centromere. The authors have already measured or imaged many of these organelles. For the other organelles, good antibodies are available. Therefore, the additional experiments should be straightforward for the authors. But the comprehensive comparison will make the work much more impactful. 

3) Line 388: The authors stated "The strong overlap between NHS ester and mCLING stains was not unexpected, given the reactivity of NHS esters towards both unreacted lysines in the mCLING molecule and antibodies." Since AcX (6-((acryloyl)amino)hexanoic Acid, Succinimidyl Ester) at high concentration was added after the mCLING staining, most of the lysines in the mCLING should be reacted by the AcX. Therefore, NHS ester dye staining should not strongly overlap with the mCLING. The authors should re-evaluate and interpret the overlap. The authors can do simple experiments like increasing the concentration of AcX, or use pH 8 for AcX treatment. If the overlap is reduced, it means the overlap was caused by the unreacted lysines in mCLING, and can be reduced. If the overlap is not reduced, there are other mechanisms which need further examination or interpretation.

Reviewer #3 (Public Review): 

Fluorescence imaging has been limited for decades by the diffraction limit. This was first eluded by the invention of different optical methods (e.g. STED, STORM, PAINT), which, however, typically require expensive instrumentation and excellent knowledge of optics. Expansion microscopy, introduced in 2015 by the Boyden laboratory (including the last author of the current manuscript) offers an alternative to this approach, by expanding the sample and then imaging it with conventional optics. 

The main strength of the manuscript is that it presents an easily-applied technology for 10-fold expansion, which seems to work both in tissues and in cell cultures. At the same time, a number of problems are apparent with the technique presented. One of the main points of the presented approach (TREx) is to reduce the gel crosslinker concentration by more than 10-fold, compared to the original expansion microscopy approach of the Boyden laboratory. This may result in expansion errors that still need to be fully explored.

Reviewer #1 (Public Review):

Sherrard and colleagues investigated the dynamics of stress fibers of the follicular epithelia during the migratory stages of egg chamber development. During the migratory stages of egg chamber development the follicular epithelium forms actin-based protrusions at the leading edge followed by a parallel array of stress fibers. As the follicular epithelia migrates along the basement membrane the encapsulates the egg chamber, the egg chamber rotates. At later stages of development the follicle cells stop migrating and the stress fibers are then used to form a contractile network which needed to help make the elongated shape of the egg. Using near total internal reflection microscopy, Sherrard and colleagues show that during this migratory period these stress fibers showed tread-milling behavior (growing in the front and shrinking in the back) and persisted for longer than the time for cell to travel at least one cell length (62%). Concomitant with these treadmilling stress fibers was the appearance of adhesions at the front of these stress fibers and their disappearance at the rear. This means that rather than just observing adhesions at the termini of the stress fibers they can be found all along their lengths. These dynamics were captured by the use of fluorescently tagged adhesion proteins (Paxillin,Talin) but could also been visualized by staining for endogenous proteins (betaPS integrin). The authors go on to correlate the treadmilling stress fibers with adhesion dynamics and observed that new adhesions are pulled reward, consistent with their maturing under tension. Blocking cell migration (with CK-666 or genetically with depletion of Abi) resulted in a conversion of these migratory (modular), tread-milling stress fibers to the conical stress fibers with adhesions on either termini rather than along the length. The authors go on to demonstrate the modular migratory stress fibers are dependent on the formin, DAAM, as depletion of this formin specifically led to 30% reduction in F-actin levels without affecting other actin-based structures. Furthermore, as the migratory phase of these follicle cells subsides, DAAM expression decreases with the more canonical stress fibers being dependent on the formin Dia.

Strengths:

The claims of the manuscript are well supported by the data presented. The analyses rely on the dynamics of stress fibers being observed in the context of organism in vivo. This is in contrast to other studies where stress fiber dynamics have been limited to tissue culture cells in 2D on a single extracellular matrix protein. The finding that stress fibers a) treadmill in this context and b) form adhesions along their length and not just their termini will likely have a big impact on our understanding of cell migration and the role of stress fibers in general. This study also elegantly capitalizes on Drosophila genetics in order to identify which formins involved in stress fiber formation and dynamics and clearly shows that there is developmental shift from DAAM to Dia as the follicular epithelium stops migrating. They also show that modular stress fibers are also dependent on behavior, and when cell migration is inhibited there is shift to canonical stress fibers, thus modular stress fiber formation is both DAAM-dependent and dependent on migration.

Weaknesses:

The 30% reduction in F-actin in the DAAM amorphic allele does suggest that there is something else contributing to the formation of modular stress fibers which is not surprising but could also be more thoroughly addressed in the manuscript. While the authors went through great lengths to quantify their observations. Many of their claims are based on qualitative observations in particular the descriptions of adhesion dynamics when correlated to modular stress fiber dynamics (Figure 4) and the shift from modular stress fibers to canonical stress fibers (Figure 5). The near total internal reflection microscopy offers them an opportunity to derive rate constants to focal adhesion dynamics, and quantify the change in stress fiber type would have strengthen the manuscript.

Reviewer #2 (Public Review):

In the present study, Sherrard et al. analyze the collective cell migration in Drosophila ovary follicle cells. The major new findings are that stress fibers show a novel form treadmilling behaviour during the migration and that the formin DAAM is required for the assembly of these stress fibers.

The authors describe the mechanism of stress fiber treadmilling and the remodeling of adhesions, in vivo, in great detail and find that is exhibits some unique features compared to cell culture models. The present study is one of the first studies addressing stress fibers in vivo in collective cell migration. Overall, this is a very well written manuscript featuring a well thought out, complete and exciting story.

There are three main novel contributions made in this beautiful study: First it is one of very few examples of studies of stress fibers in an in vivo context. Applying the wealth of knowledge about stress fibers from cell culture models requires us to gain better understanding of how they behave in the context of the whole organism. The present study does exactly that. Second, there are not many examples of studies that specifically seek to understand stress fiber behaviour during collective cell migration. Third, it combines live imaging of stress fiber with the genetic toolkit available in Drosophila, in the process identifying the formin DAAM as an important regulator of stress fiber assembly, and identifying a novel form of treadmilling. The sort of approach used in this manuscript, combining organ culture, quantitative live imaging, and genetic manipulation pushes the field forward in new directions.

This study is largely descriptive, and in that is self-contained and does not require many additional experiments. Although "descriptive" is an adjective that is typically used to diminish the importance of a paper this is absolutely not the case here. The novelty and contribution of this manuscript lies in the powerful insights derived from a high quality, genetics based, quantitative description of the behaviour of stress fibers. Although stress fibers have been widely studied in cell culture models the relation between these studies and what is actually seen in intact whole animals remains to be established. The present manuscript goes a long way towards this goal.

Reviewer #1 (Public Review):

Munc13 is a key regulator of synaptic vesicle (SV) fusion that is thought to mediate SV tethering and regulate SNARE assembly. Based primarily on Munc13 crystal structures, the authors design a set of four charge reversal mutations in the C1C2B region that are predicted to affect the interaction of Munc13 with the plasma membrane (PM). Various in vivo and in vitro consequences of these mutations are studied, leading to two main conclusions: (1) an interaction between the PM and a polybasic surface of Munc13 is likely important for SV tethering, and (2) two residues in the Ca2+-binding loops of the C2B domain are important for SV fusion.

So far, so good - I think the data strongly support the two main conclusions noted above. It is less clear that these studies support (or could falsify) the main hypothesis, stated in the title, that re-orientation of membrane-bound Munc13 controls neurotransmitter release. Primed vesicles appear to exist in dynamic equilibrium between two states, one of them "loosely" primed (LS) and the other "tightly" primed (TS). Inasmuch as this simple model is correct, one could characterize the various players - SNAREs, synaptotagmin, complexin and of course Munc13 - in terms of their ability to influence the LS/TS equilibrium, perhaps in response to Ca2+ or other small molecules. This manuscript postulates that the orientation of Munc13 relative to the membrane has a major impact on the LS/TS equilibrium, with a perpendicular orientation favoring LS and a slanted orientation favoring TS.

The authors' previous structure (Xu et al., 2017) suggested that two partially-discrete faces of C1C2B, one polybasic and the other centered around the Ca2+-binding loops of C2B, are likely involved in PM binding. In that paper they hypothesized that the polybasic face would dominate in the absence of Ca2+ whereas the 'Ca2+-binding face' [not a very good name, but the authors haven't suggested a better one] would dominate in the presence of Ca2+. Binding to the PM via the polybasic face would yield a more erect or 'perpendicular' binding orientation, whereas binding to the PM by the Ca2+-binding face would yield a more tilted or 'slanted' binding orientation.

Here the authors performed two molecular dynamics simulations, one without and one with bound Ca2+. In the Results section, they correctly point out that their findings cannot be used to support their hypothesis because, in each case, Munc13 was placed in the hypothesized orientation - perpendicular for minus Ca2+, slanted for plus Ca2+ - at the beginning of the simulation. In the Discussion however the authors argue that the MD simulations support their model. I disagree because the simulations needed to falsify the model have not yet been conducted. In addition, an opportunity was seemingly missed by not doing MD simulations on the mutants.

Of the four mutations studied, two (K603E and K720E) should specifically destabilize PM binding by the polybasic face, one (K706E) should destabilize binding by the Ca2+-binding face, and one (R769E) is expected to destabilize both. Two of the mutants (K603E and R769E) in fact abrogate priming. This result, along with biochemical experiments, implicates the polybasic face in SV tethering and thus represents the main evidence supporting the first of the main conclusions (see Evaluation Summary above). However, since an unprimed vesicle does not participate in the LS/TS equilibrium, these mutants are in this respect uninformative. Only the remaining mutants, K720E and K706E, would therefore appear to have the potential of yielding information about the LS/TS equilibrium and its relationship to Munc13 orientation.

Both K720E and K706E support normal priming but have opposite effects on vesicular release probability and evoked release. These results can be rationalized in terms of an LS/TS equilibrium. The K720E mutation, which selectively destabilizes binding by the polybasic face, would shift the equilibrium toward TS and thereby increase the release probability. Conversely the K706E mutation, which destabilizes binding by the Ca2+-binding face, would shift the equilibrium toward LS and thereby reduce the release probability.

However, the authors themselves cast serious doubt on this straightforward interpretation. In the case of K720E, they point out that the other 'polybasic mutant', K603E, has no effect of release probability. (I argued above that, perhaps, K603E is best viewed as uninformative about the LS/TS equilibrium owing to its strong upstream priming defect.) In the case of K706E, the authors point out that phorbol ester potentiation was similar for K706E and wild-type, suggesting to them "that the effects of the K706E mutation might not be related to the transition to slanted orientations but rather to another mechanism that directly influences fusion. For instance, the Munc13-1 C2B domain might cause membrane perturbations analogous to those that are believed to underlie the function of the Syt1 C2 domains in triggering release (Fernandez-Chacon et al., 2001; Rhee et al., 2005). It is also possible that the phenotypes caused by the K706E mutation and other mutations studied here reflect effects of Munc13-1 in more than one step leading to release, which complicates the interpretation of the data." If this is indeed the case, we are down to one mutant - K720E - that can be informative about the LS/TS equilibrium. (For the most part, I did not find the double and quadruple mutants informative, especially because each of them contains at least one mutation that strongly abrogates priming.)

It looks like K720E is right in the center of the polybasic surface (although it's hard to tell from a single 'projection' image) so it would have been expected to impair Ca2+-independent liposome binding, and it does. However the liposome clustering effects are very weird, displaying a much broader distribution than any other experiment, an observation which the authors disregard. However, overall, I would say that the authors' K720E findings offer modest support for their overall main hypothesis. But for me it's not enough to justify making that hypothesis the title of the paper.<br> For the most part I could not follow the discussion of figures 4 and 5. But I am struck by strong similarity between the data for K603E and K706E (comparing Fig. 4B/C to Fig. 4H/I). How can these results be reconciled with the opposite roles predicted for K603 and K706?

I'm not sure how the results of the PDBu experiments contribute to the conclusion that "two faces of the C1-C2B region are critical for Munc13-1-dependent short-term plasticity" (p. 15), since the only mutant that selectively affects one of the faces, K706E, has no impact (Fig. 6).

Why are the liposome-binding assays in Fig. 7 done with C2C present - isn't that just a confounding factor? And if Ca2+-independent binding by C2C is as weak as suggested by the results in Fig. 7, how do any of the Munc13 constructs cluster liposomes in Fig. 8? (Note that, according to my reading of the methods, V-type liposomes are simply T-type liposomes without the DAG and PIP2.)

What is the basis for the claim (p. 22) that "the perpendicular orientation of Munc13-1 is expected to facilitate initiation of SNARE core complex assembly"?

Reviewer #2 (Public Review):

In this manuscript, Rosenmund and colleagues describe new results regarding the mode of action of Munc13 in neurotransmitter release.<br> Based on molecular dynamics simulations of Munc13 (C1C2BMun) with phospholipid membranes, the authors selected promising point mutations and comparatively investigated their functional impact with electrophysiological experiments in hippocampal neurons and with a variety of in vitro experiments (lipid binding assay, liposome clustering and fusion). The results show that specific mutations in the C1C2B-domain (also referred to as polybasic face) of Munc13 (K603E, R769E) strongly inhibit vesicle priming, a property that correlates well with their re duced ability to bind to phospholipid membranes in a calcium-independent manner.

The manuscript describes comprehensive electrophysiological and biochemical experiments that are complemented and extended by thoughtful analyses. The direct combination of electrophysiological and biochemical expertise from the Rosenmund and Rizo laboratories, respectively, represents a particular strength of this study, allowing the authors to develop new insights into the function of the Munc13 protein. A welcome (but not necessary) extension of the data presented would be the demonstration that the mutants in question (K603E, R769E) also show altered phospholipid binding in the MD simulations. In any case, the presentation of the data is clear and the authors' conclusions are convincing.

Taken together, the manuscript and the results represent a significant advance in the understanding of the molecular mechanisms underlying synaptic vesicle priming.

Reviewer #3 (Public Review):

Camacho et al. have tested a model to understand the mechanism by which the active zone protein Munc-13 operates in synaptic vesicle priming and Ca2+-dependent neurotransmitter release. Their model is based on the crystal structure -previously solved by authors- of a major fragment of Munc-13-1, including the C1, C2B and MUN domains and the C-terminal C2C domains (C1C2BMUNC2C fragment) but missing the N-terminal part of the protein. An insightful molecular dynamic analysis developed to investigate the interaction of the C1C2B segment with the plasma membrane has led to refine the model and to design mutations of specific residues to test and to validate the model experimentally. A tenet of the proposed mechanism is that Munc-13-1 bridges the synaptic vesicle (SV) to the plasma membrane. The major conclusion is that Munc-13-1 adopts two conformations: (a) a conformation perpendicular to the synaptic vesicle, which is Ca2+-independent, it involves the interaction of negatively charged plasma membrane phospholipids with basic residues (K603, at C1 domain; R769, at C2B domain Ca2+-binding loop) and it is essential for SV priming and (b) a slanted conformation that occurs when Ca2+ binds to the C2B domain and it is key to progress toward the full assembly of the SNARE complex previous to neurotransmitter release. This model is well supported by studies based on the behavior of different versions of Munc-13-1 in which basic residues have been mutated to a negatively charged residue (specially K603E and R769E) to impair membrane binding. These studies include liposome interaction and fusion assays and electrophysiological analysis of synapses (measurements of the readily releasable pool (RRP), vesicular release probability and short-term synaptic plasticity) in Munc-13-1/2 DKO mouse hippocampal neurons in which either Munc-13 WT or mutant versions have been re-introduced. The results obtained based on the analysis of the K603 and R769E are clear, supports the conclusions and clearly advances the knowledge regarding the details of how Munc-13-1 mediates SV priming and Ca2+-dependent full assembly of SNARE complex.

On the other hand, the results pertaining some aspects of the contribution of other residues investigated in the study (K720 and K706, at the C2 domain Ca2+-binding loops) are less obvious to fit within a rather simple model but they open interesting perspectives for future investigations. The residue K720 seems not to be a critic residue for Ca2+-independent interaction with the plasma membrane, the mutation K720E does not interfere with priming and, surprisingly, it increases Ca2+-dependent release. Consistently with the model, the K706E mutation did not affect SV priming but it decreased the probability of release. In any case, the authors provided careful, however, open interpretations that are supported by the results obtained.

Joint Public Review:

Aggregates of the protein Tau are key pathological features of Alzheimer's Disease and several other neurodegenerative disorders (Tauopathies). Hori et al. assessed the consequences of acute elevation of soluble Tau protein levels on pre-synaptic function using the Calyx of Held as an experimental model. The experimental approach is highly complex and technically challenging, particularly the direct pre-synaptic recordings with peptide infusion. Based on paired pre-post-synaptic recordings and pre-synaptic peptide infusion, the authors demonstrate that elevated Tau levels inhibit action-potential-evoked synaptic transmission, and pre-synaptic capacitance measurements show that Tau perturbs endocytosis but not pre-synaptic calcium currents, indicating that the depression of exocytic vesicle fusion is primarily a consequence of inhibition of compensatory endocytosis. Further pharmacological perturbation experiments indicate that the effects of elevated Tau levels are likely caused by excessive microtubule polymerization or stability. Finally, evidence is provided, based on co-injection of high concentrations of a peptide derived from the lipid-binding PH domain of Dynamin-1, that Tau-induced microtubule stabilization may cause Dynamin-1 sequestration via direct microtubule binding. On aggregate, the data presented in this study support a model according to which soluble Tau impairs pre-synaptic endocytosis and neurotransmission in Alzheimer's Disease and related disorders by sequestering Dynamin-1 on microtubules.

Key Strength of the Paper

The experiments involved extremely difficult but highly informative patch-clamp electrophysiology techniques in combination with quantitative measurements to generate a highly rigorous data set.

Key Weakness of the Paper

The molecular and pharmacological perturbation experiments designed to elucidate the mechanism by which elevated Tau levels causes pre-synaptic defects are interesting and informative, but they not yet sufficient to support the firm conclusion that Tau-induced microtubule stabilization and the consequent Dynamin-1 sequestration via direct microtubule binding is at the basis of the Tau-induced synaptic defect.

Reviewer #1 (Public Review): 

Yang. et al. use computational modeling to explore how neurons can co-regulate different properties (firing rate, excitability thresholds, energy consumption) by adjusting ion channel expression. To do so, they rely on the activity-dependent channel expression model introduced in O'Leary et al. 2014 and assume that any regulation loop can be boiled down to such a model. They thus propose a parallel feedback loop regulation model, in which each loop regulates a property by chasing its target value and regulating ion channel expression according to an integral control law. 

The authors start by proving experimentally and computationally ion channel pleiotropy. This preliminary analysis is clearly developed and confirms/rediscovers in CA1 neurons known facts originally observed in invertebrate systems like the STG. They subsequently use their regulation model to provide elegant geometric explanations for the emergence of ion channels correlations and for the success or failure of homeostatic regulation, as a function of the number of regulated properties and the number of ion channels. 

The model they rely on exhibits two basic characteristics. Suppose the model possesses N different types of ion channels. 

1. As in the model in O'Leary et al. 2014, when taken in isolation, the regulator of each neural property possesses an N-1 dimensional submanifold of steady-states in the N-dimensional maximal conductance space where the target for the regulated property is reached. 

2. Regulation loops are independent of each other. 

Given these two characteristics, in the presence of M regulated neural properties, the regulation target is reached simultaneously for each of them on an (N-M)-dimensional joint target submanifold of homeostatic steady states, i.e., the intersection of M (N-1)-dimensional target submanifolds. Depending on initial conditions, homeostatically regulated maximal conductances will spread out along this submanifold, thus creating N-M dimensional correlations. This (mathematically) elementary observation leads to the various general conclusions of the paper, the main of which are i) that increasing the number of regulated properties increase ion channel correlations (by reducing the dimension of the joint target submanifold) and ii) that increasing the number of regulated properties makes homeostatic regulation more likely to fail (when the intersection between the target submanifolds is empty). This simple geometric view on multi-property regulation is neat and, most importantly, experimentally verifiable/falsifiable. 

The main drawback of this approach is that characteristics 1 and 2 (above) of the model used in the paper are non-generic and makes the model a useful but maybe oversimplified (and fragile) testbed. Let me develop. 

Property 1 reflects one of the main limitations of the model in O'Leary et al. 2014, namely, that this model provides biologically meaningful results and predictions only when initial conditions are small and in the absence of any disturbance to its regulation dynamics (including the presence of multiple master regulators). This follows exactly from the fact that, in that model, the homeostatic regulator has zero effect once the state reaches the N-1 dimensional submanifold of target steady states. Thus, different initial conditions or exogenous disturbances will arbitrarily spread conductances on this submanifold, making it unrealistically unrobust to both types of perturbations. This limitation was overcome in a simple, biologically plausible way in Franci et al. 2020 by adding a molecular regulatory network between the homeostatic sensor and the regulated conductances. The revised model still exhibits the same correlated variability between maximal conductances as the original model. But only in the revised model the correlation ray exhibits biologically-meaningful levels of robustness both to disturbances and initial conditions. In dynamical systems terminology, the model in O'Leary et al. 2014 is structurally unstable (non generic) whereas its 2020 revision is structurally stable (generic). Crucially, in the 2020 model the homeostatic target is approximately reached at a unique steady state (i.e., the target submanifold is zero-dimensional) but the presence of a slow direction v_slow in the regulation space amplifies heterogeneities and disturbances, which leads to correlated variability (along v_slow). 

Characteristic 2 of the model used in this paper is also non-generic. Consider the simple homeostatic parallel control scheme, 

output = x+y 

tau_x*dx/dt = tgt - (x+y) 

tau_y*dy/dt = tgt - (x+y) 

which (in line with the present paper) reaches the desired target output tgt on the 1-dimensional subspace of steady states x+y=tgt. Let's now introduce small coupling between the two variables as follows 

output = x+y 

tau_x*dx/dt = tgt - (x+y) - epsilon*y 

tau_y*dy/dt = tgt - (x+y) - epsilon*x 

where epsilon>0 is small. It is easy to verify that the new model has a unique exponentially stable steady state given by 

x = y; x+y= 2/(2+epsilon)*tgt ~ tgt (for epsilon sufficiently small) 

Thus, introducing an arbitrary small coupling between the two regulation loops is sufficient to change the dimension of the target subspace from 1 to zero (without introducing new regulated properties!) while only leading to a small (O(epsilon)) error in the regulated property. It is also easy to show that the uncoupled/parallel model is not structurally stable, while the weakly coupled model is. 

These observations lead to the question of whether the mathematical/computational results of the paper are realistic or whether they are artifacts of the non-generic modeling assumptions used for the regulation loops.

Reviewer #2 (Public Review): 

Yang, Shakil, Ratté, and Prescott conducted a combined dynamic clamp and modeling exploration on the geometry and dimensionality of single-output and multi-output solution sets embedded in the cellular parameter spaces of single neurons, i.e. CA1 pyramidal neurons for the dynamic clamp studies, and a generic single-compartment model with several varieties of sodium and potassium channels for the computational studies. Both types of neurons were stimulated with a randomly fluctuating current, and output measures (termed 'properties') of the neurons were measured or calculated, including rheobase, firing rate, energy consumption, and energy efficiency per spike. Ion channel maximal conductances were then varied, and the dependence of the output properties on the conductance values and their combinations were explored. 

The authors define as a single-output solution set the subset of maximal conductance space containing conductance combinations that produce a value of one of the output properties within a tolerance range around a target value. These single-output solution sets can take the shape of points, curves, surfaces or volumes in parameter space. A multi-output solution set is then defined as the subset of parameter space that produces values within a tolerance range for multiple properties simultaneously, and thus lies at the intersection of several single-output solution sets (if such an intersection exists). 

A major focus of the work is on the effect of channel pleiotropy - the impact of one ion channel type on more than one cellular output property - on the shape of solution sets, and whether homeostatic regulation schemes that adjust ionic membrane conductances to maintain and restore output properties in a target range can "find" the solution sets, and maintain the neuron within the solution set. The regulation schemes explored in this work directly use error signals of the output properties (the difference between an output property produced by a neuron and its target value) to increase or decrease maximal membrane constants, with pre-assigned regulation time constants. 

The study is systematically executed, and results support the main conclusion, that successful regulation of n independent neuronal output properties requires that at least n ion channel densities be adjustable for a unique solution to exist, and at least n+1 for degenerate solutions. This conclusion explicitly organizes previous results obtained by other modeling studies into a coherent framework, but is not surprising. 

The authors speculate that the need for neurons to regulate several of their output features may have provided the evolutionary drive for the highly diverse sets of voltage-dependencies and kinetics observed in different ion channels in nature. This speculation is intriguing, but also raises the question whether and how the unrealistically simple (and not very diverse) set of model ion channel characteristics used in this work may have impacted the extent and shape of single-property and multi-property solution sets: none of the ion channels in the model appear to have inactivation variables, and the two primarily varied ionic currents, I_Na and I_K, are identical in their voltage-dependence and activation dynamics; they differ only in their reversal potentials. It is likely that channels with such similar characteristics are more able to compensate for each other, and therefore produce more extensive and differently shaped solution sets, than more dissimilar channels. 

Further exploration of the influence of solution set dimensionality on the existence and tightness of linear correlations between pairs of maximal conductances demonstrates that higher-dimensional solution sets, and larger tolerances around output property target values, lead to fewer and weaker correlations. This again is an insight that puts an organizing perspective on previous studies. 

Finally, the paper provides examples of conductance regulation schemes that rely on multiple error signals (deviations of output properties from their respective target values) to differentially regulate different membrane conductances. These schemes are shown to successfully regulate neuron models and allow them to "find" solution sets in many circumstances (if solutions exist). While providing a proof-of-principle that echoes previous work by others, the biological interpretability of these regulation schemes is somewhat limited, because they can not be tied back to how the molecular machinery in a neuron would implement them. For example, it is not clear how a neuron would measure its energy efficiency per spike.

Reviewer #1 (Public Review): 

This manuscript describes a series of behavioral experiments in which foraging rats are subjected to a novel fear conditioning paradigm. Different groups of animals receive a shock to the dorsal surface of the body paired with either tone, an artificial owl driven forward with pneumatic pressure, or a tone/owl combination. An additional control condition pairs tone with owl alone (ie no shock is delivered). In a subsequent test, only owl+shock and tone/owl+shock animals show increased latency to forage and a withdrawal response to tone (even though owl-shock rats do not experience tone during conditioning). The authors conclude that this tone response is due to sensitization and that fear conditioning does not occur in their experimental setup. 

This approach is intriguing and the issues raised by the manuscript are extremely important for the field to consider. However, there are many ways to interpret the results as they stand. One issue of primary importance is whether it can indeed be claimed that conditioning did not readily occur in the tone+shock group. The lack of a particular behavioral conditioned reaction does not equate to an absence of conditioning. It is possible that unseen (i.e. physiological) measures of conditioning, many of which were once standard DVs in the fear conditioning literature, are present in the tone+shock group. This possibility pushes against the claim made in the title and elsewhere. These claims should be softened. 

Because systemic, group-level retreat CRs are not noted in the tone+shock condition, it would indeed be important to establish if there are any experimental circumstances in which tone paired with a US applied to the dorsal surface of the body can produce consistent reactions (e.g. freezing) to tone alone. Though it may seem likely that tone + dorsal shock would indeed produce freezing in a different setting, this result should not be taken for granted - we've known since the 'noisy water' experiment (Garcia & Koelling, 1966) that not every CS pairs with every US and that association can indeed be selective. A positive control would be clarifying. If the authors could demonstrate that tone+dorsal shock produces freezing to tone in a commonly used fear conditioning setup (ie standard cubicle chamber) then the lack of a retreat CR in their naturalistic paradigm would gain added meaning. 

The altered withdrawal trajectory seen in owl+shock and tone/owl+shock groups occurs in neither the tone+shock nor the tone+owl group, introducing the possibility that it results from the specific pairing of owl and shock. Put differently - this response may indeed by an associative CR. Do altered withdrawal angles persist if animals that receive owl+shock are exposed to owl again the next day? Do manipulations of the owl and shock that diminish fear conditioning (e.g. unpairing of owl and shock stimuli) eliminate deflected withdrawal angles when the subject is exposed to owl alone? If so, it would cut against the interpretation that fear conditioning does not occur in the setup described here, and would instead demonstrate that it is indeed central to predatory defense. This interpretation is compatible with the effect of hippocampal lesion on freezing evoked by a live predator. Destruction of the rat hippocampus diminishes cat-evoked freezing - this is thought to occur because the rapid association of the cat's various features with threatening action is not formed by the rat (Fanselow, 2000, 2018). Even though this interpretation of the results differs from the authors', it in no way diminishes the interest of this work. This paradigm may indeed be a novel means by which to study rapidly acquired associations with ethological relevance. Follow-up experiments of the type described above are necessary to disambiguate opposing views of the current dataset.

Reviewer #2 (Public Review): 

This work is dealing with an interesting question whether a simple, one trial CS+US (Pavlovian) association occurs in a naturalistic environment. Pavlovian fear conditioning contains a repetition of a neutral sensory signal (tone, CS) which is paired with a mild US, usually foot-shock (<1 mA; thus, unpleasant rather than painful) and the CS+US association drives associative learning. In this paper, a single 2.5 mA electrical shock was paired with a novel 80 dB tone to monitor the occurrence of learning via measuring success rate and latency of foraging for food. Some animals experienced an owl-looming matched with the US, just before reaching the food. The authors placed hunger-motivated rats into a custom-built arena equipped with safe nest, gate, food zone as well as with a delivery of a self-controlled US (electrical shock in the neck muscle and/or owl-looming). The US was activated by the rats by approaching to the food. Thus, a conflicting situation was provoked where procuring the food is paired with an aversive conditioned signal. Four groups of rats were included in the experiments based on their conditioning types: tone+ shock, tone+ shock+ owl, shock+owl and tone+owl. Due to these conditioning procedures, none of the rat procured the food but fled to the nest. In contrast, in the retrieval phases (next two days), the tone-shock and tone-owl groups successfully procured the pellets but not the tone-shock-owl group during the conditioned tone presentation. Rats in the latter group fled to the nest upon tone presentation at the food zone. As the shock-owl animals (conditioned without tone) also fled to the nest triggered by (unfamiliar) tone presentation, their and the tone+shock+owl group's fled responses were assigned to be non-associative sensitization-like process. Furthermore, during the pre-tone trials, all groups showed similar behavior as in the tone test. These findings led the authors to conclude that classical Pavlovian fear conditioning may not present in an ecologically relevant environment. 

The raised question is relevant for broad audience of neuroscience and behavioral scientist. However, as the used fear conditioning paradigm is not a common one, it is difficult to interpret the finding. It is based on a single pairing of an unfamiliar, salient tone with a very strong (traumatizing?) electrical shock, delivered directly into the neck muscle and an innate signal (owl looming). In addition, as the tone presentation was followed by many events (gate opening, presence of food, shock and/or owl-looming) in front of the animals, it is hard to image what sort of tone association could be formed at all. 

One could also argue that if a hungry animal does not try to collect food after an unpleasant, even a painful experience, then, it normally dies soon (thus, that is not a 'natural' behavior). The tone+shock and tone+owl groups showed similar behavioral features throughout the entire experiments and may reconcile the natural events: although these rats had had negative experience before, were still approaching to food zone due their hunger. Because of their motivation for food, the authors concluded that no association was formed. Based on this single measure, is it right to do so?

Reviewer #3 (Public Review): 

In this study, the authors aimed to test whether rats could be fear conditioned by pairing a subdermal electric shock to a tone, an owl-like approaching stimulus, or a combination of these in a naturalistic-like environment. The authors designed a task in which rats foraging for food were exposed to a tone paired to a shock, an owl-like stimulus, a combination of the owl and the shock, or paired the owl to a shock in a single trial. The authors indexed behaviors related to food approach after conditioning. The authors found that animals exposed to the owl-shock or the tone/owl-shock pairing displayed a higher latency to approach the food reward compared to animals that were presented with the tone-shock or the tone-owl pairing. These results suggest that pairing the owl with the shock was sufficient to induce inhibitory avoidance, whereas a single pairing of the tone-shock or the tone-owl was not. The authors concluded that standard fear conditioning does not readily occur in a naturalistic-like environment and that the inhibitory avoidance induced by the owl-shock pairing could be the result of increased sensitization rather than a fear association. 

Strengths: 

The manuscript is well-written, the behavioral assay is innovative, and the results are interesting. The inclusion of both males and females, and the behavioral sex comparison was commendable. The findings are timely and would be highly relevant to the field. 

Weaknesses: 

However, in its current state, this study does not provide convincing evidence to support their main claim that Pavlovian fear conditioning does not readily occur in naturalistic environments. The innovative task presented in this study is more akin to an inhibitory avoidance task rather than fear conditioning and should be reframed in such way.

Reviewer #1 (Public Review):

Short chain fatty acids produced by gut microbiota interact with the short chain fatty acid receptors FFA2 and FFA3 (formerly GPR43 and GPR41). Barki and colleagues report the results of studies designed to define the roles of FFA2 and FFA3.

Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) derived from human FFA2 modified to allow a BRET signal in the presence of agonists, 1210 compounds with structural similarity to the known agonist sorbic acid were screened in an appropriately validated assay. Reconfirmation screens identified sorbic acid and MOMBA. Assessment of 320 additional compounds identified chemicals related to MOMBA.

MOMBA did not activate human FFA2 in interaction assays. However, interaction assays could not be performed with mouse FFA2, so Gi inhibition of cAMP assays were pursued. Neither sorbic acid nor MOMBA inhibited cAMP levels using human or mouse FFA2, and the same lack of effect was seen using human and mouse FFA3. MOMBA was shown to be an orthosteric agonist of hFFA2-DREADD.

FFA2 and 3 were shown to be expressed in myenteric neurons. MOMBA increased GI transit time in hFFA2-DREADD-HA mice but not control mice. MOMBA also increased transit in animals expressing only FFA2 or only FFA3. MOMBA also increased GLP-1 release from colonic cells and tissues, an effect already reported by this group for sorbic acid. MOMBA also promoted release of PYY.

Vagal afferents in the colon were stimulated by activation of FFA3 but not FFA2. Cells from the nodose ganglion were stimulated by C3 and to a lesser extent by MOMBA. FFA2 and FFA3 also appeared to be active and functional in DRG cells.

Using wild type mice, C3 administered to the rectum activated spinal cord neurons. C3 and MOMBA activated c-Fos in hFFA2-DREADD mice. The authors conclude that there is a SCFA-gut-brain axis.

1. This paper extends findings from this group published in a very strong paper in 2019 (Nat Chem Biol 15:489-498) using knockin receptor hFFA2-DREADD mice and showing that activation of FFA2 promotes GLP-1 release, accelerates gut transit, and promotes lipolysis in adipocytes. The GLP-1 observations are confirmed here, and a new agonist for FFA2 is identified, but it is difficult to appreciate how these studies "define" a short chain fatty acid receptor gut-brain axis. Why did the authors choose to pursue detailed studies of the myenteric neurons, nodose ganglion and DRG? Other mediators of this purported axis could also be involved. What is the purpose of this axis? GI motility? If that is the focus, there are technical issues that limit the conclusions that can be drawn.

2. A strength of the paper is the elegant and rigorous screening strategy and validation of receptor agonists.

3. A weakness is that expression of hFFA2-DREADD is induced by use of a whole-body Cre mouse. Given the broad distribution of FFA2, it is likely that this receptor is being activated in multiple tissues when MOMBA is administered. How can the authors be sure that observed effects after administration of agonist in drinking water are due to local expression as opposed to an effect mediated at a distant site removed from the myentery?

Reviewer #2 (Public Review):

Strengths. Barki et al. report extensive and rigorous studies which convincingly establish that FFA2 and FFA3 are functionally expressed in dorsal root ganglia and nodose ganglia where they signal through different G proteins and mechanisms that regulate intracellular calcium concentrations. The authors further demonstrate that activation of both FFA2 and FFA3 within the gut lumen stimulates spinal cord activity and that activation of gut FFA3 directly regulates sensory afferent neuronal firing. These data support the authors' contention that their investigations define a SCFA-gut-brain axis.

The authors have employed a number of complimentary pharmacological, genetic, cell culture, and ex vivo approaches to obtain their data. The use of these diverse methodological approaches is a key strength of the work. They have employed transgenic mice where FFA2 was replaced by an altered form of FFA2 referred to as FFA2-DREADD (Designer Receptor Exclusively Activated by Designer Drugs), which can be activated by novel ligands but not SCFAs. They have further identified through screens of chemical libraries a novel FFA2-DREADD agonist, referred to as MOMBA, for use in their investigations. Using the FFA-DREADD mice, which are also HA tagged to allow for immunologic detection, and other related transgenic lines, they have been able to establish and identify distinct roles for FFA2 and FFA3 in signaling SCFA production and presence in the gut (specifically the colon) to neuronal pathways that communicate directly with the brain. Using isolated cells, the authors further establish roles for different G proteins and mechanisms that affect cellular calcium levels. Collectively, the data obtained using these diverse experimental approaches support the existence of a SCFA-gut-brain axis.

The authors' new findings significantly extent understanding of the molecular actions of SCFA's produced in the colon through bacterial fermentation of dietary fiber. Multiple publications have identified altered SCFA levels in the gut as a significant contributor to dysregulated metabolism and metabolic disease. Often such studies fail to provide insight into the molecular basis for the observed linkages between SCFAs and altered metabolic states, only reporting the association. The new data being reported by Barki et al. provide new possibilities for understanding these associations.

Weaknesses. The perceived weaknesses of the work are minor compared to the strengths. Although the authors provide a description of the general characteristics of the chemical libraries they screened to identify MOMBA, sparse other information is provided. This is especially true for the second screen of 320 compounds where the data provided indicate a number of compounds may be equally potent agonists. What similarities were there in these structures? Were any of these compounds naturally occurring or resemble naturally occurring molecules? The issue here is one of trying to understand whether endogenous substances exist that might have influenced experimental outcomes and/or data interpretation. This is far from being transparent.

In the authors' first report of the FFA2-DREADD mice (reference 15), they establish that sorbic acid is an agonist for FFA2-DREADD. Many of the studies being reported in the present manuscript are repetitive of this earlier work but using MOMBA in place of sorbic acid. The authors provide some justification for this, but this reviewer does not find these very compelling. Seemingly, sorbic acid stimulated FFA2-DREADD signaling could have been studied without the need to screen libraries and characterize MOMBA actions. This issue detracts from the overall positive feelings towards the work. Why was the identification of MOMBA needed prior to undertaking the present studies? The authors note greater activity of MOMBA over sorbic acid, but why was this important for establishing FFA2 presence and actions in signaling to the brain?

Reviewer #1 (Public Review):

Wang et al. use a multistage mathematical model to analyze incidence of advanced colorectal adenomas and to investigate if the protective effect of aspirin on adenoma incidence could be due to its effect on cellular fitness. The advanced adenoma incidence model is similar to the recently published Paterson et al. PNAS 2020 model, in that it includes the first three of the five steps to colorectal cancer from the Paterson et al. model. Interestingly, Wang et al. find that adding crypt competition to the previous model is needed to account for the observed advanced adenoma incidence curve. This is a nice contribution to the study of colorectal tumor incidence. The authors are also able to confirm previous findings that the order of mutations of the way to an advanced colorectal adenoma is determined by the crypt fission rates, and not the mutation rates.

In the second part of the paper, Wang et al. use the advanced adenoma incidence model to study the effects of aspirin on reduction of adenoma incidence. This part of the paper would benefit from more precise explanation of the assumptions used, especially how exactly is the effect of aspirin implemented at the level of individual crypts and/or crypt cells. Adding these explanations to the main text would significantly increase the clarity of the second half of the manuscript.

Reviewer #2 (Public Review):

This mathematical model of the development of advanced colorectal adenoma (polyps) stands out in terms of its biological realism and inclusion of quantitative information related to stem cell turnover and crypt biology. Early detection and prevention of colorectal cancer, be it via endoscopic screening, stool DNA tests, or use of COX2 inhibitors (Aspirin, Sulindac etc), continues to be an important public health goal, especially in view of stressed health care services under COVID and a rising incidence of CRC among younger individuals in the US. The findings are interesting and motivate further experiments to generate data that inform the model and predictions for effective chemoprevention of colorectal cancer using Aspirin.

The authors recognize that only about ~40% of non-hypermutated CRC are KRAS+. Although KRAS onco-activation may well occur after APC-/- crypts have begun to proliferate (after a 3-6 transition in the model) forming an advanced adenoma, all non-hypermutated CRCs should be carrying detectable KRAS mutations - according to this model. However, Brenner et al did not ascertain KRAS status of their adenomas and cancers. It appears that one way of finessing this problem is to assume that the prevalences of advanced adenomas used to fit this multipathway model can be represented effectively by fractions of those observed clinically. Did the authors consider/test such an adjustment? If not, why not?

To model multiple pathways efficiently the authors resort to a deterministic crypt proliferation model, forgoing a stochastic treatment. Thus, in the deterministic model, there will always be a non-zero fraction of type 6 crypts at any given time > 0. The paper does not reveal how large type 3 (APC-/-, no KRAS) crypt clones are on average when an advanced adenoma (type 6 crypt) occurs in the colon. A discussion of how exactly the advanced adenoma is defined (1 type 6 crypt or more?) and what observation thresholds were applied to model the endoscopic observations by Brenner et al, would be greatly helpful. Typically, in clinical practice, an advanced adenoma is > 1cm in caliper size, or shows signs of dysplasia.

Related to the above, the assumption of zero crypt death/fusion appears unrealistic given the findings by Baker A-M, et al. Gut 2019;0:1-8. Furthermore, given the small number of SCs in human colonic crypts, the sporadic loss of crypts cannot be zero and normal crypt fissions are clearly compensated for by crypt loss (or fusion?) in normal colon. Of further note, a recent modeling paper by Birtwell et al in (Evolutionary Applications 13, 1771-1783 (2020) also argues for crypt turnover as an important aspect of the metapopulation and stem cell dynamics in intestinal crypts and other tissue structures in multicellular bodies.

Finally, it is not clear whether the model recapitulated the relatively short Aspirin exposures used in trials. In other words, were the equations solved for the situation encountered in trials where the interventions (Aspirin) only last for a few years (typically < 10) unless long-term users are included?

Reviewer #3 (Public Review):

The work by Wang et al. demonstrates a computational approach to modeling population-level tumor incidence using a crypt-level algorithm to predict modified incidence based on differential effects on cellular dynamics. While the manuscript makes ultimate conclusions about the impact of aspirin chemoprevention and how this can be parametrized for the model, the work demonstrates a potential in silico approach towards testing putative preventive agents as well as potential factors that may accelerate tumorigenesis by accounting for epithelial cell growth dynamics under different mutation conditions. The base model operates on many relatively basic and broad-sweeping assumptions about colorectal tumorigenesis that will need to be considered for individual downstream applications.

An obvious limitation of chemoprevention studies in humans is the long timescale necessary to perform such experiments/trials and the relatively large subject numbers required to measure incremental effect sizes on relatively low incidence outcomes. Similarly, the availability of appropriate and broadly translatable in vitro (e.g. cell lines that recapitulate precancers or even 'normal' tissue within which to model protective vs. 'anti-cancer' effects) and preclinical models is limited. The strength of this approach towards being able to computationally model crypt dynamics using our best understanding of intestinal cellular proliferation is an important step towards a tool for identification and testing of putative chemoprevention agents which may assist basic and translational researchers as they consider interventions towards intercepting colorectal cancer. The authors specifically understand the constraints and limitations of their modeling approach and transparently discuss the assumptions that feed into the model. Of course, while the manuscript penultimately focuses on the effects of aspirin, the real strength is the identification of a computational model that may similarly behave to biologically relevant crypt dynamics that is able to not only consider different inputs for accelerating effects, but can also model different effects caused by preventive measures and begin to disentangle the underlying biology that is mathematically likely to be occurring in vivo. These assumptions however limit the generalizability of the individual findings as they pertain to our broader understanding of tumorigenesis and aspirin chemoprevention. As an oversimplification of this critique, the authors focus on APC/KRAS mutations, which to be fair are the most common CRC/adenoma mutations, but may not be critical to understanding aspirin's chemopreventive mechanisms, where APC/KRAS mutations have not been shown to predict the variable protective response to aspirin observed in humans. Similarly, the effect estimates on cell dynamics are derived from in vitro and preclinical work using aspirin that use high, albeit appropriately acknowledged as 'physiologically attainable', doses that do not quite represent circulating doses likely achieved through regular use of low-dose aspirin. Nonetheless, using aspirin as a 'model preventative exposure' is suitable in this setting as a way to demonstrate the adenoma models' responsiveness to this type of parameter. Appropriately, the authors do not overinterpret their findings in light of these assumptions, but readers should be similarly cautious to avoid overinterpreting the conclusions.

Reviewer #1 (Public Review):

The effects of antimicrobial peptides (AMPs) on bacteria is a major question in biology and is of great importance in medicine. Biophysical studies, such as the one described in the present manuscript, attempt to gain physical insight into the molecular mechanisms behind such effects. These may vary from structural effects of AMPs on the bacterial envelope membranes, to direct effects on metabolism from their presence in the cytosol.

In this manuscript the authors build upon a recently published seminal paper (Semeraro et al, Acta Crystallographica 2021), where they were able to fit x-ray (USAX and SAXS) and neutron (VSANS and SANS) scattering data from live E.coli cells over four orders of magnitude in length scale. In the present manuscript, they have employed time-resolved USAX/SAXS, using stopped-flow, coupled with contrast variation SANS, transmission electron microscopy, and activity assays, to study the interaction of AMPs with live E.coli cells.

The shifts in the scattering curve (Figure 1) induced by the AMP appear to be quite subtle, and yet the detailed analysis described here and in the authors' previous paper is able to detect changes in a number of structural parameters (Table 1). How much confidence do the authors have in the estimated errors cited in this table?

I think the approach outlined in this manuscript does help to answer important questions on the sequence of events, and their timescales, following the initial binding of AMPs to the outer membrane, with subsequent translocation to the cytosol, where it can interfere with DNA and other metabolic functions.

I am generally speaking confident in the modelling of the scattering developed by the authors, even though this has evolved somewhat from the approach taken in their 2017 paper. This group know how to analyse scattering curves, and while some workers in the field might be somewhat skeptical about the value of the approach, to my mind it is something of a tour-de-force, and does yield some valuable and interesting results.

Reviewer #2 (Public Review):

This article presents a novel and powerful approach, based on small angle scattering, to study the effects of antimicrobial molecules on bacterial cells in real time, obtaining information at multiple spatial scales (nm-um). As such, it is highly interesting.

The main result of the present study is that the peptides accumulate in the cytosol within a few seconds. This finding is solid and peptide accumulation inside the cell is in agreement with several previous studies. However, from this observation the authors conclude that blockage of metabolic activity and not membrane perturbation is the mechanism of bacterial killing. In my opinion, this conclusion is not adequately supported by the data, since bacterial killing over time and metabolic activity were not studied, and membrane perturbation took place essentially on the same time-scale of peptide accumulation in the cytosol. Data interpretation should be revised with increased caution.

Reviewer #3 (Public Review):

Semeraro et al. present a very interesting work on the impact of antimicrobial peptide Lactoferricin on the structure of bacteria. They use primarily small-angle neutron/X-ray scattering to look for structural hallmarks of the effect of the AMP. Based mainly on SANS/SAXS results they conclude that the peptide enters the cytosol "within seconds" and cause irreversible damage. The work is nicely carried out and well written, but I wonder whether it is a bit too ambitious and bold in its claims. In particular considering the shaky ground of which the SAXS/SANS fit analysis is constructed. I agree that TEM backs it up but the results seem only to clarify structural changes at larger scales / morphological features.

Reviewer #1 (Public Review): 

In this very interesting manuscript, Angelaki and Laurens suggest that the same climbing fiber feedback from the inferior olive may innervate functionally distinct classes of Purkinje cells. That is, the same inferior olivary signals, as assessed via the relationship between vestibular stimuli and complex spike frequency, appears to exist in multiple classes of vestibular Purkinje cells. This is an interesting and, in some ways, unexpected observation. 

Strengths: 

Overall, this is a well written and thorough manuscript. The neurophysiology data are clear, and the data analyses are presented clearly and performed rigorously. To that end, the authors' primary claim is well-supported. It is clear that "tilt" and "translation" selective Purkinje cells are innervated by the same or very similar climbing fibers (e.g., Figures 3 and 5). This observation expands the idea of functional organization in the cerebellum by climbing fibers. 

Weaknesses: 

While the primary conclusion of this study is well-supported, the model and theoretical aspects should be enhanced. It is difficult to assess whether the authors' secondary claim about the ability for a shared climbing fiber input to suitably "teach" both tilt and translation responsive Purkinje cells is justified. Much of the issue with the model likely stem from a lack of sufficient information in the methods/results sections to fully appreciate how the model functions.

Reviewer #2 (Public Review): 

Summary:

This manuscript by Angelaki and Laurens investigates the complex spike response properties of Purkinje cells in the nodulus and uvula during translation and/or tilt. The authors find that two previously identified populations of Purkinje cells, one that has tilt-selective simple spike responses and one with translation-selective simple spikes, have very comparable complex spike response properties. Using additional experimental and modeling data they propose a configuration in which the computations in translation-selective cells are used as teaching signals for both populations. These data are interesting and have an important message to add to a growing field of data on (functional) differentiation within the cerebellum. 

Review:

The manuscript is based on a subset of recordings from a dataset that formed the basis for previous work of the authors. The complex spike data are a very relevant part of Purkinje cell functioning, so it is good to see an in-depth analysis here. The recordings are of high quality and the analysis is detailed and clear. The only concern I have is in the interpretation, or perhaps the semantics of the interpretation, of the results. 

This concerns centers around the use of what, by visual inspection, indeed appear to be similar CS histograms for translation, tilt and tilt-trans, as conclusive evidence that the Purkinje cells in both populations receive the same IO input. Although I agree this is an understandable conclusion when taking anatomical data of others into account, I think the evidence, consisting of comparably shaped CS histograms, is not conclusive. For instance, it is possible that a simultaneous CS activation in groups of PCs could be provided without an identical IO projection. However, the evidence is strong and the proposed concept is very interesting.

Reviewer #3 (Public Review): 

Angelaki and Laurens examine the complex spike responses of two types of Purkinje cells (translation-PC and tilt-PC) whose simple spike responses they had previously characterized. They find that even though the simple spike responses of translation-PCs and tilt-PCs are very different from each other during 3D head movements, the two types of Purkinje cells have similar complex spike responses. These results are interpreted within the context of an artificial neural network model, leading the authors to suggest that Purkinje cells within the same cerebellar module (i.e. Purkinje cells with similar complex spike responses) can learn to generate different simple spike responses. 

The data is very exciting, and the analyses and computational modeling very revealing and insightful. The authors clearly demonstrate that Purkinje cells with (apparently) similar climbing fiber inputs can generate very different simple spike responses during 3D head movements. This is a novel finding that will stimulate new discussion about cerebellar function among researchers in the field, and it stands on its own. 

However, the authors make 2 'big' claims that are not fully supported by their analyses: 

1) Are translation-PCs and tilt-PCs in the same olivo-cerebellar loop? The authors state that both Purkinje cell populations are part of a single olivo-cerebellar loop because their complex spikes are driven by the same sensory prediction error. 

- While both translation-PCs and tilt-PCs appear to have similar complex spike responses during the specific conditions and 3D head movements tested, examining complex spike responses in other conditions/other stimuli may reveal differences that would indicate that the Purkinje cells belong to different olivo-cerebellar loops (for example, all experiments were performed in the dark and consisted of passive head movements; is it possible that some Purkinje cells, but not others, could emit visually-driven complex spikes in addition to the translation-related complex spikes observed in the dark in this study? Or could the complex spike responses of tilt-PCs and translation PCs become different from each other during active movements?). 

- Ultimately, whether two Purkinje cells belong to the same olivo-cerebellar loop is an anatomical question; it can only be demonstrated by confirming not only that (1) the Purkinje cells receive the same climbing fiber input from the inferior olive, but also that (2) the output of the Purkinje cells is sent back to the same neurons in the inferior olive. The output pathways of translation-PCs and tilt-PCs are unknown, but the correlational data in Supplementary Fig. 5S1 seems to suggest that they are different (at least with regards to their projections to the inferior olive). In the neural network model, it is assumed that only the output of translational-PCs (but not tilt-PCs) is sent to the part of the olive that projects back to both translational-PCs and tilt-PCs, whereas the output of tilt-PCs is sent to translational-PCs but not to the inferior olive. There is no anatomical data available to support this unconventional architecture. 

2) Can translation-PCs and tilt-PCs use the same complex spikes to learn their respective simple spike responses during 3D head movements? The authors mention that their computational model supports the hypothesis that a single olivocerebellar loop with a shared error signal (in the form of a complex spike) can learn the diverse Purkinje cell responses encountered in the cerebellum (page 18, lines 471-474). However, the neural network model only implements plasticity in the inputs to the tilt-PCs. There is no plasticity in the local synapses formed by the inputs to the translation-PCs. In addition, the model assumes that translation-PCs in different olivo-cerebellar loops will receive selective otolith inputs encoding a specific component of the GIA (e.g. GIAy). In the model, the weights of these highly selective otolith inputs are not learned; they are set at the beginning and cannot be changed. Thus, at the beginning of the simulation, it is these 'fixed' GIA inputs that are entirely responsible for the responses of translation-PCs (these responses are not learned), and for the responses of the inferior olive that drive plasticity in tilt-PCs. In effect, the specialized GIA inputs to the translation-PCs serve as the teaching signal for the tilt-PCs, at least at the beginning of the simulation period. There is no empirical evidence to support the idea that Purkinje cell inputs come in different flavors, including one set of inputs that is plastic and one set of specialized and highly selective inputs that is hardwired and unchangeable. It is unclear whether the network model would be able to learn the correct responses of tilt-PCs and translation-PCs if the weights of *all* the local inputs to both tilt-PCs and translation-PCs were randomly distributed at the beginning and allowed to change according to the same decorrelation plasticity rule. 

As a final comment, although Purkinje cells were classified as tilt-PCs or translation-PCs, the data makes it clear that the responses of both cell populations, both with regards to their simple spike and complex spike responses, are not 100% selective and contain components related to both tilt and translation. The gain of the response for one component (e.g. tilt) is frequently just 2 or 4 times higher than the gain of the response for the other component (e.g. translation). How this diversity of responses could emerge, or what the functional significance could be for the operation of the circuit, was not addressed in the neural network model.

Reviewer #1 (Public Review): 

This paper uses a combination of confocal and electron microscopy to localize gap junctions in the outer retina. Electrical coupling between photoreceptors is an important aspect of retinal function, and past work provides (often indirect) evidence for rod-rod, rod-cone and cone-cone coupling. The work described here indicates that rod-cone coupling dominates. The combination of techniques is quite convincing and very elegant. My concerns are primarily about the appeal of the work to non-retina readers. Some of these concerns could be mitigated by a more accessible presentation of some of the results. Suggestions along these lines, and a few other minor issues, follow. 

Introduction: 

The introduction is a bit retina-centric. I think more needs to be done to explain how each type of coupling (rod-rod, rod-cone, cone-cone) could impact retinal processing, and why it is important to resolve which are present or dominant. One issue that could get emphasized is the difference between gap junctions between like cell types (presumably involved in lateral spread of signals, averaging, etc) and between unlike cells (potentially providing an alternate path for signal flow - as in the secondary rod pathway). 

Cone-cone coupling: 

It would be helpful to put the conclusions about rod-cone and cone-cone coupling together. The paragraph starting on line 585 is a bit confusing that way. It starts by summarizing evidence that blue cones are not coupled with red/green cones. But then (in mouse) all the cones are coupled to rods, so that specific exclusion of blue cones seems unlikely to hold. You come back to this a bit later in the discussion, and there indicate that there appears to be weak cone-cone coupling. Merging the text in those two locations might help. It might also help to make the (seemingly clear) prediction that blue and green cone signals in mouse will get mixed. 

Relation to other circuits: 

Are there implications of the present results for gap junctional coupling in other circuits that could be emphasized? Things like the open probability how strongly it can be modulated seem like points of general interest - but I don't have enough expertise to know if those are established facts on other systems. Some of that is touched on in the Discussion, but quite briefly. 

Location of Cx36:

Can you speculate on why Cx36 is generally located at the mouth of the synaptic opening in the rod spherule? This was a very clear result, but it was unclear (at least to me) if it was important.

Reviewer #2 (Public Review): 

Previous studies demonstrate that modulation of gap junctional coupling in the outer plexiform layer of the mouse retina regulates the balance between sensitivity and resolution. The authors use optical and electron microscopy to structurally characterize this coupling. They find that gap junctional coupling in mouse OPL is produced by a dense meshwork of cone photoreceptor telodendrions that selectively innervate the rim surrounding the synaptic openings of rod photoreceptor spherules. The density of this coupling network is such that each cone is coupled to dozens of rods and each rod is coupled to multiple cones. Rod/rod and cone/cone gap junctions were not detected. 

The combination of antibody labeling, reconstruction of the photoreceptor terminal network, and ultrastructural analysis provides a remarkably clear view of the gap junctional connectivity that constitutes the first stage of visual processing. A few results are only weakly supported due to sample size or technical limitations. However, the overall conclusions are well supported and the data is presented with unusual transparency. The map of the network organization of photoreceptor coupling generated here is an important contribution to visual science. 

Optical imaging:

The quality of the confocal imaging is high and the images of the Cx36 distribution relative to rod spherules is convincing. There does seem to be a significant amount of processing in the images and a lack of background signal in antibody images. Whether this processing is due to the airy scan software or additional filtering and thresholding, it can be difficult to judge the distribution of signal in several images. 

Electron microscopy:

The authors perform annotations on two previously acquired volume EM datasets. The first serial blockface EM dataset is relatively low resolution and lacks ultrastructural labeling but is used effectively to reconstruct the terminal morphology and points of contacts between photoreceptors. The second EM data set uses FIB SEM to obtain smaller voxel sizes from tissue stained in such a way that the darkened membranes of putative gap junctions are distinct from surrounding membrane. Most measures of gap junction number come from the ultrastructure free dataset. In isolation, counting of gap junctions in this type of image volume could be unreliable. However, comparing the putative gap junctions in this dataset to the morphology and distribution of Cx36 antibody clusters in the confocal imaging and the darkened plaques in the FIB SEM images greatly increases confidence that the network description of rod/cone gap junctional coupling is accurate. 

Quantification:

Most quantification is presented with an unusually high degree of transparency, with scatterplots showing all data points, data source files showing the animals that data came from, and standard deviations being supplied in descriptive statistics. There are a few places where Ns are difficult to determine or the analysis is not quite clear. <br> For several results, claims are made when the sample size is too small to be sufficiently confident. The reconstruction of 5 blue cones suggests that, overall, blue cones are not radically different from other cones in their terminal morphology or gap junctional coupling to rod spherules. Claims that the blue cones are identical to other cones in most measures or that their telodendrions are smaller, but not statistically smaller are not well supported by the sampling. Similarly, the fact that the 6 nearby cones closely analyzed for cone/cone gap junctions yield no junctions, strongly suggests that vast majority of gap junctions are cone/rod gap junctions. However, the sample is too small to argue that there could not be infrequent, atypical, or region-specific cone/cone gap junctions. 

Estimate of open channels:

The authors estimate that 89% of gap junction channels are open during times of maximum rod/cone coupling and point out that this number is surprisingly high relative to previous estimates. However, this estimate appears to be subject to many significant potential errors. The estimate combines previous freeze fracture studies of the density of gap junctions from various species and various parts of the retina the measurements of the length and width of the gap junctions in the current study. Differences in tissue processing, density variation within and between systems, reconstruction error, and variation and error in the inputs to the model could all contribute to an underestimate of the total number of channels linking mouse rods and cones. Moreover, without an accounting of these issues, the real error bars on the range of possible open channels would seem to include both surprising and less surprising estimates of open gap junction fractions.

Reviewer #3 (Public Review): 

In the presented work, Ishibashi and colleagues combine immunohistochemistry, analysis of a publicly available large scale 3D EM dataset and smaller but more detailed newly acquired EM datasets to qualitatively and quantitatively study gap junctions of mouse rod and cone axon terminals. The existence of rod-to-cone gap junctions has been known before, but the use of larger 3D EM data allows to determine an average number of contacts as well as an estimate of the strength of gap junctions. This as well as the (very likely) exclusion of direct cone-to-cone coupling in the mouse as opposed to some other mammals are the main contributions of this paper and one more puzzle piece of the big picture of mouse retinal connectivity. However, while the findings are a valuable addition towards a complete picture of the connectivity in the mouse retina, the novelty of the findings is limited to the number of contacts per photoreceptor and gap junction sizes. 

In my opinion, while the authors present a thorough analysis of their data, the manuscript in its current state has stylistic flaws on the motivational side. To me, abstract and introduction lack a motivation or stronger statement of relevance for this analysis. Similarly, while each individual analysis is discussed one by one, I'm missing a broader discussion of the implications of the findings for the field and possible directions for future research to highlight relevance for a broader readership.

Reviewer #1 (Public Review): 

This study presents a comprehensive analysis of the consumptive and non-consumptive effects of predators on mosquito populations. Using meta-data has allowed the authors to explore the effects of predators across multiple families on different mosquito species. This study emphasizes the need to use empirical data on vector traits in disease models and certainly has several insights for epidemiologists and theoreticians. 

Strengths: 

The method for literature screening is robust and well explained. The criteria for study exclusion, in particular, are objective, and all requirements are reasoned out. 

The methods for data analysis are well described. The quantification of effect sizes and heterogeneity and assessment of publication bias, in particular, require a special mention. Such an elaborate methodology section will favor the reproducibility of this study and serve as a benchmark for other meta-analysis studies. 

An extensive list of published studies has been used in the discussion section to lucidly summarize the study's findings. 

Weaknesses: 

This study does not have any significant weaknesses. However, I would like to point out an aspect that the authors could leverage, given the premise of this study. The authors discuss several criteria for exclusion of studies as well as data points. In devising these criteria and curating the datasets, the authors would have had an idea of how such studies could best report data. If possible, the authors could consider including such insights briefly in this manuscript.

Reviewer #2 (Public Review): 

Russel et al. assess the consumptive and non-consumptive effects of predators on mosquito traits via meta-analysis and discuss how these effects could impact disease transmission. Their meta-analysis provides estimates of the impacts of predators on mosquito survival, size, and oviposition behavior, and how these effects are moderated by predator species, mosquito genus, and mosquito size. The ability of predators to regulate prey populations through consumption is well known, but until relatively recently, the non-consumptive effects of predators on prey traits has received less study. The authors point out that these non-consumptive effects could impact disease transmission and encourage further study on other mosquito traits related to vector competence and vectorial capacity. 

Strengths:

To my knowledge, this is the first meta-analysis quantifying the non-consumptive effects of predators on mosquitoes, showing the presence of predators negatively impacts mosquito size and deters female oviposition. The authors ultimately extracted data from 60 studies performed in laboratory or semi-field settings, and this database serves an additional benefit by providing a compilation of literature on the subject. 

Weaknesses:

Although I appreciate the meta-analysis, the implications of the work as described in the discussion may be a little overreaching, particularly the implications for WNV transmission during drought, which seems somewhat unrelated to the rest of the paper because of the weak connection to the role of predators in this phenomenon.

Reviewer #1 (Public Review): 

This study largely confirms prior observations, and the strength of the study is in its comprehensive nature rather than in shedding new insight into the effects of either FH or SDH loss. Nevertheless, there are some somewhat unexpected observations including a defect in proline synthesis, and changes in glutathione and NADPH metabolism that are interesting and incompletely explained. Some suggestions to strengthen the study include: 

1) Exactly how each perturbation affects cell proliferation is not clear. This should be considered, as whether some of the differences are a result in changes in growth or proliferation rate is possible, and will affect how they normalize their data. 

2) It is unclear why FH loss is different than SDH loss, and it is also somewhat surprising that the effects of acute and chronic loss of either enzyme are not that different. While explaining this is too much to ask, some additional speculation might be warranted. 

3) The increase in glutathione and GSSG is interpreted as a consequence of increased oxidative stress, but that will not necessarily affect total levels. 

4) The text in the Figure S1 PCA plots have legends is too small to read. This should be corrected.

Reviewer #2 (Public Review): 

In this manuscript, Ryan et al investigate how disruption of parts of the TCA cycle signals to the nucleus to drive a transcriptional response. The authors inhibited either fumarate hydratase (FH) or succinate dehydrogenase (SDH) using both pharmacological inhibitors and previously described knockdown cell lines. While there were some differences in the metabolic response between these approaches - for example in asparagine levels - the authors noted a consistent change in a number of metabolites that could be linked through their function in cellular redox homeostasis. In particular, the authors showed that a loss of TCA cycle activity through FH and SDH led to increased levels of metabolites within the glutathione synthetic pathway. This suggests that glutamate sits at a functional crossroads between the TCA cycle and glutathione synthesis, allowing the cell to rapidly compensate for mitochondrial dysfunction by increasing cellular antioxidant defences. 

Finally, the authors use transcriptional and proteomic profiling to demonstrate that in addition to the rapid metabolic response described, cells lacking SDH and FH activity respond with an ATF4-mediated transcriptional response mediated through the integrated stress response pathway. 

Strengths:

The authors' use of both genetic models - the previously published Fh1-/- and Sdhb-/- knockout cell lines - alongside pharmacological approaches to acutely inhibit these enzymes mean that they were able to identify both rapid changes in metabolism as a result of inhibition, as well as longer-term compensatory changes. The authors also use a significant array of metabolic techniques to show changes in metabolism - for example, metabolomics, stable isotope tracers, respiration measures and compartmentalised metabolite information, which provide significant confidence in their results. The final section of the manuscript, in which the authors link TCA cycle dysfunction to the integrated stress response pathway was particularly strong - including well-described functional outcome being an ATF4-mediated transcriptional response. 

Weaknesses:

Interestingly, the compounds the authors utilised to block SDH activity (Atpenin A5 and TTFA) have previously been described to inhibit the SQR activity (complex II) rather than specifically the proximal SDH activity of the complex. While in an enzyme complex where the ability to reduce ubiquinone is highly coupled to oxidation of succinate, it is possible that some SDH activity remains using this pharmacological approach, with enhanced ROS generation from the more reduced Fe-S centres. However, the use of the genetic model alongside these data provides additional confidence, as this has been previously shown to have no residual succinate oxidising activity. The use of mitoCDNB provides a novel means by which the authors could perturb mitochondrial thiol homeostasis specifically, it is probably that this reagent also covalently modifies other thiol-containing proteins in the mitochondrial matrix, including those exposed within the respiratory chain. These off-target effects could feasibly phenocopy the effect of TCA cycle inhibition independently of the mechanism the authors suggest. Additionally, some more detail in the methods would aid interpretation of certain experiments - for example the length of time for which some of the treatments were performed is not always clear.

Reviewer #3 (Public Review): 

The authors explore how modulation of TCA cycle impacts cell metabolism acutely, and they identify key links to non-essential amino acid metabolism. The authors employ broad omics analysis as well as targeted flux-based studies to confirm findings. They integrate genetic approaches with pharmacological inhibitors and ultimately describe a regulatory role for ATF4 stress response. 

On one hand, the dependence of NEAA synthesis on mitochondrial metabolism is well-established, and ATF4 is known to regulate amino acid metabolism. However, the authors explain the pathway regulation in some new and interesting ways while integrating recent findings pertaining to proline metabolism. 

There are limitations with respect to physiological relevance which should be outlined further, but the data are well presented and support conclusions.

Reviewer #1 (Public Review):

The authors examined the effect of high-intensity interval training (HIIT) on the proteome and acetylome of human skeletal muscle. HIIT altered the abundance of proteins involved in metabolism, excitation-contraction coupling and myofibrillar calcium sensitivity. In addition, HIIT increased the acetylation of mitochondrial proteins, particularly those of complex V. They further investigated the mechanism of acetylation. In addition, they found changes of some histone acetylation before and after HIIT.

One of the strengths of this manuscript is that the authors obtained the comprehensive proteome and acetylome from human skeletal muscles before and after HIIT, which are publicly available and considered to be important resources. The procedures for sample acquisition, measurements, and analyses are clearly described. Although there have been previous studies analyzing acetylome in skeletal muscle (Lundby et al., 2012), they newly found the selective acetylation of mitochondrial complex V after HIIT. A weakness of the manuscript is that there is little evidence to support the non-enzymatic acetylation.

Reviewer #2 (Public Review):

In this study, Hostrup et al., set out to determine how 5 weeks of HIIT training alters the proteome and acetylome in skeletal muscle. Some of the major findings from the proteomic data include evidence for HIIT-induced mitochondrial biogenesis and potentially a HIIT-induced "slowing" of the muscle properties, whereas the major findings from the acetylome data include evidence of HIIT-induced acetylation of mitochondrial proteins as well as HIIT-induced acetylation of proteins that are involved in the TCA cycle.

Reviewer #3 (Public Review):

This study by Hostrup et al. examines the remodelling of the proteome and acetyl-proteome in skeletal muscle following a period of high intensity interval training (HIIT). The subjects were exposed to a 5 week HIIT protocol, which produced the desired training effect, as demonstrated by physiological and biochemical changes observed at the level of the whole-body and skeletal muscle respectively. Assessment of the proteome revealed a number of 'predictable' training-induced changes, such as increased abundance of several mitochondrial proteins. Additionally, changes were observed in a number of other novel proteins and pathways, highlighting adaptive changes that facilitate the broader physiological effects and benefits of exercise training. The acetyl-proteomics revealed altered acetylation status in a range of different pathways (likely via non-enzymatic mechanisms), with prominent alterations to respiratory Complex V, TCA cycle enzymes and histone proteins.

While observational in nature, the study was performed in a robust manner, the conclusions are largely supported by the data, and the outputs collectively provide a rich resource for future hypothesis-driven examinations of mediators of the skeletal muscle response to exercise training. There are a few points noted below that would benefit from extended discussion and potentially a few additional experiments.

Reviewer #1 (Public Review): 

Baker et al. investigated the molecular evolution in primates of one protein family, the CEACAMs, that are a recurrent target of bacterial surface adhesions at epithelial surfaces. They show that multiple members of this gene family have experienced repeated episodes of positive selection in primates, especially in the N-terminal domains that are associated with protein binding. To test for the functional consequences of these evolutionary changes, Baker et al. incubated recombinant CEACAM N-domain proteins with different bacterial strains, and showed that divergence between species (and within humans) in this domain was sufficient to alter binding. Furthermore, by examining multi-gene phylogenies of just the N-terminal domain, the authors demonstrate that gene conversion between members of the CEACAM family plays an important role in this divergence. 

The major strength of this manuscript is the work the authors have done to tie together molecular evolutionary analysis with functional binding assays to demonstrate that sequence divergence in the N-terminal domain of CEACAM1 is sufficient to alter the binding profile and potentially restrict host range of particular pathogens that bind to CEACAMs. While the authors do not test every positively selected site in every species of great ape, they show that substituting single positively selected residues in humans for the bonobo amino acid is sufficient to alter bacterial binding. 

A second major strength of this manuscript is the use of population genetic data to suggest that gene conversion is ongoing in human populations, with segregating alleles that increase similarity between CEACAM1, 3, and 5 in human populations. This observation both validates the pattern observed in other primates, and suggests the importance of gene conversion as an ongoing evolutionary mechanism in this gene family. 

Overall, the major conclusions of this work concerning the role of sequence divergence in determining binding, and the presence and importance of gene conversion, appear to be well justified by the results. 

My only potential concern is that the general model of gene conversion discussed at the end of the work seems perhaps a bit too speculative. Gene conversion is a natural consequence of sequence similarity among paralogous members of a gene family, and it does not seem that special consequences for pathogen evasion need to be invoked. Instead, the presence of frequent gene conversion among members of the CEACAM family seem to indicate that maintaining some degree of sequence similarity among paralogs is beneficial, perhaps due to the role of CEACAM3 as decoy receptor.

Reviewer #2 (Public Review): 

In this manuscript, Baker et al describe the "Evolution of host-microbe cell adherence by receptor domain shuffling," which specifically examines a vertebrate-specific multigene family known as the CEACAMs, cell surface receptors and adhesion molecules that possess immunoglobulin domains. A subset of these receptors are expressed by the epithelium lining mucosal surfaces, and as such many bacteria adhere (via adhesions) to these receptors during host colonization. In this paper, the authors focus on how these receptors have been evolving in primates, and demonstrate that pathogens are engaged in an evolutionary arms race with CEACAMs, driving genetic diversification of the N-terminal domain (the main docking site for bacteria) to influence or regulate how pathogens colonize host epithelial surfaces. 

The study demonstrates that repeated adaptive evolution of primate CEACAMs is shaping host-specific cell surface adherence by pathogenic bacteria. The authors show that over half of the CEACAM paralogs of humans reveal signatures of positive selection across diverse primates, primarily within the N-terminal (extracellular) domain, or the main interaction domain. They also show that rapid evolution is occurring via sequence exchanges across a subset of CEACAM paralogs via gene conversion. And finally, the authors also show that gene conversion is impacting pathogen recognition of CEACAMs in modern humans, suggesting that diversification of CEACAM N-terminal domain sequences is significantly impacting interactions between primates and the bacteria that colonize their surfaces. 

It has been hypothesized that "exploitation of host proteins by pathogens places a significant burden on host populations, driving selection for beneficial mutations in these proteins that limit microbial invasion or virulence." But by interacting with host factors not necessarily involved in immunity (such as "housekeeping" proteins or receptors), pathogens may benefit from limited adaptive potentials, e.g., epithelial surface receptors likely have multiple roles critical to cellular and physiological functions. The authors set out to "investigate patterns of CEACAM divergence in primates and propose how CEACAM evolution and human populations have shaped interactions with pathogenic bacteria." 

The authors first demonstrate that 8 of 12 primate CEACAM genes are undergoing positive selection (likely pathogen-driven) primarily in the exons encoding the N-terminal domains. Importantly, this includes CEACAM 1, 3, 5, and 6, which have been shown to be important interaction receptors for adhesins from various human pathogens. 

The authors then demonstrate that evolution of the N-domains influences binding by bacterial adhesins. This was done using recombinant CEACAM domains, where specific mutations were introduced. First, the authors confirmed that representative CEACAM1 N-domains could bind known bacteria, via their adhesins. Specifically, CEACAMs from representative primates only bound specific bacterial strains. For example, the authors generated CEACAM1 N-domains in which a subset of human and bonobo residues were swapped; introduction of bonobo residue 44 into human CEACAM1 prevents binding to H. pylori and Opa-adhesins (e.g., Neisseria sp.), for example. These data confirm that nonsynonymous substitutions (those that change amino acids) in the N-domain are enough to shape interactions with multiple bacterial adhesins. The authors then use bioinformatic approaches to demonstrate that ongoing polymorphic changes in human CEACAM1 is likely impairing bacterial recognition; gene conversion among CEACAMs is also likely still ongoing in human populations, and that exchange of beneficial mutations may also influence binding to decoy receptors on epithelial cells. 

And finally, the authors wrap up the paper by raising and trying to address some remaining questions in the field. In particular, an intriguing conundrum exists and remains to be investigated: if many non-pathogens or commensals also depend on CEACAM-binding for colonization of host surfaces, what are the consequences of pathogen-driven polymorphisms that may impair binding of beneficial microbes to CEACAMs? 

Overall, this is simply a fantastic paper that is very well put together, very well thought out, and with a very clear writing style. The figures are nicely presented, organized, and informative. All necessary supplemental and source data are supplied. This is an important contribution to the field, and will be of broad interest. I very much enjoyed and learned from this paper; it was a pleasure to review.

Reviewer #3 (Public Review): 

This manuscript from the Barber group presents an interesting analysis of the CEACAM family of cell receptors in primates and convincingly shows that the N-terminal sequences are under strong purifying selection and have undergone both mutation and gene conversion to create diversity. The paper is clearly written, the figures are clear, and the methods used to examine the relationship are presented in a manner that a non-expert can understand their basis. As a study of the CEACAM gene family these data are very strong and the conclusions justified. The manuscript is less satisfactory in respect to the parameters driving the evolution of the CEACAM family. The authors conclude the evolutionary pressure is due to escape from bacterial (and possibly other) pathogens but the bacterial species known to bind CEACAMs are host-adapted, opportunistic pathogens (so-called pathobionts) that do not cause significant pathology in hosts and have coevolved with their hosts. Moreover, it is uncertain whether the specific bacterium used in this study, Neisseria gonorrhoeae, has been associated with primates at all and how long it's been associated with humans is under debate and suggests this organism is a major driver of CEACAM evolution can be discussed but is not solid. It is more likely that one or more primordial commensal Neisseria species have been present in primates and humanoids. They also have ignored the phase variable nature of the Neisseria Opas and the different specificity for CEACAM orthologues. While the microbe/host evolutionary arms race model is simple and easy to swallow, I believe they need to back off from concluding any microbe are the evolutionary pressure and provide a more thoughtful discussion of the possible pressures similar but expanded from that they propose for CEACAM7/8. Lastly, if you are going to rest the model on a Neisseria species, showing a spirochete form in the model figures is not accurate. In summary, I see no issues with the data and it shows that the CEACAM family of receptors has been under strong purifying selection. There are major issues with the context provided for the data and the conclusions about the pressures for the selection of CEACAM family members.

Reviewer #1 (Public Review): 

In this manuscript Ma and colleagues present a biochemical investigation into integration by the Spy Cas1-Cas2 integrase, a key component of the type II CRISPR immune response. From this work they challenge the view that disintegration (the reverse of integration) only functions as an off-pathway reaction (proof reading) and propose that disintegration-promoted integration is functionally important for integration into CRISPR arrays. 

CRISPR integration proceeds through two steps. In the first, one strand of the prespacer (fragment of foreign DNA) is integrated at the leader-repeat (R-L) junction of the CRISPR array. In the second step, the other strand of the prespacer integrates at the repeat-spacer (R-S) junction resulting in a fully integrated prespacer. The authors propose that, in vitro, water-mediated disintegration at the first site (R-L) is concomitant with second site integration (R-S). The authors present good evidence that this is happening in vitro though a series of elegant biochemical experiments using plasmid and oligo substrates. 

This is an intriguing hypothesis but its significance is unclear. As the authors themselves acknowledge it could very well be that disintegration is not required in vivo and that this is an in vitro feature of the reaction. Admittedly, demonstrating that disintegration is important in vivo would be a very difficult task. In addition, the in vitro system used here is only partially reconstituted. The substrates lack a PAM sequence, which is necessary for protospacers to be incorporated in the correct orientation and may help direct the first integration event to the L-R junction. Presumably because of this all the reactions presented do not analyze the orientation of the incorporated prespacer sequence. Cas9 and Csn2 are also absent (as are other potentially required host factors), which are necessary for correct integration in vivo. Any of these missing components could change the behavior of the system with respect to disintegration.

Reviewer #2 (Public Review): 

Ma and colleagues have show that when DNA fragments are inserted into a CRISPR array in vitro, single end integrations are the primary products of proto-spacer insertion and double-end insertions are rare. The reason for this outcome is that the second insertion event is accompanied by 3'-OH mediated disintegration or hydrolysis at the initial insertion site. A double-strand break would remain following filling of the repeat sequence gaps in vivo in the case of the hydrolysis products. The authors propose that hydrolysis of one end of the transposon DNA may be the primary mechanism for insertion of very small DNA elements (which will be difficult to bend tightly) as found for the proto spacer sequences and that cellular repair pathways would be responsible for ligating the CRISPR array back together. 

The manuscript is well written and the experiments carefully planned. The findings will be of interest to researchers studying transposition-like mechanisms in a broad range of systems. The experiments focus on analyses of Cas1-Cas2 catalyzed in vitro reactions using cleverly designed DNA substrates, leading to a hypothesis about how the system may work in vivo. The direct conclusions are well supported by the experimental data. The big unanswered question is whether the results obtained with a minimal Cas1-Cas2 system in vitro reflect what happens in the bacterial cell. More proteins are present in the cell that could modify the reaction and the native DNA array structure differs somewhat from what was used here. Overall, the results presented are intriguing, but their relationship to the cellular mechanism of CRISPR array generation is unclear, since it is not yet known whether DNA elements are inserted in vivo in the same way as observed here in vitro.

Reviewer #1 (Public Review): 

Cesanek et al. performed a series of experiments designed to reveal whether or not the encoding of motor memories for novel object weight carries categorical structure. That is, given a set of objects of varying sizes and with weights that must be learned through experience, are objects grouped into categories such that the learned weight of one object generalises to objects within the same category, but not to objects outside of that category. Their results convincingly demonstrate the presence of such a categorical encoding. They show the following: 

1) The weight of an outlier object is not learned if its weight is near the weight predicted by category membership. 

2) The weight of an outlier object is learned if its weight is far from the value predicted by category membership. 

3) The weight of an outlier object is learned if there is no category structure binding the remaining objects (i.e., there is no category against which an outlier can be defined). 

4) If an outlier object is learned, then it influences the estimated weight of the other category members. 

5) If the weight of an outlier object is learned first in isolation, it is unlearned when the remaining objects are introduced if and only if its weight is near the value predicted by category membership. 

6) The threshold that constitutes "near" or "far" from the category boundary depends on recent sensorimotor experience. 

7) Learning of the outlier is all-or-nothing on a per participant basis. 

The major strength of the paper is the persuasiveness of the behavioural experiments, which were designed soundly and yielded clear results. There is little doubt that the some motor memories carry the type of categorical encoding detailed by the authors. 

The major weakness of the paper is that it does not make strong contact with the relevant existing literature to clearly show that categorical encoding is (1) a truly novel behavioural observation in the motor learning literature, and (2) that it is inconsistent with the predictions of common motor learning theories and models. In particular, the authors own prior work frames motor learning as being governed by multiple internal models that can be switched between depending on contextual cues and environmental demands (see reference below). Insofar as contextual effects can drive similar results as categorical memory encoding, it is unclear how and why this and related models would fail to account for the present data. 

Wolpert, D. M., & Kawato, M. (1998). Multiple paired forward and inverse models for motor control. Neural Networks, 11(7-8), 1317-1329.

Reviewer #2 (Public Review): 

Using a novel 3D robotic device, the authors had participants learn to lift four training similar-looking objects whose weights were linearly correlated with the sizes and then tested how this training influenced the later memory formation for the middle-sized object with different densities. When the difference between the actual weight and the weight estimated from the linear relationship was not so large (i.e., within a family boundary), surprisingly, the training for lifting the test object was ineffective: The estimation of the object weight was constrained by the linear relationship of the family between size and the weight. The memory specific to the new object could be developed only when the difference was large enough. 

The results were unexpected from the conventional idea that object properties are encoded in an "associative map." The authors interpreted these results as evidence that the motor memory for lifting objects with different sizes and weights could be formed according to the "object family effect." All results of other control experiments were consistent with this interpretation. 

I was intrigued by the counter-intuitive results that the motor system sticks to estimate the object weight based on the family property even though this estimation is incorrect and induces the error. Although it remains unclear how such a memory for multiple objects is integrated from memory for each object, it is sure that this study has demonstrated a new aspect of motor memory while manipulating the objects with different sizes and weights.

Reviewer #3 (Public Review): 

In this paper, Cesanek et al. use a novel object lifting task to investigate the "format" of memories for object dynamics. Namely, they ask if those memories are organized according to a smooth, local map, or discrete categories ('families'). They pit these competing models against one another across several experiments, asking if subjects' predicted weights of objects follow the family model or a smooth map. This was tested by having people train on objects of varying volumes/masses that were either consistent with a linear mapping between volume/mass, or where those dimensions were uncorrelated. This training phase was either preceded by, or preceded, a testing phase where a novel object with a deviant mass (but a medium-size volume) was introduced. As the authors expected, individuals trained on the linear mappings treated novel objects that were relatively close to the "family" average mass as a member of the family, and thus obligatorily interpolated to compute the expected mass of that object (i.e., under-predicting its true mass); conversely, when a novel object's mass was a substantial outlier w/r/t the training items, it was treated as a singleton and thus lifted with close to the correct force. Additional variations of this experiment provided further evidence that people tend to treat an object's dynamical features as a category label, rather than simply forming local associative representations. These findings offer a novel perspective on how people learn and remember the dynamics of objects in the world. 

Overall, I found this study to be both rigorous and creative. The experimental logic is refreshingly clear, and the results, which are replicated and extended several times in follow-up experiments, are rather convincing. I do think some additional analyses could be done, and data presentation could be improved. I also thought the generalization analysis, as I interpreted it, was difficult to align with the initial predictions.

Reviewer #1 (Public Review):

Bohère, Eldridge-Thomas and Kolahgar have studied the effect of mechanical signalling in tissue homeostasis in vivo, genetically manipulating the well known mechano-transductor vinculin in the adult Drosophila intestine. They find that loss of vinculin leads to accelerated, impaired differentiation of the enteroblast, the committed precursor of mature enterocytes, and stimulates the proliferation of the intestinal stem cell. This leads to an enlarged intestinal epithelium. They discriminate that this effect is mediated through its interaction with alpha-catenin and the reinforcement of the adherens junctions, rather than with talin and integrin-mediated interaction with the basal membrane. This results aligns well, as the authors note, with previous observations from Choi, Lucchetta and Ohlstein (2011) doi:10.1073/pnas.1109348108. Bohère et al then explore the impact that disrupting mechano-transduction has on the overall fitness of the adult fly, and find that vinculin mutant adult flies recover faster after starvation than wild types.

The main conclusions of the paper are convincing and informative. Some important results would benefit from a more detailed description of the phenotypes, and others could have alternative explanations that would warrant some additional clarification.

1) - Interpretation of phenotypes in vinc[102.1] mutants

The paper presents several adult phenotypes of the homozygous viable, zygotic null mutant vinculin[102.1], where the fly gut is enlarged (at least in the R4/5 region). In many cases, they correlate this phenotype with that of RNAi knockdown of vinculin in the gut induced in adult stages. This is a perfectly valid approach, but it presents the difficulty of interpretation that the zygotic mutant has lacked vinculin throughout development and in every fly tissue, including the visceral mesoderm that wraps the intestinal epithelium and that also seems enlarged in the vinc[102.1] mutant. So this phenotype, and others reported, could arise from tissue interactions. To me, the quickest way to eliminate this problem would be to express vinculin in ISCs and/or EBs the vinc[102.1] background, either throughout development or after pupariation or emergence, and observe a rescue.

An experiment where this is particularly difficult is with the starvation/refeeding experiment. The authors explored whether the disruption of tissue homeostasis, as a result of vinculin loss, matters to the fly. So they tested whether flies would be sensitive to starvation/re-feeding, where cellular density changes and vinculin mechano-sensing properties may be necessary. They correctly conclude that mutant flies are more resistant to starvation, and suggest that this may be due to the fact that intestines are larger and therefore more resilient. However, in these animals vinculin is absent in all tissues. It is equally likely that the resistance to starvation was due to the effect of Vinculin in the fat body, ovary, brain, or other adult tissues singly or in combination. The fact that the intestine recovers transiently to a size slightly larger than that of the fed flies seems anecdotal, considering the noise within the timeline of fed controls. I am not sure this experiment is needed in the paper at all, but to me, the healthy conclusion from this effort is that more work is needed to determine the impact of vinculin-mediated intestinal homeostasis in stress resistance, and that this is out of the scope of this paper.

2) - Cell autonomy of the requirement of Vinculin and alpha-Catenin

Authors interpret that Vinculin is needed in the EB to maintain mechanical contact with the ISC, restrict ISC proliferation through contact inhibition, and maintain the EB quiescent. This interpretation explains seemingly well the lack of an obvious phenotype when knocking down vinculin in ISCs only, while knockdown in ISCs and EBs, or EBs only, does lead to differentiation problems. It also sits well with the additional observation that vinculin knockdown in mature ECs does not have an obvious phenotype.

However, a close examination makes the results difficult to explain with this interpretation only. If the authors were correct, one would expect that in mutant clones, eventually, vinculin-deficient EBs will be produced, which should mis-differentiate and induce additional ISC proliferation. However, the clones only show a reduction in ISC proportions; the most straight forward interpretation of this is that vinculin is cell-autonomously necessary for ISC maintenance (which is at odds with the phenotype of vinculin knockdown using the ISC and ISC/EB drivers).

Also, from the authors interpretation, it would follow induce that the phenotype of vinculin knockdown in ISCs+EBs and in EBs only should be the same. However, in ISCs+EBs vinculin knockdown, differentiation accelerates, which is likely accompanied by increased proliferation (judging by the increase in GFP area, PH3 staining would be more definitive). This contrasts with the knockdown only in EBs, which leads to accumulation of EBs due to misdifferentiation, and increased proliferation, mostly of ISCs, as measured directly with PH3 staining, but not additional late EBs or mature ECs. The authors call this "incomplete maturation due to accelerated differentiation". I think that one should expect to find incomplete differentiation/maturation when the rate of the process is very slow, not the other way around. To me, these are different phenotypes, which could perhaps be explained if vinculin was also needed in the ISC to transmit tension to the EB and prevent its differentiation, and removing it only in the EB may be revealing an additional, cell-autonomous requirement in maturation.

Another unexpected result, considering the authors interpretation, is that the over expression of activated Vinculin (vinc[CO]) does not seem to have much of an effect. It does not change the phenotype of the wild type (where there is very little basal turnover to begin with) and it only partially rescues the phenotype of the vinc[102.1] mutants, when the rescue transgene vinc:RFP does. This again suggests that there may be tissue interactions, in development or adulthood, that may explain the vinc[102.1] phenotypes. It could also be that this incomplete rescue is due to the deleterious effect of Vinc[CO]; this is another reason for doing the vinc[102.1]; esg-Gal4; UAS-vincFL experiments suggested above). An alternative experiment to perform this rescue would be to knock down vinculin gene while overexpressing the Vinc[CO] transgene - this may be possible with the RNAi HSM02356, which targets the vinculin 3'UTR and is unlikely to affect UAS-vinc[CO].

The claims of the authors would be more solid if the reporting of the phenotypes was more homogeneous, so one could establish comparisons. Sometimes conditions are analysed by differentiation index, others by extension of the GFP domains, others with phospho-histone-3 (PH3), others through nuclear size or density, and combinations. I do not think the authors should evaluate all these phenotypes in all conditions, but evaluating mitotic index and abundance of EBs and "activated EBs/early ECs" to monitor proliferation and differentiation rates should be done across the board (ISC, ISC+EB, EB drivers).

If the primary role of Vinculin is to induce contact inhibition in the ISC from the EB and prevent the EB differentiation and proliferation, one would expect that over expression of Vinc[CO] (or perhaps VincFL or sqhDD) in EBs should prevent or delay the differentiation and proliferation induced by a presumably orthogonal factor, like infection with Pseudomonas entomophila or Erwinia carotovora.

3) - Relationship between Vinculin and alpha-Catenin

The authors establish a very clear difference in the phenotypes between focal adhesion components and Vinculin, whereas the similarity of alpha-catenin and vinculin knockdowns is very compelling. Therefore I am sure the authors are in the right path with their interpretation of this part of the paper. However, some of the alpha-Catenin experiments are not very clear. The result from the rescue experiment of alpha-Cat knockdown with alpha-Cat-deltaM1b does not seem to show what the authors claim, and differentiation does not seem affected, only the amount of extant older ECs (which may be due to other reasons as this is a non-autonomous effect). Ulrich Tepass produced a UAS-alpha-catenin construct with the full deletion of the M1 region, perhaps that would show a clearer phenotype. Also, the autonomy of the phenotype is difficult to address with these experiments alone. It would be expected that the phenotype of alpha-catenin knockdown should be similar to that of vinculin knockdown in the ISCs only or EBs only.

Reviewer #2 (Public Review):

Vinculin functions as an important structural bridge that connects cadherin and integrin-mediated adhesions to the F-actin cytoskeleton. This manuscript carefully examined the mutant phenotype of vinc in the Drosophila intestine and found that vinc mutant in EBs causes significant increases of EB to EC differentiation, stem proliferation, and tissue growth. By analyzing the mutant phenotype of the cadherin adaptor alpha-catenin, the authors suggest that the vinc functions through the cell-cell junctions instead of cell-CEM adhesions in EBs. Finally, manipulation of myosin activity in EBs phenocopies the vinc mutant, suggesting that vinculin is regulated by the mechanical tension transduced through the cytoskeleton.

The authors claim that the vinculin mutant phenotype is opposite compared to the loss of the major integrin components, suggesting a function independent of the cell-ECM adhesions. However, the phenotype of vinc and integrin may not be completely opposite. Besides loss of ISCs, both mys and talin knockdown in ISCs clearly causes ISCs differentiation into EC cells (Fig.3A), suggesting a possible involvement of integrin in EB to EC differentiation. Therefore, it will be important to test the phenotype of integrin KD in EBs using EB-specific Gal4.

The authors proposed a model that the cell-cell adhesion between ISC and EBs is required for vinculin mediated differentiation suppression. However, this model is not directly supported by the data as the EB-ISC adhesion and EB-EC adhesion have not been tested separately. In addition, previous short-term manipulation of E-cadherin in ISCs and EBs shows no change in cell proliferation (Liang J. et al. 2017), which seems to contradict the authors' model. To support the authors' conclusion, long-term manipulation of E-cadherin in ISCs and EBs must be tested.

The result of MARCM analysis seems inconsistent with the rest of the data. In MARCM, no significant change of clone sizes is observed between WT and vinc mutant (Fig. 3E). However, vinc mutant in EBs clearly promotes ISC proliferation in other experiments such as esg>vinc-RNAi and the EB>vinc-RNAi (Fig. 1A, Fig. 4).

In Fig. 4H, the authors suggest that vinculin mutant prevent terminal EC formation. However, this may be simply caused by longer retention of Klu expression in the newborn ECs. To test if EB differentiation is indeed affected, the EC marker pdm1 staining will provide more convincing evidence. Another experiment to strengthen the conclusion will be the tracking of clone sizes generated from a single EB cell using the UAS-Flp system (such as G-trace).

In Fig. 6D, the survival rate of WT and vinc mutant flies were compared. However, as there is no additional assay about the feeding behavior or metabolic rate, the systematic mutant of vinc does not provide a direct link between animal survival and intestinal EBs. Therefore, an experiment with vinc level specifically manipulated in fly intestine using esg>vinc-RNAi or BE>vinc-RNAi will be more relevant.

Reviewer #3 (Public Review):

Prior work had identified essential roles for Integrin signaling in regulating intestinal stem cell (ISC) proliferation, and the authors studies were motivated by trying to understand whether Vinculin (Vinc) might participate in this. However, Vinc is involved in mechanotransduction at both focal adhesions (FA) and adherens junctions (AJ), and their results revealed that Vinc phenotypes do not match those of FA proteins like Integrin. Conversely, they do match a-catenin (a-cat) RNAi phenotypes, and together with the localization of Vinc and the phenotypes associated with a-cat mutants that can't bind Vinc, this led to the conclusion that Vinc is acting at AJ rather than FA in this tissue. The results here are convincing, with clear presentation, nice images, and appropriate quantitation. It's also worth emphasizing that initial characterization of Vinc mutant flies failed to reveal any essential roles for this protein in Drosophila, so finding a mutant phenotype of any sort is significant.

While the manuscript is strong as a descriptive report on the requirement for Vinc in the Drosophila intestine, it doesn't provide us with much understanding of the mechanism by which Vinc exerts its effects, nor how its requirement is linked to intestinal physiology.

Prior work has shown that mechanical stretching of intestines stimulates ISC proliferation (presumably through Integrin signaling), which is opposite to what Vinc does here. There is a suggestion that Vinc is involved in maintaining homeostasis, but how its regulated remains a bit murky. The authors report that reductions in myosin activity result in phenotypes reminscent of Vinc phenotypes, which they interpret as supporting a model where Vinc's role is to help maintain tension at AJ. Of course it could also be reversed - maybe they are similar because tension is needed to maintain Vinc recruitment to AJ? They lack of epistasis tests and lack of analysis of whether Vinc localization to AJ in EBs is affected by tension or the M2 deletion of a-cat leaves us uncertain as to the actual basis for the relationship between Vinc and myosin phenotypes.

Reviewer #1 (Public Review):

Zhu et al. found that human participants could plan routes almost optimally in virtual mazes with varying complexity. They further used eye movements as a window to reveal the cognitive computations that may underly such close-to-optimal performance. Participants' eye movement patterns included: (1) Gazes were attracted to the most task-relevant transitions (effectively the bottleneck transitions) as well as to the goal, with the share of the former increasing with maze complexity; (2) Backward sweeps (gazes moving from goal to start) and forward sweeps (gazes from start to goal) respectively dominated the pre-movement and movement periods, especially in more complex mazes. The authors explained the first pattern as the consequence of efficient strategies of information collection (i.e., active sensing) and connected the second pattern to neural replays that relate to planning.

The authors have provided a comprehensive analysis of the eye movement patterns associated with efficient navigation and route planning, which offers novel insights for the area through both their findings and methodology. Overall, the technical quality of the study is high. The "toggling" analysis, the characterization of forward and backward sweeps, and the modeling of observers with different gaze strategies are beautiful. The writing of the manuscript is also elegant.

I do not see any weaknesses that cannot be addressed by extended data analysis or modeling. The following are two major concerns that I hope could be addressed.

First, the current eye movement analysis does not seem to have touched the core of planning-evaluating *alternative* trajectories to the goal. Instead, planning-focused analyses such as forward and backward sweeps were all about the actually executed trajectory. What may participants' eye movements tell us about their evaluation of alternative trajectories?

Second, what cognitive computations may underly the observed patterns of eye movements has not received a thorough theoretical treatment. In particular, to explain why participants tended to fixate the bottleneck transitions, the authors hypothesized active sensing, that is, participants were collecting extra visual information to correct their internal model about the maze. Though active sensing is a possible explanation (as demonstrated by the authors' modeling of "smart" observers), it is not necessarily the only or most parsimonious explanation. It is possible that their peripheral vision allowed participants to form a good-enough model about the maze and their eye movements solely reflect planning. In fact, that replays occur more often at bottleneck states is an emergent property of Mattar & Daw's (2018) normative theory of neural replay. Forward and backward replays are also emergent properties of their theory. It might be possible to explain all the eye movement patterns-fixating the goal and the bottleneck transitions, and the forward and backward replays-based on Mattar & Daw's theory in the framework of reinforcement learning. Of course, some additional assumptions that specify eye movements and their functional roles in reinforcement learning (e.g., fixating a location is similar to staying at the corresponding state) would be needed, analogous to those in the authors' "smart" observer models. This unifying explanation may not only be more parsimonious than the author's active sensing plus planning account, but also be more consistent with the data than the latter. After all, if participants had used fixations to correct their internal model of the maze, they should not have had little improvements across trials in the same maze.

Reference:<br> Mattar, M. G., & Daw, N. D. (2018). Prioritized memory access explains planning and hippocampal replay. Nature Neuroscience. https://doi.org/10.1038/s41593-018-0232-z

Reviewer #2 (Public Review):

In this study the authors sought to understand how the patterns of eye-movements that occur during navigation relate to the cognitive demands of navigating the current environment. To achieve this the authors developed a set of mazes with visible layouts that varied in complexity. Participants navigated these environments seated on a chair by moving in immersive virtual reality.

The question of how eye-movements relate to cognitive demands during navigation is a central and often overlooked aspect of navigating an environment. Study eye-movements in dynamic scenarios that enable systematic analysis is technically challenging, and hence why so few studies have tackled this issue.

The major strengths of this study are the technical development of the set up for studying, recording and analysing the eye-movements. The analysis is extensive and allows greater insight than most studies exploring eye-movements would provide. The manuscript is also well written and argued.

A current weakness of the manuscript is that several other factors have not been considered that may relate to the eye-movements. More consideration of these would be important.

1. In the experimental design it appears possible to separate the length of the optimal path from the complexity of the maze. But that appears not to have been done in this design. It would be useful for the authors to comment on this, as these two parameters seem critically important to the interpretation of the role of eye-movements - e.g. a lot of scanning might be required for an obvious, but long path, or a lot of scanning might be required to uncover short path through a complex maze.

2. Similarly, it was not clear the how the number of alternative plausible paths was considered in the analysis. It seems possible to have a very complex maze with no actual required choices that would involve a lot of scanning to determine this, or a very simple maze with just two very similar choices but which would involve significant scanning to weight up which was indeed the shortest.

3. Can the affordances linked to turning biases and momentum explain the error patterns? For example, paths that require turning back on the current trajectory direction to reach the goal will be more likely to cause errors, and patterns of eye-movement that might related to such errors.

4. Why were half the obstacle transitions miss-remembered for the blind agent? This seems a rather arbitrary choice. More information to justify this would be useful.

5. The description of some the results could usefully be explained in more simple terms at various points to aid readers not so familiar with the RL formation of the task. For example, a key result reported is that participants skew looking at the transition function in complex environments rather than the reward function. It would be useful to relate this to everyday scenarios, in this case broadly to looking more at the junctions in the maze than at the goal, or near the goal, when the maze is complex.

6. The authors should comment on their low participant sample size. The sample seems reasonable given the reproducibility of the patterns, but it is much lower than most comparable virtual navigation tasks.

The authors have generally achieved their aim. The results appear compelling and with further consideration of the results, the findings should lead to an important research article helping advance a key aspect of navigation - how eye-movements are related to cognitive processes involved in solving the path choices in navigation.

An impact of this work is that it should encourage models of the neural system of navigation to predict how eye-movements will occur during navigation and opens the potential for eye-movements to be used in a wide range of studies of virtual or real-world navigation.

Reviewer #3 (Public Review):

In this article, Zhu and colleagues studied the role of eye movements in planning in complex environments using virtual reality technology. The main findings are that humans can 1) near optimally navigate in complex environments; 2) gaze data revealed that humans tend to look at the goal location in simple environments, but spend more time on task relevant structures in more complex tasks; 3) human participants show backward and forward sweeping mostly during planning (pre-movement) and execution (movement), respectively.

I think this is a very interesting study with a timely question and is relevant to many areas within cognitive neuroscience, notably decision making, navigation. The virtual reality technology is also quite new for studying planning. The manuscript has been written clearly. This study helps with understanding computational principles of planning. I enjoyed reading this work. I have only one major comment about statistical analyses that I hope authors can address.

Number of subjects included in analyses in the study is only nine. This is a very small sample size for most human studies. What was the motivation behind it? I believe that most findings are quite robust, but still 9 subjects seems too low. Perhaps authors can replicate their finding in another sample? Alternatively, they might be able to provide statistics per individual and only report those that are significant in all subjects (of course, this only works if reported effects are super robust. But only in such a case 9 subjects are sufficient.)

Somewhat related to the previous point, it seems to me that authors have pooled data from all subjects (basically treating them as 1 super-subject?) I am saying this based on the sentence written on page 5, line 130: "Because we are interested in principles that are conserved across subjects, we pooled subjects for all subsequent analyses."<br> If this is not the case, please clarify that (and also add a section on "statistical analyses" in Methods.) But if this is the case, it is very problematic, because it means that statistical analyses are all done based on a fixed-effect approach. The fixed effect approach is infamous for inflated type I error.

Again, quite related to the last two points: please include degrees of freedom for every statistical test (i.e. every reported p-value).

Reviewer #1 (Public Review):

Following up on their previous work characterizing acinus as an autophagy regulator, the authors have now identified a PPM-type phosphatase named nil that mediates the dephosphorylation of acinus-S437 (which they have previously shown to be phosphorylated by Cdk5/p35). This new regulatory step is shown not only important for basal autophagy levels and for clearing a pathological polyQ repeat protein, but it also mediates a neuroprotective autophagy response during cadmium intoxication because cadmium inhibits this phosphatase. The methods are up to the standards of the field and properly support the conclusions. The in situ dephosphorylation assay is not as common as using fully recombinant proteins (or perhaps phospho-acinus co-IP-d from lysates) in an in vitro reaction, but it certainly supports that it is nil that dephosphorylates acinus. This work significantly adds to our knowledge of the regulation of autophagy and especially how intoxication by the heavy metal cadmium triggers protective autophagy to promote survival of the animals.

Reviewer #2 (Public Review):

Autophagy is a key process in maintaining neuronal health. In their previous studies, authors identified role of Acinus in promoting basal autophagy and provided evidence for Cdk5 to phosphorylate Acinus at S437 to modulate its stability and subsequentially regulate basal level of autophagy. They further demonstrated enhancement of the Cdk5/Can-pS437 pathway in response to polyQ protein accumulation.

In the current study, the authors identified PPM-type phosphatase Nilkatha (Nil) crucial for controlling phospho-switch on Acn-S437. Nil counteracts Cdk5-p35 kinase complex dependent phosphorylation of Acn-S437. The authors demonstrated Acn-S437 phospho-switch as a key integrator of multiple stress signals including Cd2+ induced cytotoxicity and polyQ protein accumulation. Overall the findings are interesting and important. The conclusions are mostly supported by the data presented in this work, but some aspects of data analysis would be stronger if they were extended.

1. Throughout the study the authors demonstrated increase in autophagic flux either with an increase in Atg8a-II to Atg8a-I ratio or an increase in Atg8a puncta with or without inhibiting lysosomal acidification and degradation with chloroquine. While these methods hint towards increase in autophagic flux, it's not entirely clear if the cargo is indeed cleared up. It might have been worth doing p62 staining/western blots in addition to the Atg8a staining/western blots to validate that the cargo is indeed degraded. A decrease in p62 corresponding to an increase in Atg8a-II to Atg8a-I ratio would imply increase autophagic flux.

2. Increased Acn-S437 phosphorylation elevates the basal, starvation-independent autophagy (Nandi et. al. 2017). Here, authors demonstrate that Nil regulates Acn-S437 phosphorylation in nucleus as well as has a potential involvement in regulating components of endo-lysosomal or autophagic trafficking. Authors also demonstrate that Nil1 null mutants have increased autophagic flux. It is however not clear if the the increased autophagic flux is a result of Acn-S437 phosphorylation or a result of Nil's interaction with autophagy pathway or a combined effect of both?

3. The authors demonstrate that Acn-pS437 is necessary for autophagic response to Cd2+ exposure. It is however not clear if Acn-pS437 is sufficient for autophagic response to Cd2+ exposure. I would have included a phenotype rescue experiment here.

4. The authors also predict that Acn-pS437 induced autophagy in nil1 mutants helped the animals to cope with low levels of Cd2+ induced oxidative stress. I would have included blocked autophagy in nil1 mutants to validate this observation.

5. The authors observed an altered pattern with flw knockdown and larval lethality with mts knockdown. The authors provide an additive effect on Yorkie activity rather than direct effects on Acn as a possible explanation. Since exploring these observations further are not particularly useful for this paper, nevertheless it is an interesting result and it might still be worth discussing these observations in discussion.

Reviewer #1 (Public Review):

Rarely do I read a paper and have so little to criticize. The paper is very well written and the studies have been conducted carefully and described fully. The only comment that I would make is that the divergence of quaternary structure in CTPS filaments brings to mind some well-known parallels that should be cited and discussed. Perhaps the most prominent one is hemoglobin, where this protein can form tetramers in some species that are very different from the vertebrate tetramers. In plants, I believe that dimeric hemoglobins have been described. Another example would be the diversity of filaments formed by actin-like proteins in bacteria, while in eukaryotes the actin filament architecture has been extremely conserved.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of enzyme polymerization in enzymatic regulation and cellular metabolism. The authors use yeast CTP Synthase (CTPS) in their studies, an enzyme critical for nucleotide biosynthesis and which is also known to form large clusters (self-assemblies) in cells under nutrient deprivation (starvation) conditions. Homologs of yeast CTPS from human and bacteria are known to form linear polymers composed of multiple copies of the CTPS enzyme. Such enzyme polymerization is now known to occur in many other enzyme systems as well, though few in-depth studies such as the current work have been performed. Hence many questions remain including the effect of enzyme polymerization on enzyme activity and regulation, its role in cell biology and metabolism, and its connection to the enzyme self-assembly clusters seen in cells undergoing particular stress responses. The authors use a powerful combination of cryo-electron microscopy, biochemistry, mutagenesis, and yeast studies to investigate these questions for yeast CTPS. The authors succeed in showing that yeast CTPS polymerizes in a pH-sensitive way (low pH mimics starvation conditions), due to a particular histidine/aspartic acid interaction at the interface between CTPS tetramers in the polymer, and that polymerization stabilizes the inactive conformation of the enzyme. Hence, filamentation inhibits yeast CTPS enzyme activity, which is consistent with what would be expected for a biosynthetic enzyme when the cell is under starvation conditions. The authors also succeed in showing the importance of enzyme polymerization to cellular metabolism, as mutations that disrupt or enhance CTPS polymerization slow the growth rate of yeast cells. Interestingly, the authors speculate that polymerization may serve a function beyond merely inhibiting the enzyme under nutrient starvation, but also in controlling (slowing) the recovery rate upon the reintroduction to a nutrient-rich environment as disassembly of enzyme polymers slows reactivation of the enzyme. Other significant results include the investigation as to whether or not enzyme polymerization serves a purpose in protecting against degradation, and they find that it not, at least in a 24 hour time frame. Interestingly their results also suggestion that some "licensing" process may be required (in addition to low pH) to induce CTPS self-assemblies in cells (a possible example would be phosphorylation). Finally, to bridge the poorly understood connection between enzyme polymerization and enzyme clusters seen in cells, they analyze bundles of polymeric filaments seen in cryo-electron microscopy images. Extrapolating contacts seen in these bundles allowed for the creation of models for large super-structures, which may represent the structure of CTPS in the self-assembly clusters seen in cells, though this is still not yet known. An amino acid insert present in the sequence of yeast CTPS, but not human, is seen to bridge polymeric filaments of CTPS in the bundles but has not yet been investigated using mutagenesis.

Reviewer #3 (Public Review):

I have only one major issue:

1. Clearly, no study can do everything, but I think the authors should do a bit more to attempt to understand why their "constitutive" assembly mutant does not assemble in vivo in standard conditions.

Reviewer #1 (Public Review):

This paper seeks to address how animals use sensory feedback from multiple sensory modalities during the navigation of complex environments. In particular, the authors are concerned with the use of odor-based signals for search behaviors, as odors are sparse and do not provide reliable directional information. An additional goal of this paper is to model the general principles they extract from their experiments so that they can be applied to engineered systems. Through the use of a virtual reality (VR) system that combines visual, wind, and odor input in a controlled manner, the authors investigate the search behavior of the silkworm moth, Bombyx mori. The females of the species emit a pheromone that triggers both a search behavior as well as mating behavior in males. Whereas previous work has taken advantage of the highly stereotyped nature of this behavior, it is unclear how these animals integrate other sensory cues such as wind and vision to locate an odor source. By presenting these stimuli in different combinations where the odor is always present, the authors find that the presence of a wind source strongly influences the animal's behavior-specifically its ability to locate the target-although the direction from which it is presented is crucial when all three are together. Multimodal input also affects the moths' walking speed and turning behavior, and an updated model that incorporates wind direction outperforms other models of this behavior.

The conclusions of the paper are generally supported by the presented data, although there are some issues with the framing of the paper and the model that I feel should be addressed.

1) The introduction and discussion go to great lengths to emphasize the technical advance of the constructed VR apparatus. Although it is certainly an impressive achievement, the use of VR for exploring insect behaviors like search and navigation is hardly a new development at this point in time. Indeed, work on flies, Drosophila and otherwise, going back decades has used VR to extract principles regarding issues such as visually mediated flight control or walking. More pertinent to the ideas of multimodal sensory integration explored here, over the past decade numerous researchers have combined visual input with odor and other cues to discern the relative importance of each of these modalities during search behavior. For example, see Duistermars and Frye, 2008. Generally, I feel that the paper overemphasizes the technical advance without providing sufficient biological context. So much work has been done on Bombyx that a paper using these methods has the ability to address, but much of that literature is absent from the paper. I think focusing more on the behavior will broaden the appeal of the paper by putting it in conversation with a well-established phenomenon

2) I think that the model is well-done and fits with the goals of the paper. Asking about the role of wind direction in this behavior is an important step given the behavioral data presented. However, I am not convinced based on the data that the new model developed by the authors is much better than the surge-zigzag model. The success rates are slightly different (is the statistical difference a function of the number of runs or timesteps?) and both models search about the same amount of time before finding the source. Finally, the migration probability maps are rather similar, so it is hard for me to conclude that factoring in wind direction is necessary to get good performance out of the model.

Reviewer #2 (Public Review):

This paper uses a multi-model virtual reality system to assess which combinations of visual, wind, and olfactory information male silk moths rely on to find a female. The overall conclusion is that for the moths to search effectively, wind direction information is an important input. Vision, on the other hand, while it is used to control angular velocity, does not appear to be important for the moths to search effectively. Given what is known about other walking and flying insects these results are not surprising. Although the virtual reality system is advertised as being able to provide naturalistic and complex stimuli, the visual stimuli are limited to a traditional LED array, and the olfactory stimulus is created by projecting a 3-dimensional plume into two dimensions. The analyses of the data are rather simplistic and do not provide a mechanistic description of what the moths are actually doing on a moment-by-moment basis. The authors then proceed to construct a model for search behavior that uses frequency and relative timing information for the odor stimulus in conjunction with the wind direction information. This search algorithm is ever so slightly better than the prior surge-zigzagging algorithm. The role of relative timing information in the olfactory signal supplied to the moths in the virtual reality experiments, however, was not investigated.

Reviewer #1 (Public Review):

This work presents a new Bayesian method for detecting those patterns of neural responses that are connected to behavioral output and are worth investigating further. The manuscript contains the derivation of the approach and its test on synthetic and real neural data.

The derivation should be improved by providing additional steps. For example, it was not clear how Eq. 5 was derived and why the double derivative with respect to parameters theta_mu and theta-nu are present ( these terms appear to be missing in the definition of log-likelihood).

Parameter M should be more clearly defined as the number of samples. It is briefly mentioned on line 170, but it was difficult to connect this to equation (7) and those following that use M explicitly.

Is it possible to include multiple binary quantifications of behavior, similarly to how words are constructed from neural spike trains? For example, one can envision describing a particular song segment with respect to multiple binary features simultaneously.

Less emphasis should be made in the abstract on applications outside of neuroscience, because these are not studied here explicitly. It is fine as part of the discussion, but in its present form the abstract suggests a stronger connection to protein sequences than is actually in the manuscript.

Reviewer #2 (Public Review):

Summary:

Hernandez et al propose a new statistical tool for identifying  codeword in multivariate binary data (for instance neural activity patterns), with a small number of measurements. It demonstrates the utility of the approach on neural responses to analyzing the statistical structure of songbird responses and how they change in different contexts (during exploration vs typical song production).

Strengths:

- The approach is innovative, in that it takes advantage of clever tools from sparse linear regression, in particular a method termed Bayesian Ising Approximation (BIA), to be able to identify codewords individually, rather than directly estimating a model of their joint statistics, by comparing to a null model that assumes independence across dimensions. This approach has the advantage of resulting in a very flexible model, with very few assumptions about the statistical structure of the data, that is applicable for a range of datasets sizes; the more data is available, more of the structure underlying it can be revealed .<br> - The strong mathematical foundations provides clear bounds on data regimes in which the approximation is theoretically well justified and reasons to expect that the estimated models are minimal and interpretable.<br> - The numerical estimation procedures are fast, and computationally efficient (for a reasonably sized neural dataset, can be run on a regular laptop).<br> - The code is available on github for quick community dissemination.<br> - Application to identification of behaviorally relevant patterns of co-activity goes beyond previous Ising-based models used in neuroscience.<br> - When applied to songbird data, it reveals that the variability in neural responses during exploration has much more structure than previously thought.

Weaknesses:

- Although the paper is written as a methods paper, emphasizing the technical contributions and promising wide applicability to a range of different types of datasets, the  numerical validation of the method is very much restricted to the statistical regime of the songbird dataset. From the perspective of a potential future user of the tool it's less clear how the method would behave on different datasets, and what needs to happen in practice for adopting the tool to data with different statistics.<br> - The numerical comparison to other existing methods is minimal.<br> - The songbird analysis already reveals some challenges with respect to interpretability: in particular it is not clear how much information about the underlying neural processes can be revealed by summary statistics generated by the method, such as the number of codewords and their length distribution.

Most conclusions are reasonably supported by the data. The analysis of the irreducibility of the codewords has insufficient support based on the numerical simulations. Moreover, the generality of the tool and comparison to other methods are discussed in almost entirely theoretical terms, which makes the claim on immediate utility for other datasets less convincing, especially outside the neuroscience community.

Nonetheless, the idea is quite interesting and likely of broad interest for theorists interested in the development of unsupervised statistical tools for neural data analysis, with practical applicability for a range of modern systems neuroscience experiments that involve task specific ensembles as the building block of circuit computation.

Reviewer #1 (Public Review):

This paper investigates the function of the neurospecific 440-kDa ankyrin-B isoform in axonal branching. Previous work has shown that AnkB440 KO mice have increased branching and an enlarged corpus callosum. It was also previously shown that AnkB440 interacts with L1CAM. This paper expands this previous work to investigate the mechanism by which AnkB440-L1CAM mediates signalling. Since L1CAM is known to also interact with Neuropilin1 and Sema3A, the authors investigated whether AnkB440-L1CAM might mediate its effects on neuronal branching through modulating Sema3A collapse. Indeed, the authors find that AnkB440 is required for Sema3A-mediated growth cone collapse. AnkB440 does this by stabilizing the L1CAM-Nrp1 complex at the cell surface, as well as mediating F-actin disassembly during growth cone collapse. In addition, the authors also identify that AnkB440 and the AnkB220 isoforms exert different effects on axonal transport and cytoskeletal changes.

A strength of the paper is the careful cell biological and molecular assays which provide convincing evidence of the authors conclusions. It is also interesting that none of the autism variants could rescue the phenotype of the AnkB440 KO neurons, indicating that each of these variants affected function.

The paper represents a confirmation and advance over previous work. Many of the results follow those previously published by Yang et al., 2019. Both papers demonstrate that AnkB440 mutant mice have an enlarged corpus callosum. It is not evident if this is a more generalized nervous system effect or specific for callosal axons. It is not clear why the corpus callosum is enlarged. Increased dendritic branching of cortical neurons was observed but axonal branching was not analyzed in vivo. Callosal axons do not usually branch within the tract at the midline and no evidence is provided to explain whether callosal axons are more spread out in the tract or whether there are more branches. Since Sema3A-Nrp1 signalling also regulates the targeting of callosal axons in the contralateral hemisphere as well as other features of cortical development, it would be interesting to identify whether AnkB440 mutations may disrupt these functions.

The work nicely demonstrates that AnkB440 mutations cause increased neuronal branching by preventing Sema3A-mediated F-actin disassembly and growth cone collapse.

Reviewer #2 (Public Review):

The ANKB gene generates through alternative spiling two isoforms whose expression and functions differ. One is ubiquitous, ANKB220 while the other one, ANKB440 is neuron-specific. ANKB is a component of the sub-membrane cytoskeleton known to play important roles during brain development. This is also highlighted by the identification of variants associated with autism spectrum disorder. In their study, the authors investigated the roles exerted by the neuron-specific ANKB 440. To distinguish them with those of ANK220, they generated a mouse model specifically deleted for the ANKB440, that retains normal ubiquitous expression of ANKB220. The study, combining phenotypic analysis of corpus callosum tract, cortical cultures, functional assays and biochemistry, reveals that ANK440 regulates the branching pattern of cortical neurons and their sensitivity to exogenous signals previously found to contribute to the wiring of cortical connectivity. This study thus provides new insights into the mechanisms that shape cortical neurons during brain development, and also highlights some novel etiological causes of autism spectrum disorder.

Reviewer #3 (Public Review):

This manuscript shows that the neuron-specific giant ankyrin B isoform (AnkB440) is functioning to decrease excessive branching and plays an important role in the collapse response of cortical growth cones to the chemorepellent Sema3A. They combine a number of knockout or mutant mouse lines to show convincingly that AnkB440 specifically is involved Sema3A growth cone collapse through L1CAM/neuropilin-1 interactions. They also show that these responses, in contrast to previously shown interactions of AnkB440 with microtubules, occur through AnkB440 signaling through actin and cofilin. Moreover, they show that several ASD-linked AnkB440 point mutants do not rescue the Sema3A-induced growth cone collapse. Overall, the authors make a convincing case that AnkB440 is functioning through a well-known guidance cue pathway. The experiments often have positive and negative controls, show clear and robust differences and are analyzed in a rigorous fashion, with, for the most part, appropriate statistical tests. The one area in which the manuscript is lacking is putting these data together in the context of what is known about the Sem3A signaling pathway. If they were able to provide a strong model showing where AnkB440 fits into the Sema3A pathway, it would definitely strengthen the manuscript and increase the impact of the work.

Joint Public Review:

This study examines the possibility that Muller glia (MG) motility in the developing retina prior to eye opening may be controlled or modulated by retinal waves and neural activity. The authors carefully describe the developmental emergence of dynamic filiopdial remodeling in MG in the IPL over the period spanning eye opening and show there is initially a high rate of remodeling which is gradually replaced by stability following eye opening. They further show that motility can be pharmacologically regulated by cytoskeletal manipulations. Using careful quantitative analysis of calcium transients, they unambiguously reveal that MG calcium transients can be up or downregulated (especially in stalks) by increasing or decreasing waves pharmacologically and that mAChRs likely mediate most of the signaling from neurons to glia underlying this regulation. Finally they perform experiments to alter retinal waves and neuronal activity to test for changes in glial stability. Somewhat surprisingly glial motility was not significantly regulated by retinal neuronal activity. Thus the authors conclude that glial motility in the retina may be independent of calcium transients and neuronal activity. Overall this is a very well designed and technically impressive study. The text is also extremely clear and a pleasure to read. One criticism concerns the underpowered analysis for the morphological dynamics under conditions of neuronal activity blockade.

Reviewer #1 (Public Review):

The paper reports in a mouse study that the potent N-containing bisphosphonate, zoledronate, can directly enter alveolar macrophages and peritoneal macrophages and exercise biological activity, inhibiting the Farnesyl diphosphate synthase enzyme. The paper is technically of a high standard. 

The present work shows for the first time that an N-BP can directly internalize into non - bone cells following IV injection. It shows further that the internalized ZOL can prevent protein prenylation, with increased IL-1 and other cytokines and chemokines that constitute the inflammosome. The authors propose that their immunopharmacology studies present an explanation for N- bisphosphonate improvement of outcome of patients with pneumonia. 

These pharmacological experiments provide an exciting prospect for clinical application, and this will be especially so if the effect of N-BPs can be shown to take place with drug concentrations that might be achievable clinically.

Reviewer #2 (Public Review):

The manuscript by Munoz et al. is aimed at identifying possible mechanisms by which the clinical outcome of pneumonia can be improved by previous or concurrent treatment with aminobisphosphonates (NBP). The study was motivated by epidemiological and clinical data linking the treatment with zoledronic acid (ZOL) for hip fractures or other skeletal-related events with a reduced risk of mortality from pneumonia. The Authors conclude that zoledronic acid (ZOL), commonly used to prevent bone loss in cancer and osteoporosis, targets the mevalonate pathway in alveolar macrophages of C57BL/6J mice and boosts protective inflammatory and immune responses after bacterial endotoxin (LPS) challenge. 

The results are interesting and based on experiments well designed and well performed. ZOL is a drug commonly used in the clinics with a very safe record track. It is always exciting when an inexpensive and safe drug sounds promising for repositioning.

Reviewer #3 (Public Review): 

This is a well-written manuscript that clearly shows the ability of macrophages outside bone to incorporate bisphosphonates intracellularly and respond to zoledronic acid administration by increasing the production of proinflammatory cytokines and chemokines. The data appears solid, and the conclusions are supported by the data. The experimental design mimics the outcomes of individuals already treated with bisphosphonates when they encounter an infection, mimicked here by administration of the bacterial endotoxin, and the authors seem to imply it could also be used as a preventive measure in elderly patients. Related to the latter point, and if the reviewer's understanding is correct, authors could include a more direct statement describing the potential use as a prophylactic agent.

Reviewer #1 (Public Review): 

The papers addresses an important aspects of CNP function in chondrocytes and its signaling pathway. 

Strengths:

The hypothesis proposed for this paper. The quality of the data presented and the writing style of the paper. 

Weaknesses:

The paper lacks some clarification in some terms used in the paper such as "round chondrocytes". Additional histological data on the organ cultures would provide supporting evidence on the role of CNP in chondrocytes. 

The authors also generated NPR2 chondrocyte-specific null mice. It would be interesting to include some data on the phenotype of these mice by micro-CT, histology, etc.

The reviewer believe the authors achieved their aims, while some additional supporting data and clarifications are needed. 

The overall summary of the data provided is supportive, methods utilized are well justified, again, additional histological data would support the overall summary. 

Data generated and presented in the report will enhance our understanding on the role of CNP and possible utilization of novel therapeutic targets for CNP for the treatment of bone growth diseases.

Reviewer #2 (Public Review): 

Miyazaki et al demonstrated the role of CNP (C Type Natriuretic peptide) in the proliferation and stimulation of growth plate chondrocytes for development of bone. The author dissected the signaling axis involved in CNP mediated bone growth and discover the role of Ca+2 mediated signaling by identifying the NPR2-PKG-BK-TRPM7-CaMKII axis in bone growth. This study provides an exciting data in the field of bone biology and provided a new layer of regulation for bone growth. Overall study design is logical, and experiments were well performed and organized. Adequate number of animals were used in study and conclusions drawn from experiments are convincing. 

Strength: 

1) Experiments are well designed, performed with proper control and conclusion drawn from data is highly convincing. <br> 2) Valid animal model (floxed mice) using chondrocyte specific deletion of gene were used to show in vivo effect of gene deletion on bone formation. <br> 3) Preclinical model of OA were used in vivo using mice as an experimental animal, OA progression were characterized using established grading system. <br> 4) All the Western blots are shown along with densitometric quantification. <br> 5) Ex vivo molecular mechanism is also provided which added strength in the manuscript. 

Methods, results and data interpretation: 

1) Methods section is adequate and describes enough details to replicate in independent study. <br> 2) Relevant statistics are used to infer conclusion form data. <br> 3) There is no objective error in presenting the data, conclusions drawn from experiments are convincing.

Reviewer #1 (Public Review): 

In this paper, Suzuki et al use a combination of in vivo and in vitro experiments to characterize, for the first time, responses in the two main classes of FFIs in the mouse olfactory cortex, neurogliaform cells (NG) and horizontal cells (HZ). They find that these two cell types have different responses and different connectivity, which partially explains their different responses. This paper also helps resolve a previously perplexing result from a recent high-profile publication that claimed that FFI in the mouse olfactory cortex appears to play a negligible role in shaping cortical odor responses, presumably because those authors were only recording from HZ cells.

Feedforward inhibition (FFI) typically exerts a powerful effect shaping neural activity. This paper characterizes the odor-evoked activity and connectivity of two classes of feedforward interneurons (FFIs) in the mouse piriform cortex, horizontal cells (HZs) and Neurogliaform cells (NGs). They do this first by 2-P targeted recordings in anesthetized mice and brain slices. They find that both NGs and HZs are broadly tuned (consistent with Poo & Isaacson, 2009), but they have very different response types. NGs exhibit strong, fast, and phasic odor responses, while HZs exhibit much weaker, delayed, and prolonged responses. In slice recordings, they show that NGs inhibit HZs but that HZs do not inhibit NGs, which presumably explains, fully or in large part, their differential odor responses.

This is a timely and important piece of work. It also helps explain a puzzling result from a recent high-profile study (Bolding & Franks, 2018), and a modeling study from the same group (Stern et al, 2018), which showed negligible odor responses in FFIs, which seems odd and incongruent with previous slice experiments from multiple other labs (Bekkers, Isaacson, Schoppa, etc). Here, Suzuki et al suggest, very reasonably, that the Bolding paper probably recorded primarily from HZ cells and therefore missed the phasic odor responses in NGs. Moreover, they delicately suggest why Bolding & Franks got it wrong (L. 466: "...placement of the electrodes (close to or far from the LOT)"). Indeed, Bolding & Franks' recordings are almost certainly biased for HZ cells Furthermore, in their modeling study, Stern et al had all-to-all connectivity amongst a homogenous population of FFIs, which would yield slow, weak, and HZ-like responses.

Therefore, not only does this paper provide the first cell-type-specific characterization of FFIs in piriform cortex in vivo, it helps clarify, and possibly fully resolves, a previously confusing result.

Reviewer #2 (Public Review): 

The study is good starting point for understanding how specific inhibitory circuitry shapes cortical responses in olfaction. In these experiments, whole-cell recordings were made in vivo from identified L1 inhibitory interneurons- neurogliaform (NG) and horizontal (HZ), in anterior piriform cortex. Given the ventral location of the piriform cortex, recordings such as these are difficult and rarely performed. The main finding is that odors evoke strong, transient and short latency responses in NG neurons while HZ neurons are activated later and have prolonged and lower firing rates. Both classes are broadly tuned consistent with previous reports of broadly tuned feedforward inhibition in piriform cortex. Analysis of subthreshold currents revealed enhanced respiration phase locking during odors in NG but not HZ interneurons. Further analysis revealed that underlying synaptic inputs were consistent with spike responses, EPSPs peaked sooner and decayed more quickly in NG than HZ neurons. Interestingly, these findings seem to contradict previous in vitro work from the same group. In vitro analysis of short-term plasticity in the afferent pathway has shown that afferent EPSPs onto NGs facilitate while EPSPs onto HZs depress. This would result in opposite temporal responses to those seen in vivo. The authors directly address this and partially resolve the discrepancy for NG neurons. Additional investigation of inhibitory inputs onto NG and HZ neurons revealed that NG inhibit HZ neurons but not the reverse. This could contribute to the delayed response properties of HZ neurons. Finally, the influence of feedforward inhibition onto pyramidal neurons was investigated. Unfortunately, there is currently no means of selectively inhibiting NG versus HZ neurons in vivo. Therefore in vitro experiments were conducted to build a model of how these two classes affect feedforward inhibition. The results of these final experiments though plausible, were somewhat underwhelming. Overall, this study performs an important characterization of two understudied classes of inhibitory interneuron and provides some limited mechanistic insight into their function in the cortical circuit.

Reviewer #3 (Public Review): 

In this manuscript, Suzuki and colleagues report a careful analysis of L1 interneuron-mediated feedforward inhibitions in the piriform cortex. Using in vivo two-photon targeted patch clamping recordings, in vivo functional calcium imaging, and in vitro multiple simultaneous patch clamping recordings, the authors found that L1 neurogliaform cells mediate a strong but transient feedforward inhibition, whereas HZ cells supply a more diffuse and prolonged feedforward inhibition during odor sensation. The findings are fascinating, and experimental data are of high quality. The study represents a significant advance of our understanding of L1 neuron-mediated inhibitions in information processing, which should appeal the general readers of eLife. 

Strengths: 

1) The study is very topical. <br> 2) The study employs a combination of cutting-edge technologies that reveals unexpected differences and potential functions of the two odor-evoked feedforward inhibitions, which would not be possible for unit recordings, for example. <br> 3) The experiments employed are in general rigorously designed and executed. 

Weaknesses: 

1) The mechanisms underlying the functional differences of feedforward inhibitions is not fully illustrated. The authors did propose a few possible mechanisms based on their preliminary investigation. This is a minor weakness given the significant finding about distinct forms of feedforward inhibitions.

Reviewer #1 (Public Review):

The major strengths of this work include a comprehensive analysis of genetic and functional genomics data, such as chromatin accessibility, DNA methylation, chromatin-chromatin interaction, super enhancer and RNA-seq. The authors have also strengthened their findings through further functional assays in disease target cells. A weakness of this work is a lack of a large-scale genome-wide association study to improve the risk signal.

The authors have identified a novel risk variant, rs1047643, for systemic lupus erythematosus through GWAS together with functional evidence. The identified variant located in a risk locus, 8p23, has been established for the disease. They showed evidence that the identified variant could alter binding affinity of a susceptible factor STAT3 to affect targe genes including a known susceptibility gene BLK for systemic lupus erythematosus. These results provided further support of the functional risk variant discovery. These findings advance the understanding of the genetic susceptibility and biology of systemic lupus erythematosus risk.

Reviewer #2 (Public Review):

In this manuscript, the authors have conducted several analyses using various omics data to support their conclusion that the SNP (rs1047643) is involved in STAT3-mediated allelic imbalance as well as a B cell specific super enhancer in association with systemic lupus erythematosus.

The analytic pipeline looks reasonable, and the story looks fine for me. I am not an expert in SLE, therefore cannot evaluate the soundness of the biological story.

Reviewer #3 (Public Review):

1. By mining microarray data and ATAC-seq data, the new functional risk variant rs1047643, located at 8p23, associated with SLE.

2. STAT3-mediated SE activity and the regulatory role of SNP rs1047643 at 8p23 are associated with B-cell deregulation in SLE.

Reviewer #1 (Public Review):

In the present manuscript, Deny Cabezas-Bratesco curate a set of sequences of the transmembrane domain (TM) and TM-proximal regions from group I Transient Receptor Potential (TRP) channels (including subfamilies A, V, C, M and N) belonging to vertebrate and invertebrate animals as well as unicellular organisms. The authors generate a multiple sequence alignment, which reveals subfamily-specific patterns in the extra- and intracellular regions that connect each of the six transmembrane helices, as well as a set of 11 residues that are highly conserved across subfamilies in TRP channels from animals. Interestingly, these residues appear to be conserved, albeit to a lower extent, in TRP channels from unicellular organisms and even in voltage-gated channels that are evolutionary related to TRP channels. This suggests that the degree of conservation of these residues provides information about the evolution of TRP channels in different organisms. Importantly, the positions of these 11 conserved 'signature' residues appear to also be conserved at a structural level, and are grouped into three separate 'patches' at key regions that are known to be important for channel function. Applying direct-coupling analysis the authors find evidence that supports the conservation across subfamilies of interactions between the different channel regions included in patch 1, and suggest that interactions with lipids in this region might also be conserved. This is consistent with a wealth of information pointing to this region as a central hub for allosteric signaling in TRP channels. The authors also find that TRP channels from group I all have a cluster of aromatic residues at the core of the ligand-binding S1-S4 domain that is very different from the equivalent domains in voltage-activated channels and that would be expected to confer rigidity to that region in TRP channels. Finally, the authors find further conserved aromatic residues connecting the hydrophobic cluster in the S1-S4 domain with conserved patch 2 in the extracellular pore - this points to the existence of a conserved mechanism that communicates the ligand-binding domains with the extracellular half of the pore. Differences in the hydrophobic cluster between subfamilies and the in the connection between the cluster and conserved patches 1 and 2 could be related to mechanistic differences in signal integration between TRP channel members of each subfamily. Together, the data presented highlights a set of robustly conserved features across evolutionary related proteins that together, offer an intriguing framework for interpreting further structural information and data on channel function based on a core mechanism of function that appears to be conserved in the TRP family of ion channels.

The figures in the manuscript do not provide information to assess the diversity of organisms from which TRP channel sequences were analyzed - this is important because biases in this selection could influence some of the results, such as the extent of conservation at certain positions. Some of the patterns from the multiple sequence alignments are also difficult to assess from the figures, such as the relation between the gaps in the alignments and the assignment of each channel to one of the TRP subfamilies. In the absence of a quantitative analysis of these features, it is difficult for readers to assess the robustness of these findings.

It is also difficult to determine the validity of the proposed patterns of signature-residue conservation in non-TRP channels, because the considerable structural and sequence-level differences between these and TRP channels make it non-trivial to identify structurally equivalent positions - structural information to support these assignments is not shown for non-TRP channels.

In regards to the structural conservation of 'signature' positions in different TRP channels, only select structures are displayed showing only one TRP channel per subfamily - this introduces some uncertainty in the extent to which each of these positions is conserved at a structural level for all channels included in the multiple sequence alignment. For many TRP channel subtypes structures in different conformations have been determined, often showing considerable differences in conformation - it is unclear from the text in the manuscript whether the structural-level analysis that was performed was limited to the structures included in the figures, or whether a more systematic analysis was carried out that included a larger sample of TRP channel structures.

The data shown supporting the analysis of state-dependent changes in residue connectivity does not provide much insight - the changes in interactions between the signature residues and other regions of the channel are difficult to see, and the changes observed in the figure are largely dependent on the way each pair of structures were aligned, yielding limited mechanistic information. Furthermore, the functional state of most structures is not known, making it difficult to compare conformational changes between different TRP channel types because it is uncertain whether the pairs of structures being compared truly reflect equivalent conformations. Finally, the main text refers to analysis of TRPV1/2/3/5/6, TRPC3/4/6, TRPM2/4/8 and TRPA1, but only data for two conformations of TRPV1, TRPM8 and TRPC3 is shown.

Reviewer #2 (Public Review):

This paper highlighted a set of conserved amino acids in the larger TRP channel family and ascribed various functions to them, especially to the "aromatic core," in channel behavior.

The major strength of this paper is that it demonstrates a number of deeply conserved residues in TRP channels and proposes some ways in which these may influence TRP channel activity. Its main weaknesses are that its evolutionary analysis is lacking by the absence of some important TRP channel families and, most importantly, its lack of functional tests of the proposed hypotheses of the role of these conserved residues in channel function.

The finding of a conserved interaction between residues on the TM4 and TM5 (W549 and F589) is significant to those interested in TRP mechanisms, but there is no functional data for F589 or structural comparison analysis of the two sites in the apo and bound states.

In addition, without access to the supplementary data, the reviewers cannot adequately assess the quality of the evolutionary analyses. Specifically, the authors did not include reviewer links to the Dryad repositories, and the DOIs have not yet been activated, so I cannot access the data or attest to its quality.

Reviewer #3 (Public Review):

This study focuses on finding common evolutionary and structural characteristics of the wide and elusive superfamily of transient receptor potential (TRP) ion channels, present in almost all animal species. The aim is to use standard sequence- and structure-based bioinformatics techniques to find conserved residues and analyze their structural and functional importance in the TRP families.

The manuscript is written in a clear and accessible way, and provides an interesting overview on the different pieces of information about structural and functional relevance of TRP proteins. The references to the literature are not only helpful for the reader, but also important for the comparative analysis performed. The bioinformatics techniques used are very well-established and applied with care, which guarantees the reliability of the analysis. On the other hand, more recent and advanced methodologies that could have offered even more insights have been disregarded.

Through database cross-referencing, multiple sequence alignment, phylogeny studies and comparison with literature, the authors identify a group of 11 (mostly) aromatic amino acids with >90% conservation. Structurally, they are grouped in three regions (patches) of functional relevance. Structural properties of each patch are identified and compared with previous studies.

This work is an important reference to any researcher who studies structural and evolutionary aspects of TRP families. It includes an impressive amount of well-organized information, which the authors are able to complete with their own original analyses.

Reviewer #2 (Public Review):

In all vertebrate species investigated, cerebrospinal fluid contacting the cerebrospinal fluid express the channel PKD2L1 (in macaques, mice and zebrafish: Djenoune et al., Frontiers in Neuroanatomy 2014; in lamprey: Jalalvand et al., Current Biology 2016b; Jalalvand et al J Neurosci 2018). However, in all species investigated these cells fall into two functional types based on their axial sensitivity to detect spinal curvature (in vivo for zebrafish: Bohm et al., Nature Communications 2016; Hubbard et al., Current Biology 2016), expression of neuropeptides and neuromodulators (in lamprey;: Christenson et al., Neurosci Letter 1991; Schotland et al., JCN 1996; in zebrafish: Djenoune et al., Scientific Reports 2017) or their firing patterns (in mouse: Petracca et al J Neurosci 2016; Di Bella et al., Cell Reports 2019).

While the microscopy techniques used here are outstanding and bring without a doubt important evidence on the location and density of DSVs, there are concerns to address regarding the consolidation and interpretation of the physiological recordings of the ciliated neurons and pharmacology based on evidence that only ASIC1 channel is expressed in lamprey (see phylogenic analysis: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047259/), and that the lamprey ASIC1a is proton insensitive (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464184/).

Reviewer #1 (Public Review):

In this paper Jalalvand et al. employ various imaging techniques to investigate the function and physiology of cerebrospinal fluid contacting (CSF-c) neurons in lamprey. By combining expansion microscopy techniques with lattice light sheet microscopy, they can reveal the spatial organization of somatostatin and dopaminergic neurons along the spinal cord. Zooming into these neurons with STED super-resolution microscopy they can show that somatostatin and dopamine are stored into dense core vesicles. Combining STED with alteration of extracellular pH and electrophysiology the authors could show that somatostatin, but not dopaminergic neurons are responsible for pH regulation. Further, they employ expansion microscopy combined with STED to investigate the architecture of cilia in the two neuronal types and discover that somatostatin neurons have mainly sensory cilia while dopaminergic neurons have motile cilia.

Based on these observations, the authors suggest that pH/mechanosensitive somatostatin-expressing CSF-c neurons are sensory neurons. On the other hand, non-pH- but mechanosensitive dopaminergic CSF-c neurons may act as cerebrospinal fluid flow generator. I believe the data presented nicely support these claims.

Data is of excellent quality, considering the technical challenges or working with expansion microscopy and STED super-resolution in tissues. Expansion microscopy STED is extremely challenging in cell monolayers and the data in expanded tissues is of extremely good quality. Additionally, application of the same work pipeline to other tissues/cell types promises to help us gain insights into whole organism physiology down at the single cell level.

Reviewer #3 (Public Review):

This manuscript uses a variety of optical superresolution techniques to explore the structure and function of different cerebrospinal fluid contacting (CSF-c) neurons. First, Expansion Microscopy and Lightsheet Microscopy are combined to image large volumes of tissue from lamprey and to demonstrate the known organization of somatostatin-expressing and dopaminergic CSF-c neurons (Fig. 1). The authors then used STED to explore the subcellular location of somatostatin and dopamine in CSF-c neurons and demonstrate their presences in vesicle-like structures ranging between 60 and 200 nm (Fig. 2). Subsequently, the relation between GABA and somatostatin is examined in somatostatin CSF-c neurons. The authors show that there is no obvious colocalization between these molecules and that only somatostatin levels are altered in response to changes in the extracellular pH (Fig. 3). The authors furthermore demonstrate that dopamine levels with dopaminergic CSF-c neurons are also insensitive to pH changes (Fig. 4).

The authors have previously shown that somatostatin CSF-c neurons are mechanosensitive and now also demonstrate this for dopaminergic CSF-c neurons (Ext Data Fig. 1). They also show that their mechanosensitivity is mediated differently (ie. Not through ASIC3). To further explore this difference in mechanosensitivity, the authors set out to explore ciliary structure of these ciliated neurons. By combining expansion and STED, the authors succeed in resolving ciliary ultrastructure and demonstrate that they can distinguish between between motile (9+2) and primary cilia (9+0) (Fig. 5). They find that the majority of cilia on somatostatin-expressing CSF-c neurons is primary, whereas all cilia on dopaminergic CSF-c neurons were motile (Fig. 6).

Overall, this is an interesting imaging study that reports a number of technical steps that enable tissue-imaging with exquisite detail, such as discriminating between motile and primary cilia. It also nicely demonstrates what sort of new data can now be obtained in tissue, e.g. changes in vesicle numbers upon certain stimuli. However, as explained below, both the composition of the main text and the reproduction quality of the figures make it hard to judge the biological significance of this work.

Comments:<br> 1/ Overall, I feel the paper needs a thorough rewrite. The introduction should give more insights into the underlying biology and also clarify which questions are being asked and why these are important. Currently the introduction is mostly a long summary of all results, but it doesn't help to understand the biology that underlies this work. Because of that the different experimental pieces currently feel a bit random and disconnected.

2/ The reproduction quality of Figures 1, 2, 3 and 6 in the merged PDF is not great. In many cases, I cannot read the annotations or appreciate the content of the images. This make it rather impossible to judge the quality of the work. The data shown in Figure 5 is very impressive and I am sure the raw data for the other figures is equally great, but I can only judge what I see for myself.

3/ Page 8: the conclusion that PKD2L1 is the mechanosensitive receptor for dopaminergic CSF-c neurons is only based on its presence in this cells. To really demonstrate this loss-of-function experiments would be needed.

Reviewer #1 (Public Review):

In this paper, the authors study "recruitment" (see below) of spinal motoneurons over a 3 week developmental period. They show that properties change, and that properties of late-firing vs immediate firing MNs diverge over this period. With respect to firing, they show that the development of "persistent inward currents" and currents mediating sag could account for these changes. They suggest that the divergence of properties mediated by these currents leads to a wider range of recruitment and contributes to orderly recruitment of MNs.

Overall, this is a very good manuscript with experiments well done. The large numbers of recordings are very helpful. The data are convincing that the range at which action potentials are recruited broadens over this time period, and that the currents studied contribute to this broadening. This is of fundamental interest to neuroscientists studying movement.

However, there are a few limitations:

1. The authors use a surrogate marker for "slow" vs "fast" MNs: immediate vs delayed firing in response to rectangular current injection. They switch their language to call these motoneurons slow and fast, but they should be more cautious about doing so, given that firing is a surrogate marker that has not been fully studied and characterised across this time period. We do not know if developmental changes in Kv1 might also contribute to the effects on spiking seen here. I agree, there is delayed firing from the outset, which is interesting in itself given the rather homogenous other properties. But that doesn't meant that Kv1 expression is stable and thus non-contributory to the changes in firing described.

2. The focus of the manuscript is on MN recruitment, but recruitment is never defined, despite being used in the title, through the abstract and key points, as well as throughout the manuscript. What they are looking at is response to current injected at the soma vs recruitment during a behaviour when synaptic inputs are bombarding an extensive dendritic tree. Thus, this manuscript does not look at recruitment per se, but rather activation of action potentials in response to intra-somatic stimulation. Accordingly, the term "recruitment" would be best kept to the Discussion. (I also note that the "size" principle relates more to electrical than physical size in the 21st century; I agree that the two are correlated, but the authors may not want to stick to arguments about physical size.)

3. Figure 6 is problematic, possibly in the way it's presented. It seems to me, but not clear, that the authors suggest that Ih is active at rest, more so in "F" than "S," and that therefore "F" are more depolarised and have smaller mAHPs. So (a) how come RMP didn't seem to come out in the PC analysis earlier? (b) would that not suggest that "F" would be "recruited" earlier? (Or is it that there is reduced sodium channel availability because of the depolarisation - what are the differences in spike amplitude and rate of rise?) (c) shouldn't the mAHP be larger if the cell is more depolarised and further from the potassium equilibrium potential? On the last note, maybe it's that Ih makes the mAHP smaller, but with the kinetics of Ih, wouldn't the decay be faster, but in Fig 6 the decay seems faster when Ih is blocked? Finally, Fig 6E suggests changes in the fAHP and delayed depolarisation (and spike width?) - how to these come into the picture? If the fAHP is thought to result from a high conductance state, and if therefore one were to align the voltages based on this potential, then the mAHPs would be about the same amplitude? The authors likely have explanations, but I'm afraid that I can't follow it.

4. A number of labs have looked at development of MN properties, and it would be useful to compare properties seen across different labs, for example Quinlan (e.g. PMID: 21486770) and Whelan (e.g. PMID: 20457856, which is only mentioned in the manuscript) (and for that matter, mine - e.g. PMID 10564356, PMID: 32851667 although I don't want to self-promote).

5. In the Discussion, the authors might want to discuss propensity of F vs S MNs to express PICs / sustained firing as described in the Heckman lab (albeit particularly in cat; see PMID 9705452, for e.g.). How do these data correspond?

Reviewer #2 (Public Review):

The manuscript deals with a fundamentally important tissue in motor control, the mechanism governing the orderly recruitment of spinal motoneurones. It is widely accepted that this is a consequence of excitability differences intrinsic to the different functional classes of motoneurones. For decades, the dogma has been that the recruitment order of spinal motoneurones is governed primarily by cell size which influences input resistant and this has been the standard textbook explanation. With an increasing number of reports not finding a convincing size difference between slow and fast motoneurones in rodents, it is now clear that this explanation does not suffice. This manuscript expertly addresses this issue with a set of elegant experiments to investigate how the intrinsic properties of slow and fast motoneurones become tuned during development to ensure the later recruitment of fast motoneurones. They show that the size is only a primary determinant of recruitment currents at developmental stages. With further development by post natal week 3 the difference in recruitment currents of fast motoneurones have diverged from slow motoneurones as a consequence of increases in the activation threshold of sodium persistent inward currents combined with an active hyperpolarization activated inward current (Ih) at resting membrane potential in fast motoneurones.

These experiments were expertly designed, executed, analysed, presented and discussed with the results clearly supporting the conclusions. The experiments carefully balance the need for the recordings to be made from functionally mature mice with the benefits of an in vitro preparation allowing pharmacological manipulations to test the hypotheses. This is made possible by the group's expertise in spinal cord slice preparations and the authors should be particularly commended for this as very few laboratories have succeeded in obtaining viable healthy slice preparations of adult spinal cords for motoneurone recording.

The data convincingly demonstrate a casual role for the persistent sodium inward currents and Ih currents in determining the recruitment currents for fast versus slow motoneurones. The only minor caveat is that due to the potential loss of dendritic L-type calcium channels in a slice preparation combined with the use of intracellular current injection to the soma to recruit motoneurones, this makes it difficult to conclude that L-type calcium do not also play a role in influencing recruitment order under normal synaptic activation. However, the influence of the sodium and Ih conductance is so convincing that these results alone mean that we must finally re-write the text book explanation for the fundamentally basic principle of recruitment order of motor units.

Reviewer #3 (Public Review):

There are different types of motoneurons based upon the muscles they innervate. The recruitment order of these motoneurons has long been believed to be due simply to differences in the size of these motoneurons, which leads to differences in input resistance and threshold currents. The authors used recent electrophysiological signatures of fast versus slow motoneurons to study whether active conductances in these neurons developed differently during the early postnatal stages of the mice when these animals start to weight bear and show more coordinated movements.

The experiments consist of electrophysiological recordings of motoneurons in slice preparations of the spinal cord. Motoneurons are categorized based on several electrophysiological properties previously shown to differentiate motoneurons. The recordings are well done and the data analysis shows strong support for their conclusions that during the second week of development, fast motoneurons and slow motoneurons start to differentiate themselves due to the properties of a persistent inward current and a hyperpolarization-activated current. The main weakness of the paper is a confirmation of the type of motoneuron recorded with a technique that does not rely solely on the electrophysiological signature.

As stated above, the results are convincing. There are clear differences in the properties of the currents that are being studied, and their analysis suggests that cell size is a weak predictor of possible recruitment order.

The findings are likely to push those in the field of motor control to reconsider how the maturation of motor circuits can also happen at the level of intrinsic properties of spinal neurons

Reviewer #4 (Public Review):

Motoneurons (MNs) are classified in 3 subtypes according to contractile properties of the innervated muscle fibers [Fast-contracting Fatigable (FF); Fast-contracting fatigue-Resistant (FR); Slow contracting fatigue resistant (S)]. To cover the full range of locomotor frequencies, MNs are recruited in the sequence S, FR and FF. According to Henneman, this orderly recruitment correlates to the size of MNs : S-MNs are the smallest, FRs-MN have an intermediate size and FFs-MN are the largest. The rationale is that the smallest MNs are activated prior to the largest as a consequence of their high input resistance. However, because the excitability of motoneurons is also regulated by nonlinear conductances of membrane ion channels, differences in ionic conductances between MN subtypes (related to differences in the expression of channel subtypes) may contribute to the Henneman's principle. In this framework, the authors performed a longitudinal study of the electrical properties of MNs in mice to distinguish between passive and active properties that secure the orderly recruitment of MNs.

Based on extensive analysis of electrophysiological intracellular recordings, the authors suggest that the recruitment of fast and slow MNs is similar and mainly based on size during the first postnatal week, then differs partly due to a differential maturation of active properties between motoneuron subtypes. Specifically, the authors show that fast motoneurons display a more depolarized threshold for persistent inward currents (PICs) during the 2nd postnatal week and a prominent hyperpolarization-activated inward current during the 3rd postnatal week, both delaying the recruitment of fast MNs relative to slow motoneurons. The present study provides significant insights on the maturation of electrophysiological properties in diverse MN subtypes, but key claims on ionic mechanisms contributing to the orderly recruitment of MN subtypes appear not enough supported by data presented. In particular:

1) During the 1st postnatal week, authors suggest that fast and slow MNs cannot be distinguished neither on their passive properties nor the rheobase and therefore their recruitment is mainly based on the size. The conclusion that the recruitment is not linked to MN functional differences is difficult to follow since the main distinction between MN subtypes is based upon the presence of a delayed firing, an active property that regulates the recruitment of MNs (Leroy et al., 2014). However, the square current pulse adopted to discriminate between delayed and immediate firing in Figure 1 was replaced with a ramped depolarization protocol on which the authors measured the rheobase (Figure 3A1). This suggests that the slow depolarization in immature motoneurons might minimize the activation of ionic conductance(s) responsible for the delayed firing and thus may bias the measure of the rheobase (the minimal current amplitude of infinite duration). In line with this, the recruitment of a motoneuron has been shown to depend on the rate of membrane potential depolarization preceding a spike (Krawitz et al., 2001). Rather than using a slow ramp depolarization, it therefore seems more appropriate to assess MN rheobase with the current pulse protocol used to distinguish between MN subtypes. With this kind of measure, differences in the excitability of MN subtypes related to active conductances may come out earlier during development.

2) During the 2nd postnatal week, the study suggests that "PICs contribute to the emergence of orderly recruitment amongst MN subtypes". This interpretation appears too definitive because the study did not provide direct evidence for that. The authors describe a more hyperpolarized activation of PICs in slow MNs suggesting that the early recruitment of PICs in slow MNs may help them to fire before fast MNs. By a pharmacological approach the authors show that the sodium PIC mediated by Nav1.6 channels sets the activation threshold of PICs and that their blockade increases the rheobase (recruitment current). However, since pharmacological investigations have been done only in fast MNs, it is not very informative on the putative role of the sodium PIC (and PICs in general) on the orderly recruitment of MN subtypes. Similar experiments should be extended to slow MNs to compare the effects with those observed on fast MNs. If sodium PIC plays a significant role in the differential recruitment of MN subtypes, its blockade should induce an overlap in the recruitment of slow and fast MNs. Furthermore, voltage clamp recordings to characterize PICs in MN subtypes have been done without blocking potassium conductances. Therefore it is difficult to determine if differences in PICs between MN subtypes are related to inward currents or opposing outward currents.

3) During the 3rd postnatal week, the authors suggest that fast MNs display a prominent Ih current at rest that provides a depolarizing shunt delaying their recruitment compared to slow MNs. However, data appear not enough conclusive for such interpretation. First, the relationship between the resting membrane potential (RMP) and the amplitude of Ih (the larger the Ih, the more depolarized the RMP) depicted in Figure 6B from a small sample of MNs is not consistent with values reported in the supplementary table 1. Indeed, fast motoneurons supposed to have a prominent Ih current display a more hyperpolarized RMP compared to slow MNs. The opposite would be expected according to the authors' hypothesis. A similar concern can be raised regarding the strong relationship between the amplitude of Ih and that of the AHP illustrated in Figure 6F, which is not in line with the lack of difference in the amplitude of the AHP between slow and fast MNs in week 3 (see supplementary table 1). Second, the inward current recorded in fast MNs to hyperpolarization at -70 mV appears not significantly affected by the Ih blocker ZD7288 (Figure 5J,and 5L) suggesting that Ih is not recruited at rest in this class of MNs. On the other hand, ZD7288 hyperpolarizes the RMP in fast MNs (Figure 6A) and reduces the amplitude of their sags recorded at -70mV (Figure 5M). Similar discrepancies are more striking for slow MNs. Slow MNs did not display inward current sensitive to ZD7288 above -80 mV (Figure 5N). However, ZD7288 unexpectedly hyperpolarizes their RMP (Figure 6C). How the authors can explain such discrepancies? Finally, there is a mismatch in values reported in supplementary table 2 and figures 5G and 5I. In the table, both Ih amplitude and Ih density (at -70mV) appear significantly different between slow and fast MNs in week 3, but not in figures 5G and 5I. Altogether, these results appear inconsistent. Regardless of inconsistencies, data should be replicated at least with a second Ih blocker such as Ivabradine hydrochloride or Zatebradine hydrochloride.

Minor concerns:

1) Does the pharmacological blockade of Nav1.6 channels 4,9-AH-TTX induce changes in the spiking threshold as already reported in cortical neurons (Hargus et al., 2013)? Such an effect may contribute to the higher rheobase observed in fast MNs under 4,9-AH-TTX (Figure 4M).

2) The study reported a more depolarized PIC in fast MNs during the 2nd postnatal week but the acceleration onset voltage in response to a current ramp depolarization (attributed to the activation of PICs), is similar between slow and fast MNs at the same age (Figure 4C). This is in discrepancy with figure 4G, where a significant effect on PIC onset voltage is shown within the same time points

Reviewer #1 (Public Review):

Understanding why some immune stimuli (e.g. influenza vaccination) induce worse immune responses than others is important both as a matter of fundamental biology, and in order to improve vaccine design. The authors provide significant new insight into this: they measure the prevalence of evolution in B cell lineages responding to a variety of stimuli, and compare these to similar measurements in influenza vaccine response. They show that evolution is more prevalent in cases where it is expected (e.g. HIV, prime vaccination) vs not (e.g. boost vaccination), and that while lineages in blood with detectable evolution are rare after influenza vaccination, they are likely present, and are more prevalent in GC samples. They apply an existing metric to B cell receptor repertoires for (I believe) the first time, while adding an improvement to it (polytomy resolution).

The basic idea – when you have longitudinal data, focusing on lineages whose SHM increases with time – is on the one hand quite obvious, but on the other, the authors demonstrate both that it's a more accurate technique than I would have expected, and that the particular method they use of quantifying "SHM increase" is a good one. Both of these things are of significant value. Those interested in understanding poor immune responses, as well as those looking to find evolving lineages in B cell receptor sequencing data more generally, will likely find the publicly available software useful (although note that I did not test the software).

Reviewer #2 (Public Review):

The manuscript by Kenneth Hoehn et al. investigates the evidence for detectable evolution across longitudinal samples of BCR (B cell receptor) repertoires. The authors set out to quantify the degree to which such continued evolution can be observed in existing high-throughput BCR repertoire sequencing datasets. They focus in particular on the context of influenza vaccination with corresponding recent datasets (Turner et al. 2020) with blood and lymph node samples.

The authors applied a robust statistical framework that they tested extensively on synthetic data and proved to be a relevant tool to analyze immune repertoires. Over 20 distinct datasets were analyzed to cover a wide range of conditions of acute and chronic infections as well as healthy individuals and their response to vaccination. This comprehensive analysis is valuable in the field where consistent analysis of different datasets is not found often. The key findings, that is an enrichment in certain conditions, notably HIV infections, and lack of thereof in others, support previously known findings.

The method of detecting measurable evolution between timepoints adapted to B cells sheds new light on long-standing questions about affinity maturation, particularly ones of formation of the memory repertoire and its reentry to germinal centers upon vaccination. Due to a limited number of datasets and donors in each cohort, as well as limited sequencing depth, this approach has a number of limitations. New experiments are necessary to fill in the gaps in our understanding of the evolution of B cell repertoires. The important contribution of this work consists not only in a useful review of existing datasets but also in suggesting new directions in studies based on high-throughput sequencing.

Reviewer #3 (Public Review):

Hoehn et al. studied the somatic evolution of human BCRs after immune stimulation. Using a phylogenetic approach to analyze publically available longitudinal BCR repertoire sequencing data, they examined whether and how mutations accumulate in BCR clones over time after primary or secondary immune stimulus. They focused particularly on the important open question of whether seasonal influenza vaccination elicits de novo somatic mutation and clonal evolution. The main conclusion of the study is that germinal center-associated B cell clones undergo continued evolution after seasonal influenza vaccination.

On the whole, the work addresses an important question, the analysis approach is innovative, and the conclusions are generally well supported. However, there is one major problem consisting of an alternative explanation for the central result, which must be ruled out in order for the result to be compelling.

Major concerns:

1. The most substantive concern is that there is an alternative explanation for the data which must be ruled out in order to conclude that mutations are occurring during the study period. Consider the following scenario. Suppose that B cell clones expanded and diversified through somatic hypermutation prior to the study period (that is, prior to the secondary vaccination event which is the focus of the study). It seems that preferential expansion of highly mutated subclones during the study period could bias detected sequences towards more divergent sequences, even without ongoing somatic mutation during the study period. Preferential expansion of divergent sequences would give rise to higher average divergence as the study period goes on, giving the appearance of accumulation of additional mutations, but in fact these mutations had occurred prior to the study period and are simply more readily detected in the sparsely sampled repertoire sequencing data after their expansion. Far from being simply a pathological counter-example, this scenario seems biologically plausible, given that B cells harboring more divergent, affinity-matured sequences should generally have higher affinity antibodies that allow them to better compete for limited antigen and thus provide stronger division stimulus. This model predicts that some highly divergent sequences exist at early timepoints and would occasionally be detected. An example of this is seen in Fig 3C, where a divergent tip from an early sample time is present (labeled PB and colored blue, in the middle of the diagram), indicating that this divergent sequence was present early. While the authors' model of ongoing evolution is supported, this alternative model also appears to be consistent with the data and must be ruled out in order to conclude that clones are accumulating mutations during the study period, which is the central claim and most interesting and impactful finding of the work. The authors must provide evidence that their approach can distinguish between these scenarios. This could potentially be accomplished using simulations of the two scenarios to determine the power of the approach to distinguish between them.

2. Statistical support for measurable evolution appears to be lacking in several key examples. The reported percentages of measurably evolving lineages in several scenarios (7.2% for primary hepatitis B vaccination; 6.5% for allergen-specific immunotherapy; 5.9% for HIV infection) are near the false positive rate of the test (5% of lineages measurably evolving). The authors have performed this test on datasets from ~21 studies, raising a concern that multiple hypothesis testing could give rise to false positives in some of the datasets. These results are interpreted as evidence of measurable evolution, even though they could seemingly be explained by the false discovery rate combined with multiple hypothesis testing. The authors should clarify how these results can be interpreted in light of the false positive rate of their test and multiple hypothesis testing, and must consider whether more conservative conclusions are warranted in these scenarios.

Minor comments:

1. The authors define measurably evolving populations as systems undergoing mutation and selection rapidly enough to be detected. Despite this definition, the test employed for measuring evolution appears to focus purely on accumulation of mutations without examining selection per se. Mutations accumulating neutrally could be detected as measurable evolution. For the sake of clarity, the authors should explain more clearly whether their test examines selection and ensure that initial definitions are consistent with later usage. It may be interesting to further examine whether the mutations detected as measurable evolution in antibody lineages are neutral or selected using classical tests for selection, such as the dN/dS statistic or summary statistics of the site frequency spectrum.

2. Statistical support for the association between GC B cells and measurable evolution should be clarified. On p14 L7-8, it is reported that "6.5% of lineages containing sequences from GC B cells were measurably evolving, compared to only 3.7% of lineages with no identified GC sequences." However, this does not constitute convincing evidence for the association because this difference of proportions is not significant. The proportions are 3 lineages measurably evolving among 46 lineages containing sequences from GC B cells, and 4 lineages measurably evolving among 107 lineages not containing sequences from GC B cells. Applying Fisher's exact test for a difference of proportion yields P = 0.43. While the evidence based on the trend in Fig 3A (of fraction measurably evolving against GC sequence percentage) is compelling, the authors should clarify whether each difference of proportions is significant. Providing statistical support for the trend itself, such as through bootstrapping or simulation, would seem most direct.

Reviewer #1 (Public Review):

In this manuscript Lin et al. report the effect of a Traditional Chinese Medicine, the water extracts of GE (WGE), to suppress neurodegeneration in LRRK2-G2019S flies. They argue that WGE suppresses the accumulation and hyperactivation of LRRK2-G2019S proteins in dopaminergic neurons. WGE also activates the antioxidation and detoxification factor Nrf2 in astrocyte-like and ensheathing glia. This in turn antagonizes LRRK2-G2019S-induced Mad/Smad activation in the glia. Moreover, the authors show that the role of WGE to suppress the hyperactivation of LRRK2-G2019S proteins and regulates Smad2/3 pathway are evolutionally conserved in the brain of the LRRK2-G2019S transgenic mice. Furthermore, WGE also suppresses the locomotion declines, the loss of dopaminergic neurons, and the up-regulation of the number of the hyperactive microglia in the LRRK2-G2019S transgenic mice.

In summary, the authors provide compelling genetic, biochemical and behavioral data to reveal the function and the mechanism of WGE and its bioactive compounds, gastrodin and 4-HBA, to suppress neurodegenerative phenotypes in two LRRK2-R2019S animal models (flies and mice). Given that the function of WGE is evolutionally conserved in flies and the brain of mice, their data suggest that WGE and its bioactive compounds can be potential therapeutic drugs for PD. Moreover, this manuscript is well written and the authors provide sufficient data to support most of their arguments.

Reviewer #2 (Public Review):

In this study, Lin et al. utilized a well-established fly model of Parkinson's disease (PD) carrying the LRRK2-G2019S mutation to demonstrate the mechanisms underlying the neuroprotective effects of Gastrodia elata (GE). They found that G2019S flies treated with water extracts of GE (WGE) or its bioactive components (Gastrodin and 4-HBA) exhibited an improvement in locomotion as well as protection of dopaminergic neurons. They then continued to elucidate the mechanisms of WGE beneficial effects in PD flies. WGE exerts its function via two distinct modes. First, it downregulates the total and active forms of G2019S proteins in dopaminergic neurons. Second, as nicely demonstrated by the use of reporters and tissue-specific drivers, WGE activates the antioxidant factor Nrf2 in the astrocyte-like and ensheathing glia, rather than in dopaminergic neurons. Activation of Nrf2 is essential for the antagonization of G2019S-induced Mad/Smad signaling in glia. Importantly, the findings from the fly model are applicable to the mouse G2019S model, and similar pathways were implicated in WGE-treated mice. LRRK2-G2019S transgenic mice treated with WGE also show reductions in activation of G2019S proteins and Smad2/3 pathways. This study elucidates the molecular mechanisms whereby GE confers neuroprotection in G2019S-induced PD and provides a potential therapeutic treatment for PD patients. The experiments were very well designed; the images are high-quality and convincing. The conclusions are well supported by multiple of lines of genetic and biochemical evidence. It is a pleasure to read this beautiful manuscript.

Reviewer #1 (Public Review):

The authors propose two main advances. First, they present a novel mechanistic model of metabolic cost that is proposed to account for phenomena not explained by current models. They demonstrate using a combination of elegant experiments and simple models that the limitation of current models is in their inability to account for the energetic cost associated with the rate of developing force. Second they demonstrate that this novel model of metabolic cost is capable of reproducing stereotypical arm reaching trajectories, and thus provides an energetic basis for smooth movements. The novelty of this second advance is that thus far reaching movements have been oft explained as the consequence of maximizing accuracy. This new metabolic model, suggests that energetics play a role in determining movement control.

The first advance is compelling and will add significantly to our current understanding of the fundamental basis of metabolic cost. The experiments are thorough, the cross-validation is impressive, and the model is simple, yet elegant. The second advance, energetic cost as a basis for movement control, could be strengthened. Firstly, energetic cost is already generally assumed to play a role in movement control, it is the quantification that has been difficult. This paper thus provides such a quantification (with its first advance). Demonstrating that it can explain bell-shaped profiles, while nice, feels lacking given the number of cost functions that can generate those same profiles and the fact that other factors such as accuracy likely play a role. Further tests of the metabolic model's ability to explain features of movement control will help strengthen the work. Similarly, examples of where the model fails will also help determine its limits and identify future directions. Taken together, the authors hope to demonstrate the significance of this model of metabolic cost on movement control, yet support is lacking.

Reviewer #2 (Public Review):

This paper shows that the rate of muscle force production as cost function term predicts the metabolic cost of reaching movements better than other more commonly used terms. This an important contribution to the field and will be of interest to the motor control and biomechanics field.

The work is an initial attempt to unify two theories in motor control: minimum-variance (focused on endpoint accuracy) and energy minimization as in optimal feedback control. This work shows that a new energy minimization term (the rate of muscle force production):<br> 1) more accurately metabolic cost on their experimental data;<br> 2) produces smooth movements; thereby offering an initial attempt to unify energy minimization with minimum-variance theory.<br> Their first contribution is clearly important and sufficient for this work to have broad impact. The second contribution, while interesting and potentially very important, is more speculative and not fully fleshed out. For instance, I believe that optimal feedback control (Todorov 2002) also predicts smooth motions, and, more importantly, minimum-variance theory predicts more than smooth motions (i.e., the speed-accuracy tradeoff). The new cost function term has no impact to the speed-accuracy tradeoff (energy impacts movement duration, but movement duration does not increase when accuracy requirements increase). Hence, the unification proposed in the abstract remains to be accomplished, which doesn't take anything out of this important contribution to the field.

Reviewer #1 (Public Review):

This manuscript begins with the larger notion that comparing item similarity is an important principle that guides human behavior, and that these similarity representations can have both a general and an idiosyncratic component. While individual-specific representations have been identified in some visual processing areas using personally meaningful object stimuli or simple stimulus features, this study looks at complex real-world stimuli with no personal meaning. They observe that even using the same stimuli across people, there are differences in how people judge the similarity of same-category items, and these differences correlate with performance on comparing the identities of images presented in sequence. Further, they examine the visual stream – specifically early visual areas like the early visual cortex (EVC) and lateral occipital complex (LOC), and late visual areas like the perirhinal cortex (PRC) and the anterior lateral entorhinal cortex (alErC), to see how representations in the brain relate to these behavioral representations. They observe that while EVC and LOC show correlations with behavior, PRC and alErC really show the strongest links to the individual-specific representation and to fine-grained differences across stimuli.

The analyses in this paper are very methodologically sound. They rely on well-controlled and well-tested analyses (e.g., testing if representational similarity is indeed higher for comparisons with one own's behavior, versus someone else's). They also replicate their results using classification-based analyses. My only key methodological question is more about experimental design. Given that participants performed the object arrangement task right before entering the scanner, I wonder if similarities in the brain to their own behavior could be due to memory for the representation they created just prior (especially given the role of PRC and alErC in memory). So, if instead, participants were shown and interacted with someone else's similarity arrangement, I wonder if these regions would show more similarity to that other person's arrangement, or still show similarity with one's own representations. It is thus currently unclear if the current findings are due to some deep-seated individual, internal representations, or memory for a recently performed task.

The results presented in this work are very clear and fit in well with previous findings on idiosyncracies in visual areas (Charest et al., 2014), and various work on the PRC as it relates to oddball tasks and object representational similarity. One question I am stuck with in this work is whether these current results show us something surprising or new. I'm unsure if we would have expected anything different for these generic real-world stimuli (versus the personally meaningful stimuli, or limited visual features tested previously). The manuscript frames the study around the idea that similarities are an important guiding principle of behavior. But this statement is not necessarily so obvious to me – is judging similarity itself an important ecological behavior, or is it just that looking at similarity structures can give insight into underlying relationships in how we represent information? (The latter is how I often see these sorts of representational similarity analyses.) What is this similarity task really capturing about our representations for these objects, and why do these idiosyncrasies emerge? My main hesitation about the current work is that I struggle with seeing a scope beyond a replication (e.g., finding behavior-correlated idiosyncracies in the brain, but with a different stimulus set, and in a slightly similar but expected region). I really want to know what factors are driving these idiosyncracies (e.g., is it visual? mnemonic? semantic?), and what this implies about the mechanisms of the PRC and ERC.

Reviewer #2 (Public Review):

The current manuscript describes a novel application of RSA to an fMRI dataset comprising task-related responses to real-world object stimuli. The authors scanned younger adults (n=23) while they discriminated between 4 different object exemplars for 10 different categories; participants also completed subjective similarity ratings between all pairs of objects, based on a validated iMDS approach (Kriegeskorte & Mur, 2013). The value of this work rests on 2 main findings. First, perirhinal and entorhinal cortices predicted subjective similarity, even when item comparisons were restricted to exemplar comparisons of different levels of similarity. Second, the subjective similarities predicted discrimination performance in an observer-specific manner, such that Brain-Model similarities are higher within- than across-individuals-and only in PRC and ERC. There are many follow-up and supplementary analyses (whole-brain searchlight analysis, SNR calculations), all of which help to support the main findings.

Reviewer #1 (Public Review): 

The manuscript by Ahmad et al. reports on the high-resolution crystal structures of sugar and a β-lactam molecule binding to an allosteric site of Mycobacterium tuberculosis L,D-transpeptidase (LdtMt2). The structural changes upon dual ligand binding highlight the structural alterations that span across the allosteric pocket and the catalytic site. The authors systematically mutate residues in both, the allosteric site and the catalytic site and use biochemical assays to show how they are linked. 

Strengths: <br> Structural, biochemical and Mutagenesis experiments <br> (a) High-resolution crystal structures of LdtMt2 with ligands in both allosteric site and orthosteric site. <br> (b) The study unequivocally demonstrates that residue S351 forms a part of the catalytic triad and not S337, as previously reported in the literature. <br> (c) Residue R209 in the allosteric pocket is directly linked to the catalytic activity. 

Weakness: <br> Computational studies <br> (a) How exactly are the two sites structurally linked across a distance of >20Å is not explained.

Reviewer #2 (Public Review): 

In this work, authors seek a better understanding of the L,D-transpeptidase class of enzymes. They investigate Ldt-Mt2, 1 of five L,D-transpeptidase paralogs rom Mycobacterium tuberculosis. They determine a crystal structure of LdtMt2 and identify a pocket between two domains YkuD and IgD2. This pocket is hereafter referred to as the S-pocket. In summary, authors conclude that the S-pocket is an allosteric site that controls catalytic activity of Ldt-Mt2. 

This study is potentially very exciting and very important, as the report of this allosteric site is novel and might be a prevailing mechanism across other L,D-transpeptidases. 

There are two major issues that should be addressed. 1) Because of the nature of the evidence presented to support the existence/role of the S-pocket (i.e. computational modeling, ligands modeled into electron density, a single disruptive mutation), it would be helpful for the authors to provide an additional piece of data to support the existence of the S-pocket. 2) The computational studies as currently presented, are not rigorous enough to support the claims that are made.

Reviewer #3 (Public Review): 

This study reports on a novel site termed as the 'S-pocket' that is located at a hinge region of the LdtM2 transpeptidase of M. tuberculosis. Building on observations of the presence of a glucose molecule within the crystal structure at this site, the authors hypothesise that this may also serve as a binding site for peptidoglycan precursors such as NAG and model a pentameric peptidoglycan molecule across the S-pocket and the inner cavity of the catalytic site. However, Thermofluor data of the effect of NAG binding is weak. It would be helpful for the readers to compare this data with similar data for glucose. Additional data for other point mutations within the S-pocket as well as a point mutation in the active site (e.g. at the inner cavity) will be needed to fully justify these conclusions. 

The remaining bulk of the manuscript is devoted to understanding the interaction of beta-lactams with the S-pocket. The authors present convincing Thermofluor data to demonstrate a clear synergy between non-covalent interactions at the S-pocket and covalent binding at the catalytic site. However, the MD simulations have not been performed with the same rigour and at this stage, do not add to the strength of the evidence presented. Furthermore, inclusion of animations or videos from the MD simulations performed for this study would significantly help the readers grasp these points in an intuitive fashion versus the snapshots presented here. The authors directly apply this knowledge to understanding the interaction of an experimental carbapenem T203 with the M. tuberculosis Ld transpeptidase, showcasing how this improved understanding of the functioning of LdT transpeptidases in this pathogen can translate to developing new tools to combat this globally relevant pathogen.

Reviewer #3 (Public Review):

Wild-type Chlamydomonas cells possess a pool of ciliary precursors that, in conjunction with newly synthesized precursors, are used to build a new cilium after de-ciliation. The cellular and molecular mechanisms the underly retrieval of the pool are unknown. Given the role of the Arp2/3 complex in endocytosis in other systems, it was reasonable to test whether it also functions in reclaiming of the pool of precursors. The central finding is that cells bearing a mutation in an essential Arp2/3 component, ARPC4, fail to assemble cilia in a timely fashion after de-ciliation. Although the failure of assembly is well-documented here, the manuscript lacks evidence that cells missing ARPC4 actually establish a pool of ciliary precursors in the first place. Without such information it is not possible to determine the cellular function that fails to occur after de-ciliation in the mutant: Retrieval of a pool of ciliary precursors? Establishing the pool during the ciliary assembly that occurs after cell division? Synthesis of the precursors as new cilia are formed? Sensing loss of cilia and activating the events needed for re-ciliation?

Other conclusions also were not sufficiently supported by the experimental results, including the following: Clathrin function in endocytosis: Pitstop2 was described as specifically blocking clathrin-mediated endocytosis, but reports in the literature indicate that Pitstop2 also blocks nuclear functions and clathrin-independent endocytosis. Experiments with an anti-clathrin antibody were interpreted as showing mislocalization of clathrin in the arpc4 mutants. The antibody was raised against a peptide near the N-terminus of human clathrin, but the antibody was not validated, and it was not reported whether Chlamydomonas clathrin even has that peptide.

Internalization of a protein from the plasma membrane: In the experiments to use protease-sensitivity to examine SAG1-HA relocalization induced by db-cAMP, the authors assumed that all of the SAG1-HA was on the cell surface in untreated cells and the 2 chemically treated cells, but they never experimentally documented this assumption.

Arp2/3 relation to actin dots: Structures termed actin dots that stained with Phalloidin were reported to undergo changes after de-ciliation of wild-type cells and were missing in the arpc4 mutant. The conclusion that the properties of the dots in the wild-type cells changed with de-ciliation were not supported by statistical analysis. Also, without experiments showing localization of Arp2/3-V5 at the dots, it was not possible to assess whether, as the text asserts, Arp2/3 functioned at the dots.

Cilia resorption induced by BFA in arpc4 mutants: In the experiments with the Golgi-active agent BFA, the percent ciliation in the arpc4 mutants, but not wild type, fell rapidly after drug addition. The authors concluded that BFA induced ciliary resorption, but they did not determine whether the lack of cilia on cells was a consequence of cilia resorption or cilia detachment.

Reviewer #1 (Public Review):

In this manuscript Bigge et al. use chemical inhibitors and a mutant in ARPC4arpc4 mutant to investigate the role of the Arp2/3 complex in regulating cilia length and assembly in Chlamydomonas. The authors have previously shown that the actin cytoskeleton is required for ciliary assembly and maintenance in this organism, but the precise mechanism(s) involved were unclear. Furthermore, while previous studies targeted the actin cytoskeleton in general, the current study focuses on branched actin networks regulated by the Arp2/3 complex. The authors first demonstrate that chemical inhibition of the Arp2/3 complex leads to shortening of existing cilia, a phenotype that is recapitulated in the arpc4 mutant and which can be rescued be reintroducing V5-tagged ARPC4 in the latter mutant. Next, using similar approaches they show that initial stages of cilium biogenesis are also impaired upon Arp2/3 complex inhibition. They next use a variety of approaches, mostly involving chemical inhibitors and F-actin- or membrane dyes, to assess the mechanism by which Arp2/3 complex affects ciliary biogenesis. They provide evidence indicating that Arp2/3 complex specifically promotes endocytosis at the plasma membrane to support lipid and protein for the growing ciliary membrane. This is an interesting discovery that advances our understanding of how ciliary membrane biogenesis is regulated, especially in Chlamydomonas.

Reviewer #2 (Public Review):

Previous studies have demonstrated that interfering with actin polymerization leads to the shortening of flagella in Chlamydomonas cells, indicating that F-actin is important for ciliary/flagellar elongation. However, the precise roles of F-actin, and especially of branched actin networks nucleated by the Arp2/3 complex in cilia formation have not been elucidated. Here, the authors aimed to examine one of the mechanisms that may be involved in F-actin-dependent cilia elongation/formation, namely, actin- and clathrin-dependent endocytosis of membrane proteins.

The authors used both pharmacological inhibition and genetic disruption of the Arp2/3 complex to demonstrate that interfering with the activity of the Arp2/3 complex reduces the ability of Chlamydomonas to form or elongate cilia. The authors then showed that incorporation of existing (not newly synthesized) proteins into cilia is perturbed by the genetic or pharmacological inhibition of the Arp2/3 complex, and that new membrane for building cilia may be derived via an Arp2/3-mediated membrane retrieval pathway.

These experiments are carefully performed and convincing. A description of the Arp2/3 complex components and clathrin-containing structures in Chlamydomonas is novel and will be of interest to the cell biologists working on this model organism.

The authors propose that the Arp2/3-mediated actin assembly promotes cilia elongation/formation due to the contribution of the branched actin to clathrin-dependent endocytosis. This is an intriguing idea that ties together previous findings showing that F-actin is needed for cilia elongation and that some ciliary proteins are internalized from the plasma membrane and then redistributed to cilia. One concern regarding this portion of the manuscript in that the experiments addressing the role of endocytosis rely solely on the use of a pharmacological inhibitor of clathrin-dependent endocytosis, PitStop2. PitStop2 was previously shown to have non-specific effects on endocytosis, suggesting that its mechanism of action may not be directly related to disrupting clathrin heavy chain interactions (see, for example, Willox et al., 2014).

Intriguingly, a previous paper by Kim et al., 2010 demonstrated that interfering with the Arp2/3-dependent actin assembly resulted in cilia elongation in mammalian cells, suggesting that branched actin assembly was counteracting growth of cilia. Similarly, actin depolymerization is known to promote ciliogenesis in mammalian cells. The difference between mammalian actin organization/cilia growth regulation vs. the new observations in the Chlamydomonas system should be discussed by the authors to help the readers understand whether the findings can be generalized to the diverse ciliated cell types or are unique to algae.

Reviewer #1 (Public Review): 

In humans, aversive associative learning occurs through experience or instruction. In this paper the authors examined how instructed and directly experienced aversive learning and reversal impacted subjective pain and used fMRI to study the neural systems underlying these processes. They identified cortical and striatal regions which followed reversals and thalamic and midbrain regions which maintained the original learning contingencies. They then used quantitative modeling to show that expected value changed more quickly with instructions compared with experiential learning and revealed a dissociation in the brain regions whose activity was correlated with these processes. 

These results extend previous work from this group on instructed learning during fear conditioning into the subjective experience of pain and the effects of expectation on this. However, the methods used for analyzing the data are not described in enough detail for this reviewer to understand what the BOLD signal is integrating in the different analyses. More broadly, there are important differences found in the brain regions mediating instructed vs. directly experienced aversive learning between this study and previous work. The reasons for these differences are unclear and without a deeper understanding of these differences the impact of this line of research and the potential impact of the current paper could be compromised. If the reasons for these differences could be addressed in some conceptual way, the current paper could be much more compelling.

Reviewer #2 (Public Review): 

The study sets out to compare behavioural and neural measures of instructed and experiential reversal learning in health volunteers. The results identified brain regions with distinct response patterns that partly dependent on the type of learning. The exploration of differences between instructed and experiential learning is of great importance - from a general learning perspective but even more so because of its immediate clinical relevance. The study is well designed, the analysis is sound, and the complex findings are presented with a clear structure. I would particularly like to commend the authors for their diligent reporting of both Bayesian and frequentist statistics which allows the reader to assess the findings and their relevance in detail. However, I have concerns regarding the robustness of the data and the interpretation of the findings.

Reviewer #3 (Public Review): 

Atlas and colleagues use a reversal-learning task in which two auditory cues predict painful heat stimuli calibrated to three levels of intensity. One group of participants (n=20) and is not informed about the initial contingencies and about the reversals, whereas a second group of participants (n=20) is explicitly instructed about the initial contingencies and about the reversals. The authors provide detailed and sound analyses of behavioral ratings of subjective pain intensity and pain expectations as well as (mediation) analyses of fMRI signals during the receipt of painful stimuli. I find it laudable that the authors use Rescorla-Wagner (RW) models, which are very common in studies on appetitive learning and have also been used in studies on aversive learning. 

In my view, one of the most interesting findings of this study is a difference between the uninstructed and the instructed groups of participants in the relation between signals in the rostral anterior cingulate cortex (rACC) and the expected value of painful stimuli (see Figure 7). One strength of this paper is that this finding of a differential involvement of the rACC between uninstructed and the instructed learning situations might give a better idea of the general role of the rACC. This can inspire new studies that test the generality of the reported finding in a rather straightforward way by using different types of aversive and appetitive stimuli as well as different types of instructions and contingencies between cues and stimuli.

Reviewer #1 (Public Review): 

The manuscript entitled, "Early evolution of beetles regulated by the end-Permian deforestation" by Zhao et al. is a strong, interesting, and well-written study worthy of publication after revision. 

The authors met their goal of documenting and analyzing the diversity of Paleozoic beetle taxonomy, morphological disparity, ecosystem roles, and phylogeny. This, in my opinion, is the strongest portion of the paper as it brings several lines of evidence to show the high diversity of xylophagous beetles, up until the EPME, followed by a distinct extinction of xylophagous beetles and the expansion of ecological roles into a more modern component of beetles. 

A distinct weakness of the paper is the reliance of correlation between biochemical cycling and the evolution of beetles. To address this, we would ideally see isotopic data associated with these statements. My overall suggestion is to make clear that this is speculative and bring other hypotheses to the table, and hopefully rule them out. It isn't very helpful to say something like xylophagous beetles were the main source of nutrient cycling in the Permian without discussing fungus at greater length. Or similarly, implying a drop in O2 was caused by beetles, without describing any of the other biotic/abiotic things going on at that time.

Below are some more detailed suggestions: 

-It would be helpful to address the evolution of lignin-consuming fungi. Whether or not you can tie fungal symbiosis into the evolution of these beetles, fungal decomposition may (or may not) have accelerated in the Early Permian due to the timeline of particular clades of fungi. Worth a quick sentence or two. See relevant references below. 

Nelsen, M.P., DiMichele, W.A., Peters, S.E. and Boyce, C.K., 2016. Delayed fungal evolution did not cause the Paleozoic peak in coal production. Proceedings of the National Academy of Sciences, 113(9), pp.2442-2447. 

Floudas D, et al. (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336(6089):1715-1719.Abstract/FREE Full TextGoogle Scholar 

Kohler A, et al., Mycorrhizal Genomics Initiative Consortium (2015) Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nat Genet 47(4):410-415.CrossRefPubMedGoogle Scholar 

For the concluding paragraph in the Discussion, there is no acknowledgment to modern studies of xylophagous beetles in relation to climate change. There are many studies of the effects on climate change and xylophagous beetles, ex the North American pine bark beetles. Might be worth saying that the diversity and abundance of xylophagous beetles are extremely sensitive to climate change and can cause forest collapse too.

Reviewer #2 (Public Review): 

The study aims at providing a general hypothesis about the early evolution of the megadiverse beetles (Coleoptera) in the Late Permian and Early Triassic, i.e. in the periods of major environmental changes connected to large-scale extinctions in all lineages of organisms. This is achieved by complementing the data about all known fossil beetles ever recorded from these periods, their critical revision, and the subsequent analysis of their taxon diversity over time, morphological diversity over time, and the phylogenetic relationships of the early beetle lineages. 

The principal strength of this study is, in my opinion, in the combination of the multiple evidence and multiple views on the early beetle evolution. The usual phylogenetic view, based on an updated phylogenetic hypothesis, is combined with the taxon diversity measures over time at different levels (family, genus, species), with the formal taxa included or excluded. The latter is a very important improvement to earlier studies in which these two categories were usually mixed, and hence the contribution of the hard-to-grasp formal data was difficult to evaluate. The third view is the one analyzing the morphological diversity over time, which, to my knowledge, is the very first trial to evaluate the evolution of beetles in this way. All these analyses separately show significant and important results helping us to better understand how was beetle fauna looking like in this deep past and why so many ancient groups did not survive until today. Additional insight is, however, gained by combining the results of all these three views, and putting it in the context of the known data about environmental changes, paleobotany, and geology of the P-T period. As a result, the study provides a well-supported view on the evolution of early beetles and their role in the ecosystems, and set new hypotheses which may be further tested in the future. 

There are naturally several weaknesses of the study, partly based on the fact that the studied organisms are fossils (that is, unique, often fragmentary preserved specimens) and that the number of beetle species known from the examined period is already quite high. This is reflected mainly in the phylogenetic analysis, which is reduced into very few terminal taxa, usually with a single species representing the whole family. I would also prefer to see the dataset to be newly compiled and not based on those published previously, and based on the actual examination of the respective specimens (although this is not stated anywhere in the study, my feeling is that not all included taxa were actually re-examined in detail for all characters coded). In this aspect, I strongly believe that a detailed restudy of selected fossils, resulting in a revised (rather than adopted) morphological matrix, is the desired way for future research. Also, including multiple species per family (naturally, only those in which the fossils provide enough characters) may provide a more detailed view on the phylogenetic relationships among the principal clade, and understand the morphological diversity and disparity within each clade. In this aspect, the phylogenetic analysis presented in the study can be significantly improved. However, the detailed genus-level phylogeny of early beetles was not the main purpose of this study, and I agree that the simplified analysis presented in the study provides the information needed for combining the phylogenetic data with those of the other analyses. 

Despite these limitations, the study definitely brings a novel view on the early evolution of beetles, strongly supported by multiple means of evidence, and will set a new starting point in the studies of insect diversity, beetle evolution, and environmental and climatic changes. The authors definitely reached the goal in providing the very clear hypothesis about the early evolution of beetles!

Reviewer #1 (Public Review):

The manuscript by Mast et. al. describes the generation and characterisation of a collection of nanobodies against the SARS-CoV-2 Spike protein. The data presented here comprise a large body of work with some detailed characterisation and functional analysis. The generation of nanobodies against non-RBD epitopes, and particularly against the S2 subunit is novel, and has potential to be of interest in a therapeutic setting.

There is considerable effort taken to demonstrate that the collection includes nanobodies that are functionally competent with the SARS-CoV-2 variants of concern (VoC) Alpha, Beta and to some extent Gamma. The authors also test the ability of multiple nanobodies to bind simultaneously to spike and act synergistically in viral neutralisation as a strategy to avoid loss of potency with variants. Nanobody combinations are further demonstrated to reduce the ability of escape variants to arise in selection assays, though their efficacy is not actually tested against existing VoCs.

A stated aim of the paper is to show novel mechanisms of neutralisation, and although there is much speculation about the neutralisation mechanisms of the nanobodies presented, and given their predicted binding sites several may indeed have interesting mechanisms of neutralisation, there is very little experimental work undertaken to demonstrate that the suggested mechanisms are actually occurring.

Much of the discussion of results here revolves around demonstrating the differences between nanobody and antibody engagement of spike that could give nanobodies an advantage as therapeutics in their monomeric, unmodified format. However, due to rapid clearance in vivo, this is not a format currently used and the authors do not demonstrate that their nanobodies are able to prevent or treat in vivo infection in the manuscript.

Overall this work adds a well-characterised collection of nanobodies to the body of antibody-like molecules targeting SARS-CoV-2 and introduces several neutralising agents outside of the highly targeted RBD domain.

Reviewer #2 (Public Review):

The manuscript 'Highly synergistic combinations of nanobodies that target 2 SARS-CoV-2 and are resistant to escape' by Mast et al. describes an impressive collection of new nanobodies binding SARS-CoV-2 spike. The authors employ a comprehensive method to identify novel nanobodies from immunized camelids, which was initially described by the same lab and relies on proteomic analysis of the serum and correlation with the sequence repertoire found in purified B cells.

Strengths:<br> The extended number of new nanobodies as well as their meticulous characterization on a biophysical and functional level (i.e. neutralization data) provide a rich resource for the community. The nanobodies fall into 10 groups of nanobodies with differential binding sites/modes and include the first examples of neutralizing nanobodies binding S1 independent of the RBD, as well as the S2 subunit of spike. As such, the study likely provides one of the most complete coverages of the possible epitope space of SARS-CoV-2 spike described to date. Characterizing a representative collection of nanobodies, the authors provide detailed affinities (including kinetics), neutralization data (including data on the emerging variants alpha, beta, and gamma), escape variant data, epitope mapping, and the systematic analysis of synergy between different nanobodies and their epitopes.<br> Most conclusions are backed up by solid data and the authors provide a fairly complete functional characterizations despite the large nanobody collection (mostly on the basis of methods that can be conducted in high and medium throughput). A particular strength is the identification of escape variants for most representative nanobodies as well as the systematic analysis of synergy, which is typically backed up with much less complete data in other publications.

The new nanobodies add some previously missing epitopes and therefore make an important contribution, although the list of described SARS-CoV-2 nanobodies is ever growing and no completely new type of neutralizing nanobodies or novel mechanism of action was identified.

Weaknesses/Room for improvement:

A substantial number of SARS-CoV-2 nanobodies has meanwhile been described and it is therefore challenging to extract genuine novelty about the biology of SARS-CoV-2 spike and the fusion it catalyzes, as well as the neutralization mechanism of nanobodies or antibodies. The field (and translational efforts) are aiming at neutralizing nanobodies targeting well-conserved regions of S2 that potently interfere with the fusion mechanism per se and therefore allow broad neutralization activity with little room for genuine escape variants. Although the authors describe the first neutralizing nanobodies targeting S2 and the NTD of S1, these expectations are not met as neutralization by the identified nanobodies can also be avoided by single point mutations. This underlines that efforts to identify broadly neutralizing nanobodies that do not allow mutational escape are not trivial (and may be difficult to achieve with a typically RBD-dominated immune response).<br> The strength of such an impressive and comprehensive analysis of large collection of nanobodies lies in the comparison with existing nanobodies. To fully benefit from the publication of this latest collection of nanobodies, the authors should publish all the sequences and have to make the best efforts to provide comparisons with existing nanobodies described in the literature:

- Values obtained for neutralization potency differ substantially between different techniques and labs. A good reference point for neutralization data is the use of ACE2-Fc, which is commercially available and widely used in earlier publications. It is hard to compare the described nanobodies with the control nanobodies from the literature mentioned (Wrapp et al., Xiang et al.), as they are not identified in detail. Differences between the potency described in the original description and the values determined here are not discussed.

- Epitope mapping defines a new list of epitope groups, although similar efforts had been undertaken earlier. It would make the comprehensive list of SARS-COV-2 nanobodies even more helpful if information about representative existing nanobodies targeting the same epitopes is included throughout the paper (in particular taking advantage of determined nanobody-Spike structures). Comparison to nanobodies with structural information would permit more meaningful predictions with regards to the mechanism of action and help explain the synergistic behavior observed.

- The manuscript substantially refers to previous antibody publications (with important contributions from the institution of the last author). Yet, important earlier publications of SARS-CoV-2 nanobody are barely mentioned/discussed. Many of these publications defined structures and epitopes, contributed to an understanding of spike activation, and determined modes of neutralization. These publications should be appropriately acknowledged.

Reviewer #3 (Public Review):

By combining in vivo antibody affinity maturation and proteomics, the authors report the discovery of hundreds of high-affinity nanobodies that target the whole SARS-CoV-2 spike. The authors produced, bioengineered, and comprehensively characterized a repertoire of the spike nanobodies and their combinations by complementary biophysical, structural (epitope mapping), and functional assays. While other papers have been published on the related topic, this work can be distinguished by 1) identification and characterization of non-RBD nanobodies (including novel neutralizers that target the highly conserved S2 epitope) 2) extensive bioengineering to substantially improve potency and resistance to escaping variants, 3) demonstration of synergistic activities using nanobody cocktails. Overall, the science is thorough, solid, and of the highest caliber.

Reviewer #1 (Public Review): 

This study aims to find the genetic mechanisms underlying sex-ratio distortion through male-killing in Drosophila melanogaster flies infected with the endosymbiont Wolbachia. The endosymbiont carries the prophage WO, which is in the center of interested in this study. The key result of this study is that a synonymous mutation in a prophage gene can explain the differences between sex-ratio distorting and not distorting symbionts. The study uses transgene technology to modify phage genes and to investigate which changes in the gene is involved in the phenotype. The finding, that a synonymous SNP plays a key role is not entirely novel in biology, but there are only few examples known of this type of genotype - phenotype associations. The study does not include experiments to show that the main finding is not limited to one particular background of the fly line used. An experiment including multiple genotypes would be needed to show this. 

The study is mostly clear and easy to follow, but requires a lot of attention. The authors choose to build up the story as I guess it was carried out in the lab. Thus, the reader is guided through every step of the process. While I see that this is appealing from the way the study was carried out, it results in a very long manuscript with a lot of material that would be much better placed in a supplement. 

The introduction seems unfocused. It meanders around, jumping from topic to topic and does not give the reader a sense of where things will go. Fig. 1 gives an overview about the different aspects addressed here, but it is not used to guide the reader through the different lines of thought addressed in the introduction. If Fig. 1 will stay (I actually think it is not needed) it should be introduced earlier and used as a road map for the paper. Alternatively, the introduction could stay more general and only in the last paragraph the different ways the system is studied will be summarized. Along these lines, it would be good to have a better reasoning for the combination of experiments conducted. It is left to the reader to understand why certain types of experiments have been done. On the other hand, the introduction misses a section on the biology of the phage and its interaction with the host(s). It is hard to understand the biology of the system without getting an understanding of the insect - Wolbachia - phage interactions. For non-specialist, understanding the role of the three players is essential for the system. 

The result section could be easily shortened by focusing on the essential experiments. Experiments that do not contribute to the final result can go into the supplement. 

Also the discussion is much too long. I suggest to reduce it to half and focus on the important points and the take-home messages. Currently the discussion follows the way the results are presented in the result section. However, this is not needed. The important finding should be discussed first. Findings that are important in the development of the project, may not be important for the biology of the system overall. And they may not be important for the reader.

Reviewer #2 (Public Review): 

This study aims to unravel the genomic basis to wmk-induced male killing by transgenically expressing homologs of varying relatedness, with synonymous nucleotide changes, and predicted alternative start codons in D. melanogaster flies. The study builds on previous work showing that expression of wmk in fly embryos recapitulates several aspects of male killing. While more distantly related homologs did not induce male killing when expressed in D. melanogaster, more closely related wmk homologs induce either killing of both sexes or male killing only. However, the male-killing phenotype was not due to amino acid differences, but associated with RNA structural differences of the different wmk homologs. In addition, only one synonymous nucleotide change was sufficient to ablate the killing phenotype. These findings suggests that minor and even silent nucleotide differences impact on the expression of male killing in D. melanogaster. It is concluded that a new model incorporating the impacts of RNA structure and post-transcriptional processes in wmk-induced male killing needs to be developed. 

The strength of the study lies in the systematic and carefully controlled approach to quantify the phenotypic effects of both sequence and structural changes to various wmk homologs for inducing the male-killing phenotype. Detailed dissection of the phenotypic impact of minor changes to the wmk homologs including sequence variation, silent nucleotide changes, and RNA structural differences was quantified. This approach reveals a complex genotype-phenotype relationship, but highlights the importance of including post-translational processes. The data is novel in that previous work have largely ignored structural changes and assumed that synonymous differences in codons has no effect on protein function, whereas the current study based on updated codon optimization algorithms reveal that this assumption is incorrect. The finding highlights the importance of considering also structural genetic variation for phenotypic expression differences. This suggestion is further corroborated by the lack of difference in wmk homologue expression levels, indicating that the functional differences are due to post-translational effects. 

There are limitations to the findings of this complex genotype-phenotype relationship. The current study only examined the phenotypic impact by expressing the different homologs in one D. melanogaster genetic background. Given the variability of the phenotypic pattern revealed based on minor changes to the wmk homologs, it will be critical to repeat some of the main findings in other D. melanogaster genotypes to determine the importance of the variation in the wmk homologs more generally. It is entirely plausible that the observed changes in the effect and strength of killing is due to an interaction between host and wmk genotype. This has implications for unravelling the underlying genetic basis to the male-killing phenotype more widely. It is as yet to be demonstrated whether wmk is involved in male killing in natural population, and to what extent there are shared patterns and mechanisms of male killing induced by other bacterial endosymbionts such as Spiroplasma.

Reviewer #1 (Public Review):

Garcia-Souto, Bruzos, and Diaz et al. analyzed hemic neoplasia in warty venus clams at multiple sites throughout Europe. They identified cases of disease in two locations, in Galicia and in the Mediterranean. They then use Illumina sequencing to discover that the samples with cancer DNA had reads which mapped to the mtDNA reference sequences from a different clam species in the same family, suggesting a cross-species transmissible cancer. By mapping reads to both the V. verrucosa and C. gallina mitogenomes they showed that more reads mapped to C. gallina in cancer samples compared to matched host tissue samples, and this was consistent across the whole mitogenome. Phylogenetic analysis of mtDNA genes of the host and cancer samples as well as identification of SNVs at a short region of one single-copy nuclear locus suggest that all cancer samples come from a single C. gallina transmissible cancer clone. All data agree that a single lineage of cancer from C. gallina is responsible for all identified cancers in V. verrucosa.

There are a few sections where there are either unclear methods or the methods do not quite match the descriptions of the results.<br> 1. Regarding mapping of reads to different reference Cox1 sequences (for Figure 2a): "Then, we mapped the paired-end reads onto a dataset containing non-redundant mitochondrial Cytochrome C Oxidase subunit 1 (Cox1) gene references from 137 Vererid clam species." I do not see where this is explained anywhere in the methods, where this list of references comes from, or what is in it.<br> 2. Regarding de novo assembly of mitogenomes: "Hence, we employed bioinformatic tools to reconstruct the full mitochondrial DNA (mtDNA) genomes in representative animals from the two species involved....Then, we mapped the paired-end sequencing data from the six neoplastic specimens with evidence of interspecies cancer transmission onto the two reconstructed species-specific mtDNA genomes." In contrast to this, the methods say, "Then, we run MITObim v1.9.1 (Hahn, Bachmann, & Chevreux, 2013) to assemble the full mitochondrial genome of all sequenced samples, using gene baits from the following Cox1 and 16S reference genes to prime the assembly of clam mitochondrial genomes." It is unclear which method was used.

There is one minor claim which may not be fully supported by the data: the statement that, "The analysis of mitochondrial and nuclear gene sequences revealed no nucleotide divergence between the seven tumours sequenced." If I am understanding the filtering of the SNVs from the nuclear gene correctly, only the presence or absence of the 14 SNVs that were fixed within each of the two species were analyzed. Therefore, it is unclear whether the authors looked for any additional somatic mutations within the cancer lineage that would have occurred at other positions. For mitochondria, the authors state that sequences were "extracted from paired-end sequencing data," but it is not explained how this was done. The data suggest that there are no differences between cancer samples in the 13 coding genes and 2 rDNA genes, but data on possible SNVs in the intergenic regions is not shown.

Reviewer #2 (Public Review):

In rare but well-documented instances, certain types of cancers can transmit horizontally. These transmissible cancers have a clonal origin and have adapted to bypass allorecognition. A form of marine leukemia (hemic neoplasia or HM) belongs to this class of transmissible cancers and has been detected in several bivalve species (oysters, mussels, cockles and clams). Although HM mostly propagates within the same bivalve species, instances of cross-species transmission have been reported. To better understand the mode of transmission of HM, Garcia-Souto et al. analysed mitochondrial DNA (mtDNA) by next generation sequencing in different bivalve species collected in the Mediterranean Sea and the Atlantic Ocean. The authors found that HM isolated in Venus verrucosa contained mtDNA that actually matched Chamelea gallina. Analysis of the nuclear gene DEAH12 also showed single nucleotide polymorphisms (SNPs) matching C. gallina DNA. Based on mtDNA and DEAH12 sequences, the authors use Bayesian inference to generate phylogenetic trees showing that HM found in V. verrucosa is much closer to C. gallina than the host species. They conclude that HM propagated from C. gallina to V. verrucosa.

Overall, the study is well performed with enough samples analysed. The results are quite convincing but there are also some concerns.

1. Transmissible cancers are known to split into clades based on mtDNA differential rate of evolution and also to incorporate mtDNA from exogenous sources, so one has to be extra careful that the results prove cross-species transmission and not HM divergence into two clades and/or exogenous acquisition. Samples HM ERVV17-2997 and EMVV18-376, both at the N1 stage, appear devoid of C. gallinae mtDNA and do not appear to have been screened for DEAH12. One explanation for this result is that there are too few HM cells in the samples (but supplementary Figure 1 shows some HM cells in ERVV17-2997. However, a different explanation is that these samples contain V. verrucosae mtDNA. ERVV17-2997 and EMVV18-376 could have been analysed in greater depth to verify that they also contained C. gallinae mtDNA and typical DEAH12 SNPs.

2. To strengthen their argument, the authors could have analysed a few more nuclear genes for specific SNPs, although the sensitivity of this approach will depend on the depth of sequencing.

3. It would have been interesting to have more information in the Discussion on the potential immunological barriers that this tumour needs to overcome for cross-species transmission.

Reviewer #3 (Public Review):

The authors first investigated by cyto-histological examination the prevalence of hemic neoplasia, a leukemia-like disease of bivalves, in 345 warty venus clam V. verrucosa from six sampling regions in the Atlantic and the Mediterranean coasts of Europe. Eight specimens from two sampling points in Spain were diagnosed infected by hemic neoplasia. Electron microscopy and karyotyping confirmed abnormal cells suggestive of neoplasia. They secondly carried out whole-genome sequencing of eight tumoral haemolymphs and host feet. They also sequenced seven genomes of healthy V. verrucosa. Preliminary investigation of mtCOI sequences suggested cancer mitochondria could be related to a sister species, the striped venus C. gallina, and the authors sequenced two genomes of this species together with one genome of its sibling species Chamelea striatula. Mitogenome analysis confirmed cancerous clams were infected by the same clonal lineage of transmissible neaoplasia that have emerged in a C. gallina host. The analysis of a nuclear gene, DEAH12, confirmed the result. To find out whether this transmissible cancer lineage was still present in the donor species, the authors conducted histological inspection of 166 striped venus clams but found none with evidence of hemic neoplasia. The donor species could possibly have become resistant to this transmissible neoplasia, or more sampling would be needed to find infected individuals (8/345 versus 0/166, P=0.06).

Overall the study provides convincing evidences of a new transmissible cancer that crossed species boundaries in venus clams. This is an important finding that should make a nice contribution to Elife. My concerns are minor and mostly about methodological issues. I'd mostly like to understand how the authors came with a single nuclear gene while they have high coverage genome data. However, they do not need more data to support their conclusions and it's mostly about clarifying the methodology.

Transmissible cancers are important study systems in many points, somatic evolution, metastasis, self recognition, host-parasite co-evolution, clonal interference, comparative oncology etc.. although they were thought rare exceptions. The field has long remained limited to the two mammals known cases, but bivalve transmissible neoplasia have recently emerged as a new fascinating playground to study the ecology and evolution of transmissible cancers. One new observation is host species shift, as reported here in venus clams. This study comes at an early stage of an emerging research program on unique living entities we know very little about, and will undoubtedly have a strong impact.

Reviewer #1 (Public Review):

This research presents compelling evidence that PcTx1 induces a conformational change of the extracellular domain of mASICa that is distinct from PcTx1 binding and channel pore opening. The data, analysis, and transparent presentation are all of high quality. Strengths of this work are the use of fluorescence detection of conformational change coupled with electrophysiology that enable PcTX1 binding to be distinguished from multiple conformational changes. The use of fluorescently labeled concatemers with appropriate controls allows the stoichiometry of PcTx1 effects to be determined. The use of a binding site mutation F350L to further probe the coupling between binding and conformational changes adds mechanistic depth to the study, although there is some question concerning the mechanism underlying effects of PcTx1 on F350L. This study succeeds in identifying conformational changes that are coupled to, yet distinct from channel conductance that impact ASIC function. The interpretation of results is carefully nuanced and well supported by the data. Overall this study is an inspiring conceptually-driven mechanistic characterization of coupling between protein conformational changes. This work is likely to be valuable to the field of ASIC research, with the advances in methodology of interest to a broader community studying coupling between protein conformational changes.

Reviewer #2 (Public Review):

Borg and colleagues set out to study the mechanism and stoichiometry of acid-sensing ion channel modulation by PcTx1. They do this using voltage clamp fluorometry in conjunction with concatenated subunits. The principle finding is that PcTx1 induces a conformational change, as reported by a delta F, which persists beyond the toxins' apparent effect on channel gating. The authors explain this striking observation by proposing that PcTx1 binds in three distinct modes: a low-affinity non-modulatory 'Loose' mode, a high affinity modulatory 'Global' mode and a high affinity non-modulatory 'ECDonly' mode. The 'Global' mode is the one typically studied using electrophysiology. They argue that given time and some pH-stimulus, the channels accumulate into the 'ECDonly' mode where the influence of PcTx1 is uncoupled from the pore but not the ECD. They further explore these observations using a prior mutation, F350L, which reduces the channel's sensitivity to PcTx1. By pairing this mutation with VCF in concatenated ASICs, they show that a single F350L does not detectably alter PcTx1 inhibition as measured by electrophysiology but does destabilize the ECDonly state, further arguing for a distinction between these conformations.

The paper is well-written with high quality data and generally excellent figures. The persistent PcTx1 effect itself is quite striking and will undoubtedly motivate subsequent study. A further strength of the work is the authors take care to measure the pH responses of their mutants and calibrate the conditioning and test stimuli accordingly. This is critical for ASICs and it's great to see such consideration become more common place. However, an inherent weakness in the VCF approach is the fluorescence signal reflects the occupancy weighted sum of signals from all states. A small fraction of channels moving to one state with a big delta F could occlude signal from a larger fraction with a smaller delta F. VCF is further complicated in this case by the use of pH as a stimulus (which can alter local environment by protonation instead of motion) and a Trp-containing toxin as a ligand. The authors do an excellent job mitigated these latter concerns by reproducing all data at structurally distinct labelling positions. Moreover, they are fairly conservative in their interpretation of data. As a result, the most innovative conclusions (that PcTx1 induces a novel long-lived state and it produces inhibition with two intact binding sites) are well supported by the data.

Overall, this is a deeply interesting study with the characteristic high quality of the Pless group. The stoichiometry experiments are a great addition to the literature as is the observation that PcTx1 induces a novel, long-lived conformation. However, in the present form the more intricate assertions (that PcTx1 is bound in a ECDonly state with weak ECD/pore coupling) require more evidence. Nevertheless, this paper provokes a reassessment of PcTx1 effects on ASICs, motivating much more in-depth examinations of PcTx1 specifically, and other ASIC modulators in general.

Reviewer #3 (Public Review):

In this study Borg et al. explore the mechanism of PcTx1 inhibition of ASIC1a using TEVC fluorometry. They detected a robust change of a fluorescence signal when PcTx1 was added, and based on this finding, propose that the toxin has three different binding modes: 'loos', 'global' and 'ECDonly'. In addition, using concatamers they conclude that damage of a single PcTx1 binding site out of the three sites present in ASIC1a destabilizes the conformational changes but disruption of two or three binding sites is required to prevent PcTx1-mediated inhibition.

The main weakness of the study is the lack of additional experiments to confirm that the proposed three PcTx1 binding modes are actually happening.

Reviewer #1 (Public Review): 

The study is elegantly done, with the outstanding questions clearly laid out and the results presented in a clear and informative fashion. I have only a few suggestions to strengthen some of the results. 

1) Determination of layers: The CSD based method used to determine the layers seems a bit ad hoc, although other studies have often used a similar approach. Some histological evidence would be great. If that is not possible, the authors should provide some more details to determine the layer specificity. For example, where were the supragranular-granular and granular-infragranular borders for different penetrations (i.e., which electrode(s) marked these boundaries)? These could be expressed as fractions of the shaft length, and from that, we would approximately know the depth. Also, were these results affected by how the CSDs were smoothed? 

2) Another important factor is the orthogonality of the penetrations. This can also be better quantified based on the variation of the RF centers with depth.

Reviewer #2 (Public Review): 

Identifying and representing the object structure of a scene is a fundamental visual function that needs to be clarified. It is this function that allows the brain to detect object structure, to link the emerging visual properties of objects (like shape, color and movement) to the individual detected structures, to establish object representations that are stable across eye movements, and to focus selective attention on objects. Previous studies have shown that border ownership selectivity, that is, object-based coding, emerges in visual cortex shortly after the onset of feature signals. The unsolved puzzle is where the necessary context information comes from and how the cortex computes object structure from it so fast. Previous studies have ruled out feed-forward mechanisms and intra-cortical horizontal connections of V1/V2, the former because they do not have the context, the latter because they are too slow. 

The present study, showing that border ownership selectivity emerges in the deep layers of area V4 earlier than in the granular layers is significant in two ways. First it rules out the possibility that V4 simply inherits border ownership selectivity from V1/V2. Second, it points to the deep cortical layers as the origin of border ownership computation. This points to several possible sources of the context input, including cortico-cortical connections and thalamic input. 

There is one previous study (Bushnell et al., J Neurosci, 2011) that also concluded, based on the latency of the effect, that border ownership selectivity in V4 cannot be simply inherited from upstream areas. They tested curvature selective V4 cells and found that the responses to the optimal shape were suppressed when the critical curved contour was not intrinsic to the shape but owned by an occluding object. They found latencies of suppression as short as 46 ms, shorter than the latencies of V2 cells reported in Zhou et al. 2000. The present study should mention this finding. 

The present study also demonstrates that the cell's spatial tuning for direction of border ownership is somewhat independent of their orientation tuning. That is, their border ownership preference is not simply for one or the other side of their preferred edge stimulus; and even cells without orientation tuning can be direction-of-object selective. This is further indication that object location is computed by an independent mechanism. 

It would be very interesting to see a similar laminar analysis applied to area V2. Perhaps the computation of border ownership there occurs also in the deep layers, using similar sources of context information. Alternatively, the V2 border ownership selectivity could be the result of back projection from V4. Because of the importance of representing object structure for many visual tasks, as pointed out above, I would not be surprised if similar fast border ownership computation would be generally found in the deep cortical layers, in V1 and V2 and in areas of the ventral stream beyond V4. 

The new findings support the idea of the grouping cell model, namely that the context information is provided by an external grouping signal that modulates the activity of the feature neurons in the visual cortex. The grouping signal is supposed to represent a 'proto-object', a computational structure that (1) links the visual feature signals, (2) is being remapped across eye movements, and (3) serves object-selective attention. Compared to the large number of studies of feature selectivity in visual cortex, the question of the representation of object structure has prompted relatively few studies, despite its theoretical importance, and the big question of where and how grouping signals are generated still awaits an answer.

Reviewer #3 (Public Review): 

The authors ask a simple but important question related to our ability to assign borders to objects, namely whether these are computed at early stages of cortical processing (and inherited at mid-level), whether they are organized in columnar manners, and whether they match to orientation preference. They report that border-ownership in V4 is not inherited from upstream areas. They speculate that it is computed de novo by infragranular neurons, but there is no proof for that. It could also be is due to feedback from higher areas. Border ownership is organized in columns, and while it often aligns with preferred orientations, there is often a surprizing mismatch. The experiments are performed to high standard and convincing, and answer a relevant question.

Reviewer #1 (Public Review): 

Podinovskaia et al report a simple method to enlarge endosomes, and possibly the TGN, using cultured transformed cells. They report that a brief treatment of the cells with the ionophore nigericin results in a rapid swelling of endosomal compartments. It is shown that, despite this, perturbation, swelled endosomes mature via rab5 to rab7 conversion. It is further shown that the kinetics of rab conversion on swelled endosomes is approximately the same as for native endosomes, suggesting that endosomes that are swelled in this manner can be used to infer functional aspects of endo-lysosomes. Using this approach, the authors find that Rab11, a regulator of a plasma membrane recycling pathway, and Snx1, a component of multiple cargo export pathways from the endosome, persists throughout endosome maturation. They report that endosome re-acidification (ie, after nigericin washout) correlates with the appearance of Rab7 of the endosome. Finally, the authors report that the TGN is reversibly swelled similarly by nigericin and that this swelled compartment receives endocytosed material. 

Overall, this is an excellent study that provides an interesting new experimental approach for investigating endosome dynamics. The validation studies convincingly demonstrate that rate of rab conversion on nigercin-swelled endosomes is similar to the rate of maturation of untreated endosomes.

Reviewer #2 (Public Review): 

The manuscript by Podinovskaia focuses on a new method to visualize and measure endosome maturation in common cell lines by enlarging early endosomes. This was achieved by producing acute insult to the cells by ionophore treatment, leading to budding of abnormally large post Golgi vesicles that fuse with early endosomes. Endosome maturation of these enlarged endosomes containing Golgi-derived cargo (GalT) proceeding with apparently normal kinetics, ultimately leading to lysosomal delivery. Taking advantage of this assay, the authors investigate Rab5-to-Rab7 conversion, acquisition and loss of PI3P, acquisition and loss of Snx1 on apparent endosomal subdomains, interaction of early and late endosomes with Rab11-positive recycling endosomes, and lumenal pH changes. The new maturation model presented here will likely be quite useful to the field with continuing impact. The current state of the endosome field in many ways remains fragmentary, with various processes studied extensively in isolation, but with little information on their relative timing and potential interactions as endosomes mature. This new assay should help understand the relationships between these processes, some of which are investigated in this manuscript. 

Concerns: 

1) The data and conclusions related to Rab11 interaction with early endosomes in Fig 8 are not convincing. There are simply too many Rab11 endosomes in the cell to know if their short term proximity indicates meaningful interaction with the early endosomes, or if the data simply reflects random collisions of small recycling endosomes with the enlarged early endosomes. No data is presented to show that the interactions are meaningful, e.g. that recycling cargo transfer occurs during these interactions. Conclusions from this analysis are overstated. 

2) Lack of information on endocytic cargo acquisition by the enlarged early endosomes: to really establish this endosome maturation model the authors would need to establish if the enlarged endosomes contain endocytosed cargo, as opposed to Golgi-derived cargo, and determine how long it takes to acquire such cargo. This could be accomplished using Tf, EGF, or perhaps dextran at early timepoints after nigericin washout. 

3) Figure 7 - It was not convincing that data in panels F and G are different from each other. 

4) Figure 11 - it is unclear how we can interpret this as connected to Rab conversion when even the labeled compartments at the earliest time point in the czz1 knockout have abnormally high pH, and during the time-course even the last timepoint for czz1 KO is higher than that of the earliest timepoint for WT. 

5) Figure 12 - The criteria used to determine which GalT structures are Golgi or lysosomes seems questionable. Morphology alone is not sufficient to identify the compartments with high accuracy, especially after perturbation. Also, it is unclear to what extent GalT-CFP labels lysosomes without nigericin treatment.

Reviewer #3 (Public Review): 

The authors describe a useful new way to track the maturation of dramatically enlarged endosomes in animal cells. Ionophore treatment is a significant perturbation, but a variety of findings indicate that the enlarged endosomes follow the normal maturation pathway. This system was coupled with additional technical advances and targeted perturbations to perform functional tests. The results reveal that recycling to the plasma membrane is uncoupled from the Rab5-to-Rab7 early-to-late conversion process, whereas acidification seems to be linked to Rab conversion. 

My sense is that this work makes a valuable contribution to our knowledge of a fascinating cell biological process. However, the manuscript would benefit from a clearer emphasis on how the data inform our views of outstanding questions.

Reviewer #1 (Public Review): 

The authors provide strong evidence for how MakA interacts with membranes. The structural studies are, in general, sound, and provide a high resolution picture of how MakA undergoes a pH-dependent conformational change.

Reviewer #2 (Public Review): 

Prior work identified the V. cholera MakA/B/E tripartite toxin. The present study reports, however, that even in isolation the MakA subunit alone has pore-forming and cell-killing properties. This activity depended on acidic pH; above neutral pH MakA is soluble. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms high molecular-weight oligomers, binds and remodels membranes into high-curvature tubes, and eventually leads to loss of membrane integrity. Especially exciting, the authors report a helical reconstruction of MakA filaments with lipids that appears unprecedented. Nadeem and co-authors succeeded in showing us how low pH leads MakA to refold and embed a hairpin of transmembrane helices into a distorted, thinned, and discontinuous lipid bilayer. The conclusions are supported by the data and I have only minor comments for the authors to consider in preparing a final version for publication.

Reviewer #3 (Public Review): 

The main strength of the manuscript is the structural analysis of the MakA-membrane nanotubes, which corresponds to a new type of supramolecular assembly with a very unsual lipid and protein distribution. While the authors provide solid evidence about the concentration- and pH-depdendence of the induction of these protein/lipid nanotubes by the addition of MakA, the main weakness of the study lies in the fact that two main issues question the phyisiological relevance of these structures. First, the topology of the protein with respect to the lipid bilayer is opposite to the one that the protein would encounter upon endocytosis and acidification in the lysosomes. Meaning the protein in this case would be inside the lysosomes and not outside. Second, if the protein were to be secreted by the pathogen into the extracellular medium of the host, it is unclear that the pH could be acidic and thereby enable the conditions used in this study.

Reviewer #1 (Public Review): 

In this manuscript, the authors make use of next-generation sequencing to provide a preliminary inventory of tribe Metriorrhynchini, a hyperdiverse group of beetles with intricate systematics mainly due to likely morphological convergence of their Millerian rings. The authors provide an admirable sampling within Africa, Asia and Oceania, with about 700 successfully sampled localities and thousands of specimens. 

The main result of the manuscript is the curated database of Metriorrhynchini that will be useful in future research. In addition, different statistical methods are used to provide an idea of the undescribed species within the tribe, the astonishing species richness in New Guinea or the use of phylogenomic data to explore major phylogenetic relationships. However, some of the author's claims should be questioned: 

- Surprisingly, the authors rely on a very low threshold to identify mOTUs (2% in the manuscript). The authors refer to Hebert et al. (2003) and Eberle et al. (2020) to justify the threshold, but still, they are likely overestimating the number of mOTUs and thus, considering putative species what it may be different populations. Figure S17 provide estimates of mOTUs with different thresholds (1 to 10%), which rapidly decrease their estimates (a decrease of 25% mOTUs is found when 6% was considered). Still, an overwhelming sampling effort but a more realistic estimate. 

- I think the phylogenomic tree did not receive the required attention (for example, the FcLM analysis is barely mentioned). 

- It is not clear why should be important to mention the "person-months of focused field research" across the manuscript. Each study group has a unique sampling technique (also not found in the manuscript), preferred localities or traits, which make comparisons impossible. The authors' effort is remarkable, but it is not an important result/finding to be highlighted all over the manuscript.

Reviewer #2 (Public Review): 

Conservation efforts must be evidence-based, so rapid and economically feasible methods should be used to quantify diversity and distribution patterns. The principal objective of this study is to demonstrate how biodiversity information for a hyperdiverse tropical group can be rapidly expanded via targeted field research and large-scale sequencing. The authors have attempted to overcome current impediments to the gathering of biodiversity data by using integrative phylogenomic and three mtDNA fragment analyses. As a model, they sequenced the Metriorrhynchini beetle fauna, sampled from ~700 localities in three continents. The species-rich dataset included ~6,500 terminals, >2,300 putative species, more than a half of them unknown to science. It is an amazing finding. Their information and phylogenetic hypotheses can be a resource for higher-level phylogenetics, population genetics, phylogeographic studies, and biodiversity estimation. At the same time, they want to show how limited the taxonomical knowledge is and how this lack is hindering biodiversity research and management.

Reviewer #1 (Public Review):

Autoantibodies that bind nucleic acids and nucleic acid-binding proteins are a hallmark feature of SLE. In this study, Rosen and colleagues identify AIM2, a dsDNA responsive inflammasome forming sensor as a new autoantigen in SLE. Further they show that anti-AIM2 antibodies are associated anti-IFI16 Abs previously linked to SLE and athese ALR Auto antibodies correlate with anti-dsDNA Abs and SLE disease severity. The authors also provide compelling evidence that AIM2 and the ALR family member IFI16 bind NETs and shield NETs from Dnase-mediated nuclease degradation. The finding that AIM2 like IFI16 are autoantigens and that these ALRs might contribute to SLE disease pathogenesis by protecting NETs from destruction thereby enhancing extracellular nucleic acid driven immune stimulation and IFN responses is striking and directly relevant to disease. This is a very interesting study. The identification of AIM2 as an auto antigen and the role of AIM2 in promoting the interferogenic activity of NETs by preventing their clearance has important implications for SLE pathogenesis.

Reviewer #2 (Public Review):

Antiochos et al. analyzed plasma from 131 Lupus patients, 133 Sjogren's syndrome patients and sera from 49 healthy controls for binding to AIM2, a DNA sensor, IFI16, a related cellular protein, and dsDNA, a characteristic Lupus antigen. These 3 reactivities tended to overlap and their presence tended to correlate with a more severe disease. In addition, the authors considered whether the proteins would be able to associate with NET chromatin from healthy control neutrophils treated with PMA and whether they could protect the DNA from degradation by DNAse 1. Further, the authors identify structures in kidneys from Lupus biopsies that stain with antibodies to AIM2, IFI16, or MPO, a marker for neutrophils. These are visualized relative to DNA, and areas of more decondensed chromatin are considered as NETs. Binding to each is observed and also colocalization is performed using confocal microscopy. The statistics of data analysis are valid and correlations with disease scores are appropriate. Negative controls for microscopy data presumably have been done by the authors (not shown).

An inherent conundrum is present in the initial data on plasma reactivity determination. The AIM2 protein is prepared in vitro and radiolabeled during in vitro translation. This is then incubated with patient plasma and the counts in the immuno precipitate are used to determine antibody reactivity to AIM2. This would seem to be a sensitive and quantitative way to measure antibody binding. However, the additional observations made by the authors call the straight-forward interpretation into question. The authors show that AIM2 strongly binds to dsDNA and protects it from nuclease digestion. Previous studies from other authors have shown that cell-free DNA is present in plasma and more elevated in severe cases of Lupus. Moreover, immune complexes between autoantibodies and nucleic acid complexes are also found in the plasma of lupus patients. Thus, it is difficult to exclude the possibility that rather than measuring the binding to AIM2, the assay detects other antibodies that bind to a complex to which the AIM2 protein binds as well. This complication does not apply to all samples, as clearly there are plasmas that are anti-dsDNA negative but anti-AIM2 positive. This is also a clear benefit of the SS samples, where binding to DNA is rare. Nevertheless, it is difficult to suggest an alternative route of approach, as the binding of AIM2 to DNA is DNAse resistant. Once a complex of DNA and the recombinant AIM2 forms, it would presumably be able to survive an IP procedure. This represents a major obstacle, at present, to the clear identification of "bona fide" antibodies to AIM2 in the Lupus samples.

Reviewer #3 (Public Review):

The authors developed an assay to quantify Aim2 autoantibodies and demonstrated that SLE patients with detectable Aim2 autoantibodies, on average, had more severe clinical disease. Previous studies had demonstrated that Ifi16, another cytosolic DNA sensor, was also an SLE autoantigen. There was a correlation between higher AIM2 antibody titers and high SLEDAI scores.

They found that patients frequently made autoantibodies reactive with Aim2, Ifi16 and DNA and, given the recent interest in neutrophil NETS and SLE, decided to explore a potential structural explanation for this observation. Their immunofluorescent data convincingly documented the ability of both AIM2 and Ifi16 to multimerize into filaments that bind neutrophil NETS, generated in vitro by PMA activation. They also demonstrated co-localization of NET DNA and their DNA sensors in renal sections obtained from patient populations. Their imaging of renal sections from lupus nephritis patients is particularly impressive. Importantly, they further reported that NET DNA bound by AIM2 and Ifi16 was protected from DNase1-mediated degradation. As far as nuclease protection, there is one point that could be addressed further - the images shown in Figure 2D appear to indicate that AIM2 and Ifi16 are not binding comparably across the NET structure. Quantification of sensor binding in additional images would therefore be informative. The authors should also mention other studies in which DNA-binding proteins protect neutrophil NETS from nuclease degradation (eg. Hakkim 2010).

The authors further documented the presence of AIM2 autoantibodies, but not Ifi16 autoantibodies, in the sera of Sjogren's Syndrome patients. As an explanation for the difference between SLE and SS, they point out that neutrophils are often found in the kidneys of lupus nephritis patients but do not normally infiltrate the salivary glands of SS patients. They raise the possibility of a different scaffold for AIM2. The novelty of the current study could be considerably bolstered if they could detect and identify AIM2 structures by immunofluorescence in tissue from SS patients.

Overall, this is a highly significant study that establishes the connection between high AIM2 autoantibody titers, resistance of AIM2/Ifi16 bound DNA to nuclease degradation, and the presence of AIM2-bound NETS in renal biopsies of SLE patients. The report could be further strengthened by addressing mechanisms by which autoantigen-bound NETS might promote autoantibody production and immune activation.

Reviewer #1 (Public Review):

In this manuscript, the authors present well-designed experimental data to demonstrate a bacterial T3SS effector, XopQ, encodes a phosphoribose-hydrolase to be involved in endogenous cytokinin (iP and tZ) biosynthesis. Inactivation of xopQ resulted in decrease of biofilm development and virulence, whereas addition of exogenous cytokinin rescued the phenotypic deficiencies of the mutant. They also found an ipt gene mutant of Xoo phenocopied xopQ mutation, reinforced the notion that Xoo synthesizes cytokinin with important role in the bacterial pathogen. In addition, the authors found that activation of bacterial cytokinin sensor pcrK and its cognate response regulator gene pcrR decreased virulence and biofilm, but addition of cytokinin cannot suppress the deficiencies of the pcrK or pcrR mutations. This work, together with previous studies carried out in Xcc and M. tuberculosis, reveals that bacteria produce cytokinins, which were originally recognized as plant hormones, and modulate essential physiological processes of these pathogens. Cytokinin is an adenine derivate, and further studies will shed more light on this interesting topic.

Major comments:

1. The regulatory logic among XopQ, ipt and PcrR-PcrK is somewhat loose. Although XopQ and IPT seem in a pathway to synthesize cytokinin, the relationship between them and PcrRK is not established, especially with regard to endo- and exogenous cytokinin.

2. The author showed that bacterium Xoo synthesize more iP than tZ, but does tZ rescue deficiencies in producing biofilm and virulence?

3. The authors suggested that the IPT gene of Xoo is likely originated from HGT; are there more data on its possible origin?

Reviewer #2 (Public Review):

Prior to this work, it was known that plants and bacteria produced cytokinins, which affect plant physiology. This work provides new evidence that cytokinins produced by a plant pathogen affects bacterial physiology controlling free-living and biofilm states, which is a novel finding.

The strengths of the work include the use of genetic and quantitative assays to demonstrate that proteins associated with cytokinin biosynthesis and cytokinin sensing are required to maintain planktonic growth. The work is however descriptive and does not provide a mechanistic understanding of the role of cytokinin in the regulation of biofilm formation and type III secretion. The weakness is that the study does not include experiments addressing when cytokinin is produced (exponential growth, motility, stationary phase and viability) and biologically relevant concentrations of cytokinin that lead to the different states (free-living, biofilms, infectious state). More evidence supporting the concentration-dependent activity of cytokinin on bacterial physiology will strengthen the main observation reported, and help evaluate data where exogenous treatment with cytokinin led to unexpected phenotypes (e.g. wild-type bacterial cells + cytokinin produced more biofilm than untreated wild type bacterial cells).

The importance of this work is that it reveals new roles for cytokinins outside of the plant kingdom and highlights an additional complexity by which these hormones contribute to pathogenic and nonpathogenic states.

Reviewer #1 (Public Review):

Originally identified as a component of the bacterial adaptive immune response, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have found widespread biotechnology applications in genome editing, the detection and imaging of nucleic acids, gene silencing, and chromatin engineering. The focus of this paper is on the CRISPR-Cas9 system, which is paradigmatic for this class of protein complexes. Thus, understanding allosteric communication in CRISPR-Cas9 in relation to its ability to recognize and excise targeted nucleic acid segments is a topic of great current interest. Specifically, this study combines solution NMR with molecular dynamics simulations and employs graph-theoretical analysis of the protein network to uncover the effects of three key mutations in the HNH domain (K810A, K848A, and K855A) that increase Cas9 specificity and reduce off-target activity. These mutations were found to disrupt the main communication pathway between the RuvC and REC modules, suggesting a direct link between changes in the allosteric network and the observed increase in Cas9 target specificity. Notably, the magnitude of decrease in allosteric communication (as measured by mutation-induced edge betweenness difference) correlates perfectly to the order of specificity enhancement (K855A > K848A ~ K810A). Thus, the paper sheds light on an aspect of Cas9 function that may have important implications for the design of new gene editing tools with improved specificity. The findings are novel and well supported by the data. In particular, the molecular dynamics simulations and analysis appear to have been done using appropriate simulation protocols. Both the experimental and computational methods are described in detail. Approaching allosteric effects in Cas9 from multiple angles, using multiple experimental and computational techniques is a notable strength of this manuscript.

Reviewer #2 (Public Review):

The CRISP-Cas9 complex has revolutionized genomic editing techniques. The widespread application of this new molecular tool enables a precise and accurate DNA cleavage that has been impossible to achieve. Yet, in some cases, the system suffers from a lack of specificity. In this paper, the authors present a new study on the characterization of the allosteric communication within the CRISP-Cas9 complex. They identified three different mutations that disrupt the complex's internal allosteric communication, affecting the cleavage reaction's specificity to different extents. The authors argue that the various degrees of perturbation are correlated with the Cas9 specificity. Given the size of the complex, the authors utilize a divide and conquer approach to studying the structural-dynamic changes of the isolated HNH endonuclease using NMR spectroscopy. Then they used molecular dynamics simulations to relate the changes in the isolated enzyme to the entire complex. As marked by the authors, the effects of the selected mutations (K855A, K810A, and K848A) are minimal. The HSQC spectrum in Figure 2B shows only marginal chemical shift changes in the protein fingerprint. The latter is supported by the CD spectra that show no significant perturbations in the dichroic profiles. However, the lineshapes reveal substantial changes in the enzyme dynamics apparent from the broadening of several signals. The chemical shift perturbations, although small, show that K855A has the most pronounced spectroscopic changes followed by K810A and K848A. As expected, the most significant differences are revealed by relaxation studies. The authors performed T1, T2, and heteronuclear NOE experiments to characterize the fast dynamics of the protein in the NMR time scale, revealing the most significant differences in the K855A mutant.

Additionally, they used CPMG dispersion experiments to analyze the dynamics in the micro-to-millisecond time scale. From these measurements, the authors conclude that the relaxation characteristics of the mutants do not change significantly, i.e., the mutants possess conformational flexibility similar to the wild type. To interpret the dynamic behaviors of the different HNH variants, the authors performed MD simulations and analyzed the allosteric network using community analysis. The computational work revealed the connections between the communities and how the mutants affect interdomain communication (figure 5).

Overall the paper is exciting and shows how NMR and MD simulations can be used synergistically to dissect the intra- and inter-molecular allosteric communication in highly complex systems. However, there are a few shortcomings that the authors need to address. One significant concern is the lack of a direct comparison between the NMR studies and the MD simulations. Additionally, it is unclear how these dynamics or structural perturbations caused by these selected mutants are converted into the enzyme's increased or decreased specificity.

Other technical concerns:

A) The authors performed relaxation measurements for fast dynamics. However, they did not calculate the order parameters for the protein backbone. Usually, the order parameters from the protein backbone can be directly compared to the calculated values from MD trajectories. How do the S2 values from the two techniques compare?

B) The authors state that the differences in the relaxation dispersion profiles are less than 1.5 Hz, indicating small changes in dynamics. Did the author compare all residues or a subset of residues?

C) In the discussion, the authors refer to the synchronous motions that may be responsible for specificity. How did they deduce that the motions are synchronous? From MD simulations or the global fitting of the CPMG curves? Do motions need to be synchronous for effective allosteric communications?

D) Finally, the authors claim that mutations can target sites identified in this study (hotspots) to improve CRISP-Cas9 function. Can the authors elaborate more on this point? How do they envision mutations to tune the function of the complex?

Reviewer #3 (Public Review):

In the associated manuscript, the authors aim to combine solution state NMR and MD simulations to characterize the structures and dynamics of three mutations within the HNH domain of the CRISPR-Cas9 system. These previously described mutants increase the specificity of the endonuclease through a proposed mechanism of altering the allosteric signaling of the enzyme. A precise understanding of this mechanism could allow for the production of next generation CRISPR-Cas9 systems with increased specificity.

The authors use a 3.6 us MD simulation and interesting analysis methods to characterize the changes in structure and dynamics of the three mutants. Unfortunately, the NMR data plays a very minor supporting role and is not fully analyzed or described. Thus, many of the conclusions from these results are weakened, and it is unclear whether the results support their conclusion.

If this work is predictive, it could be very interesting for the field. Off-target effects are a problem with gene editing via the CRISPR-Cas9 system, and this study could suggest novel mutants which may help further increase specificity. That promise remains to be seen.

Reviewer #1 (Public Review):

The authors propose several ways of leveraging single-particle tracking experiments to distinguish between intracellular phase separation and an alternative model of clustered binding sites. The first proposed scheme is particularly intuitively appealing: in the binding site scenario, the local density of binding sites both increases particle density and slows effective particle diffusion, leading to a definite relationship between these two quantities, while the phase separation scenario would not necessarily couple these two quantities. The additional schemes based on particle movement near a cluster boundary, angles between consecutive steps, and search times add to the arsenal of potential analysis tools. Overall, the work is timely, rigorous, and generally clearly presented and given the growing list of reported observations of phase separation, will appeal to a broad audience.

Reviewer #2 (Public Review):

Heltberg et al. investigate two possible mechanisms for the formation of nuclear foci and how these mechanisms can be distinguished experimentally, based on single-particle tracking of molecules that are up-concentrated in the focus. First, liquid-liquid phase separation (here: Liquid Phase Model, LPM) is treated as one of the major mechanisms currently hypothesized. Second, as an alternative mechanism, a polymer-bridging model (PBM) is investigated, in which the focus is held together by polymer bridges and contains binding sites, which can lead to local enrichment, appearing as a focus.

The theory is presented in a clean way, and while the Langevin equation for single molecules in a phase-separated liquid comes without derivation, it is plausible, and in fact backed up by our own calculations. A similar Langevin equation is found for the PBM and it is subsequently shown that both models can lead to very similar displacement distributions, thus showing that this simple observable cannot always distinguish between PBM and LPM.

Subsequently, the authors derive an effective description of the PBM, based on the experimental observation that potential binding sites on the DNA (proxied by Rfa1, a DNA-binding protein) diffuse much more slowly than a typical repair factor (represented by Rad52). Thus there is a separation of time scales between the two relevant diffusion processes, which is used to constrain the possible parameter combinations for the PBM. Based on these constraints, the authors shown that PBM is incompatible with their previous experimental results.

The remainder of the paper deals with a number of interesting observables, such as the angular distribution of displacements and search time to find a repair target, which can also be used to distinguish PBM and LPM with an ideal setup.

Strengths:

Heltberg et al. present a clean way to distinguish LPM on the one hand, and a realization of PBM on the other hand, based on theory. This is validated by comparison to data they obtained in previous work. The theory is rigorous and the data analysis is well carried out, save for minor ambiguities, which can likely be eliminated during revision. The paper draws its main strength from its interdisciplinarity.

(Minor) Weaknesses:

While the PBM presented here seems like a reasonable model if one were to think of alternatives to LPM, it is always possible to think of more specialized models with additional parameters and mechanisms to account for the same observations. In interpreting this study and its conclusions it is thus important to keep in mind that the PBM presented here is not the only possible realization of a model that can give rise to focus formation. It would be interesting to explore alternatives that are less dependent on the precise form of the potential chosen here and which might treat the polymer bridges more directly. Also, thinking of possible multi-species extensions might be an interesting future direction.

It would be helpful to back up the rates which were not measured experimentally by suitable references or discuss more transparently where assumptions were made that have not been investigated in the literature.

Conclusions and Discussion:

The authors have achieved their goal of distinguishing LPM and PBM. The corresponding theory will be of great use for everyone in the field aiming to make this distinction based on single molecule tracking, a strategy that has been attempted numerous times, but eventually always failed due to the lack of an appropriate theoretical framework. Heltberg et al. have gone on to show a striking difference between experimentally constrained PBM realizations and the experimental measurements themselves, rendering the PBM much less likely than the LPM.