A conserved Arf-GEF modulates axonal integrity through RAB-35 by altering neuron-epidermal attachment

  1. Igor Bonacossa-Pereira
  2. Sean Coakley
  3. Massimo A. Hilliard

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Abstract

Neurites of sensory neurons densely innervate the skin and are embedded within it. These delicate structures are exposed to acute and chronic mechanical strain and yet their integrity is maintained throughout life. Although evidence suggests a neuroprotective role for the skin, the molecular pathways involved are still poorly understood. In C. elegans , the cytoskeletal molecule UNC-70/β-spectrin functions in synergy with the small GTPase RAB-35 within the skin to stabilize neuron-epidermal attachment structures against mechanical strain and prevent movement-induced damage to mechanosensitive axons. However, the full suite of molecules regulating these specialized attachments remains elusive. Here, through an unbiased forward genetic screen we have identified a guanine nucleotide exchange factor (GEF) previously associated with the endocytic-recycling machinery, AGEF-1a, that impacts axonal maintenance. We show that AGEF-1a functions selectively within the skin to regulate the integrity of the embedded axons. Mechanistically, we reveal that this effect is achieved through an interaction between AGEF-1a and epidermal RAB-35 to facilitate its activation, which in turn modulates the neuron-epidermal attachments. Finally, we demonstrate that the function of this GEF is highly conserved, with the expression of its human ortholog, BIG2 capable of replacing AGEF-1a. Together, we reveal the specific molecular machinery responsible for fine-tuning neuron-epidermal attachments and maintaining axonal integrity during life.

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    Reply to the reviewers

    Manuscript number: RC-2025-03064

    Corresponding author(s): Massimo, Hilliard; Sean, Coakley

    1. General Statements

    We are grateful to the reviewers for taking time to review our manuscript and for providing such clear, insightful and actionable suggestions. The consensus between 4 independent reviewers that this story is of general interest to cell biologists, neurobiologists and clinical researchers is remarkable. In addition to our mechanistic insights into the regulation of GTPase activity, we think that the experimental systems we have developed will be of great value to study how GTPases their associated GAPs and GEFs function to maintain the nervous system, especially due to the demonstrated conservation of these molecules. We believe that our data provides a powerful and tractable model to study such molecules in a physiological context.

    We agree with the reviewers' concerns and propose the following plan below to address them.

    2. Description of the planned revisions

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

    __Summary Stability of the PLM axon in C. elegans is maintained through interactions with the epidermis. Previous studies by this group found that loss of the tbc-10 Rab GTPase Activating Protein strongly enhanced the PLM axon break phenotype of unc-70/beta-spectrin mutants. TBC-10 is a GAP for RAB-35 and thus loss of rab-35 suppresses the tbc-10 phenotype. Of the two RAB-35 GEFs, loss of RME-4 partially suppressed the tbc-10 phenotype and FLCN-1 was not involved suggesting that there may be an additional GEF involved. Here Bonacossa-Pereira et al identify a point mutation in agef-1a (vd92) as a suppressor of tbc-10 PLM axon break phenotype (all experiments also have a dominant allele of unc-70) and confirm that point mutation is causative by replicating the mutation via genome editing (vd123). Rescue experiments demonstrate that AGEF-1a is required in the epidermis and not PLM as previous demonstrated with tbc-10 and unc-70. Rescue is dependent on a functional SEC7/GEF activity. AGEF-1a is a functional ortholog to human BIG2/ArfGEF2 as its expression fully rescues tbc-10. AGEF-1a functions upstream of RAB-35 as expression of activated RAB-35 can suppress loss of agef-1. AGEF-1a functions in parallel to RME-4 as the double has stronger suppression of tbc-10. AGEF-1a is an ARF GEF, however it functions independently of ARF-1.2 as loss of arf-1.2 does not suppress tbc-10. They demonstrate that AGEF-1a interacts with RAB-35 through colocalization experiments suggesting that AGEF-1a could directly activate RAB-35. Finally, they demonstrate that AGEF-1a regulates the localization of the LET-805 epidermal attached complex component as it restores localization in a tbc-10 mutant.

    Major comments

    The manuscript is well written and easy to understand.

    The experiments are well done and controlled.

    I enjoyed reading this paper. However...

    Some of the claims are not supported by the data.__

    __1) The claim that AGEF-1a directly interacts with RAB-35 was not demonstrated. The evidence provided to support a direct interaction are colocalization experiments in Figure 3. AGEF-1a does partially colocalize with RAB-35 in the epidermis. However, colocalization does not indicate a physical interaction direct or indirect. A simple fix would be to change the claim to that they partially colocalize. Optional, a physical interaction could be done with the split-GFP since they already have the AGEF-1 strain or they could perform co-IP experiments, though neither of those are proof of direct interactions.

    __

    We agree that the biochemical co-IP experiment could provide some answers, however, using a full length AGEF-1a would not only represent a significant technical challenge but will also not prove a direct interaction in a physiological context. To overcome this limitation, and to directly test their interaction in vivo, we propose to use a split-GFP approach as suggested by the reviewer. In this experiment, we will generate an endogenously tagged GFP1-10::rab-35 allele and combine it with the previously generated and available tagged agef-1a::GFP11x7. If AGEF-1 and RAB-35 closely interact, we should observe the reconstitution of full length GFP. It is possible that the endogenously tagged versions only provide a very weak GFP signal that will be difficult to detect. As an alternative approach, we will generate the same tagged molecules as overexpressed transgenes under epidermal-specific promoters (such as Pdpy-7). If the results are still negative, we agree to temper our claim that these molecules physically interact and rephrase the manuscript to reflect the new data.

    2) The claim that AGEF-1a facilitates RAB-35 activation is not supported. While it is likely that AGEF-1a facilitates RAB-35 activation based on the epistasis experiments as well as studies in mammalian cells there were no experiments to demonstrate that modulating AGEF-1a activity resulted in a change in RAB-35 activity. I would suggest tempering this claim to something along the line that the data are consistent with AGEF-1a regulating RAB-35 activity as shown in mammalian cells. An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081).

    We welcome this suggestion and acknowledge the limitations of these experiments. While we might be able to determine if AGEF-1 and RAB-35 physically interact in vivo with the experiments proposed above, screening for the relevant *rab-35 *effector in this context and/or doing effector pull-down/cell based GEF assays would be a significant technical challenge. We propose to temper our claim as suggested.

    3) The claim that AGEF-1a functions independently of ARF-1.2 is not well supported. The fact that the ARF-1.2 mutant does not suppress tbc-10 suggests that ARF-1.2 may not be involved but does not eliminate the possibility that ARF-1.2 functions redundantly with ARF-5 or WARF-1/ARF-1.1. This can be resolved by toning down the claim. Alternatively, this can be tested by RNAi of arf-5 and warf-1 in tbc-10 and arf-1.2; tbc-10 mutants.

    We agree that warf-1 and arf-5 could be functioning redundantly with arf-1.2. We have attempted to generate an AID::arf-5 allele to test the effect of cell-specific degradation, but homozygous AID::arf-5 animals were lethal. We have not yet examined warf-1. We believe the best way to test these two molecules is through RNAi knockdown, and we propose to do this experiment and adjust our interpretation and discussion according to the new data.

    Minor comments

    Figure 1C the CRISPR generated allele (vd123) is referred to as [S784L] and then in 1E vd92 is referred to as [S784L]. Perhaps it would be clearer if the allele name was used instead of the amino acid change.

    We will reformat the manuscript to include the allele names instead of amino acid change.

    Page 6 "We reasoned that if the S784L mutation we isolated causes a similar loss of the GTPase activation function, then SKIN::AGEF-1a[E608K] would not have the capacity to restore the rate of PLM axon breaks to background levels in agef-1[S784L]; tbc-10; vdSi2 animals." It was unclear to me whether you were testing if the S784L mutation could be disrupting a GEF independent function or might disrupt the nucleotide exchange activity as might be tested in a biochemical assay. There are many reasons this change could cause a loss of function phenotype (ie. Improper folding, mislocalization, etc.). The most clear explanation would be that you were testing if GEF function was required for rescue rather than testing if the S784L mutation disrupted GEF activity.

    Indeed, this experiment reveals that reducing the activation of the AGEF-1 target phenocopies the effect of S784L and does not further enhance the effect of S784L. However, it does not answer if, specifically, the GEF function is affected by S784L. We propose to rewrite the quoted sentence as follows: "We asked whether the GEF function is required for axonal damage. If that is the case, then SKIN::AGEF-1a[E608K] overexpression should phenocopy the effect of AGEF-1a[S784L]."

    Page 13. It was unclear how testing if AGEF-1, RME-4, ARF-5 and RAB-35 form complexes in vivo (I assume you are suggesting colocalize based on figure 3 interpretation) would resolve how AGEF-1 was regulating RAB-35.

    We apologize that our phrasing was not clear. We will rewrite this section to better reflect the following idea. Given literature data showing an allosteric interaction between RME-4/DENND1 and ARF-5/Arf5, and our own data showing that AGEF-1 regulates RAB-35, we believe these molecules could form a complex. Considering that we do not have data to support this notion, mostly due to the inability to test the effect of ARF-5, we will present this possibility in the discussion section.

    __**Cross-commenting**

    I agree with the comments made by the other reviewers and I stand by my own as well. I will echo that it is important to know the nature of their agef-1 allele.

    Reviewer #1 (Significance (Required)):

    Bonacossa-Pereira et al identify AGEF-1 as a regulator of axon integrity that functions in a pathway with RAB-35 in the epidermis is an exciting finding. As pointed out in the discussion, mutations in the human ortholog cause neurodevelopmental defects which leads to obvious characterization of BIG2/ArfGEF2 in neurons while this study indicates that this protein can have cell non-autonomous roles in regulating neurons. These findings could have important implications for understanding the etiology of these defects that would be of interest to neurobiologists and clinical researchers.

    The finding of this paper would also be of interest to cell biologists and particularly those studying the roles of Rab and Arf GTPases in membrane trafficking, such as myself. The idea that AGEF-1 might function as a Rab35 GEF is provocative and would generate a lot of interest and skepticism from the field. However, there is no data to support that AGEF-1 would be a direct regulator of Rab35 over the previously demonstrated cross regulation of Rab35 by Arf GTPases. Therefore, it would be fine to speculate in the discussion a direct interaction, but I would refrain from suggesting this as a model and elsewhere in the manuscript.

    __

    Although we agree that current evidence is not sufficient to support the model where AGEF-1 is a direct regulator of RAB-35, our data points to the direction where there is an important genetic relationship between these molecules in a physiological context in a living animal, with a defined phenotype relevant to the nervous system maintenance. We think that the proposed revision experiments will provide a better understanding of how AGEF-1 functions with RAB-35 and we agree with the suggestion to rephrase our manuscript to reflect the limitations of our results.

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

    This interesting manuscript reports the outcome of a fruitful C. elegans genetic screen with a complex but clever design. Through it, the authors identify AGEF-1 as a GEF that likely regulates the active state of the GTPase RAB-35 in the skin to protect touch receptor axons from mechanical breakage.

    Major points:

    1. Based on localization experiments, the authors claim "AGEF-1a interacts with RAB-35 in the epidermis" (Results heading) and state "these data demonstrate that AGEF-1a interacts with a subset of RAB-35 molecules in the epidermis." In general, localization studies cannot be used to conclude physical interaction (with some exceptions such as single-molecule kinetics). In this case, the data in my view do not even make a compelling argument for co-localization. There is a lot of AGEF-1 and RAB-35 signal everywhere and it may not be meaningful that the signals sometimes overlap. A more quantitative approach with controls would be needed to conclude meaningful co-localization. Importantly, this would still not demonstrate interaction.__

    We thank the reviewer for the comment. Indeed, co-localization does prove a physical interaction, and we appreciate the concern about our imaging data not making a compelling argument. To address this notion, we plan to perform an experiment using a more robust, quantitative and physiologically relevant strategy. We will generate an endogenously tagged mScarlet3::rab-35 allele for precise endogenous localization. In addition, as a positive control, we will generate an endogenous rme-4::GFP11x7 allele to cell-specifically demonstrate the level of colocalization of RME-4 with mScarlet3::RAB-35 within the epidermis. To address the possible interaction between AGEF-1a and RAB-35 we will leverage a split-GFP approach to assess their interaction in vivo, in the context relevant to the phenotypes we observed (see reply to reviewer #1 point 1).

    __2. The effect of the AGEF-1(S784L) mutation is not clear to me. Naively, as the S784L mutation lies in the auto-inhibitory domain, I would have expected AGEF-1 to become constitutively active, not inactive as the authors seem to suggest. Is the idea that it is constitutively auto-inhibited? The main evidence for a loss of function effect seems to be that a putative dominant negative mutation AGEF-1(E608K) does not further supress axon breakage when co-expressed in trans to AGEF(S784L), but in my view this only shows that, once the defect is suppressed, it cannot be suppressed any further. Defining the nature of the S784L allele is important. Some suggestions, although the authors may come up with different approaches: use of an inducible or cell-specific depletion system like AID/TIR1, Cre/lox, or FLP/FRT to circumvent the lethality of agef-1(0) and reveal what a true loss-of-function looks like; testing if deletion of the auto-inhibitory domain phenocopies S784L to test if this mutation impairs autoinhibition.

    __

    This is an very insightful comment. To address this point, we will follow the reviewer's suggestion and deplete AGEF-1 cell-specifically in the epidermis using the auxin-inducible degron system. Specifically, we will generate an agef-1::AID allele to degrade this molecule in a spatially and temporally controlled fashion, which will allow to circumvent the lethality of agef-1(0) and determine whether the S784L allele mimics the depletion of AGEF-1.

    Although it would be interesting to further dissect the effect of this mutation on AGEF-1 activity, we believe that this falls outside of the scope of this manuscript. As an alternative, we propose to elaborate more in the discussion the implications of the possible roles for the S784L mutation to clarify our model of its function. Our data supports a model in which this mutation reduces AGEF-1 function leading to a reduction in the activity of its downstream target GTPases. It is possible that this is due to AGEF-1 becoming constitutively autoinhibited, or that this mutation affects the structure of the molecule in a way that it reduces its affinity towards its downstream effectors.

    __Minor points:

    1. I am not able to see the "vesicle-like structures with a clear luminal space" or RAB-35 being "notably enriched at the membrane near the epidermal furrow" in Fig. 3. The "3D surface rendering" in Fig. 3e is grossly oversampled and should not be included.__

    We will rectify this section and include new super-resolved images using Airyscan confocal microscopy. We hope these will yield a better-quality representation of these concepts. __

    1. As the agef-1a isoform is specifically referenced throughout, please describe the different agef-1 isoforms somewhere to save readers from having to look this up.__

    Yes, we will include a description of the isoforms. In C. elegans there are two: AGEF-1a which has been confirmed by cDNA and AGEF-1b which is predicted and partially confirmed by cDNA. The mutation we isolated exclusively affects AGEF-1a.

    3. The authors include an interesting speculation in the Discussion: "Future investigations of BIG2-associated neurological disorders should consider... hyper-activity of BIG2 as a driver of neuropathology." If the authors have the tools to test the effect of hyperactive BIG2 in this system, it could be an exciting addition.

    This is an exciting idea that we would like to keep in the Discussion. The biology of BIG2 activity regulation is a nascent field of research and we believe that to accurately generate and characterise a hyperactive BIG2 would be beyond the scope of this manuscript.

    __ On a personal note, since GEFs act oppositely to GTPase Activating Proteins (GAPs), I had to stop and re-read carefully whenever the authors referred to a GEF "activating" a GTPase. I understand their meaning (i.e., putting the GTPase in its active GTP-bound state, not activating its GTPase function) but I wanted to point out this potential confusion in case there is a way to better define terms in the Introduction or change word choice. I realize this may be a standard jargon in the field.__

    Indeed, this is confusing nomenclature and a difficult concept to deliver in an accurate and succinct manner. We propose to include a clearer, more didactic explanation of their function. In a simple explanation, GTPases perform cellular functions when bound to GTP. GAPs terminate GTPase activity by catalysing GTP hydrolysis, generating GDP. GEFs initiate GTPase activity by catalysing the release of GDP and allowing GTP binding.

    __ Please check the correct nomenclature for CRISPR/Cas9.__

    We will rectify where appropriate.

    __6. p.7 "these molecules act in synergy", consider replacing with "redundantly".

    __

    We will rectify where appropriate.

    __Reviewer #2 (Significance (Required)):

    The significance of this story is to show that GEF-GTPases pairing can be highly context-dependent. Previous studies have identified GEFs that pair with RAB-35 and GTPases that pair with AGEF-1, but the authors find that these factors have at best a modest role in the context of skin-axon interactions. Instead, the authors suggest a novel GTPase-GEF pairing of RAB-35 with AGEF-1 and provide evidence that this relationship is conserved in the human homolog of AGEF-1. These results suggest that GTPase-GEF pairings depend not only on chemical affinity but also cellular context.

    The main strength of the study is its clever genetics. For the screen, the authors looked for suppressors of a synthetic defect in axon integrity caused in part by elevated activity of RAB-35 due to loss of its GAP TBC-10. It is satisfying that this screen isolated a mutation in a GEF that in principle could counterbalance the loss of a GAP.

    The main weakness of the study is the lack of direct evidence for an AGEF-1/RAB-35 interaction. While not necessary for publication, the inclusion of biochemical data to support the role of AGEF-1 as a GEF for RAB-35 and the effect of the S784L mutation on this activity would strongly elevate the study. The genetic data for this interaction are consistent with the model but not conclusive, and in my view the colocalization data are not compelling. Nevertheless this is a solid genetic story with a clever screen.__

    __ __We appreciate the feedback and are grateful for the positive comments on the significance of our study. As explained in the significance section related to Reviewer 1, if we find evidence of a direct interaction between AGEF-1 and RAB-35 in the proposed new experiments, we will include it in the manuscript; alternatively, we will present it as a possibility in the discussion section, as suggested. We agree that a more nuanced understanding of the effect of the S784L is interesting and that our colocalization data can be improved, and we have proposed experiments to address these concerns.

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

    This paper investigates the mechanism by which molecular pathways in the skin protect the processes of nerves that innervate them from damage. The authors previously showed that spectrin and the small GTPase RAB-35 act in the epidermis of C. elegans to protect mechanosensory axons from breaking. In this paper they used a suppression screen to identify another gene involved in this process, an ARF-GEF called AGEF-1. Partial loss-of-function mutations in agef-1 suppress the axon-breakage phenotype of spectrin mutations, and genetic experiments by the authors are consistent with the possibility that AGEF-1 could act directly as an exchange factor for RAB-35. Consistent with this model, they show that AGEF-1 and RAB-35 colocalise in the skin.

    Major comments: The experiments in this paper are well-designed and well-controlled, and the interpretations of the results are all reasonable. On the other hand, I don't think the authors' hypothesis that AGEF-1 acts directly as an exchange factor for RAB-35, or that these two proteins directly interact, is definitively proven. This is not an issue of the authors overinterpreting their data--the paper is very carefully and thoughtfully written. However, the most interesting and counterintuitive finding--that an ARF-GEF could also be a RAB-GEF--might be strengthened with more experiments (for example, could they more directly show protein-protein interaction through co-IP or mass spec?).__

    We thank the reviewer for the suggestion. We propose to further investigate the notion that AGEF-1a might be a direct interactor of RAB-35 using a split-GFP approach to assess whether these molecules closely interact, in vivo, in the physiological context that is relevant for the maintenance of the touch sensing neurons (please see reply to reviewer #1 major point 1 and reviewer #2 major point 1 for more details).

    Minor comments: There are also two places where the fact that null mutations are lethal (for agef-1 and arf-5) prevented the authors from addressing the effect of agef-1 loss of function in the skin, and addressing whether ARF-5 could be an AGEF-1 target, respectively. In principle, they could have tried to make a CRISPR line in which these genes could be cell-specifically deleted in the skin (using a dpy-7-driven recombinase). I don't think either of these experiments are essential, but if it is feasible to make these lines it would tie up a couple of loose ends.

    We agree to explore the roles of agef-1 and arf-5 loss-of-function. We propose to tissue-specifically degrade agef-1 using an auxin-inducible degradation strategy (please see reviewer #2 major point 2 reply for more details). For arf-5, we propose knocking-down its function using RNAi to overcome lethality (please see reviewer #1 major point 3 reply for more details).

    __Reviewer #3 (Significance (Required)):

    Overall I think this is an interesting paper on a topic of general interest. The most interesting finding is that an exchange factor for an ARF (a small GRPase involved in vesicle coating/uncoating) could also be an exchange factor for a RAB (a small GTPase involved in vesicle tethering). The evidence presented is suggestive and intriguing, though as noted above not completely definitive. In summary, I think it is an interesting paper in its current form, and anything it could do to more firmly establish a direct interaction between AGEF-1 and RAB-35 would increase its impact and importance.

    __

    We thank the reviewer for the positive evaluation of the significance of our study.

    __ Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    Summary: In this study Bonacossa-Pereira et al. identify AGEF-1a, an Arf-GEF, as a factor that functions in the epidermis through RAB-35 to regulate axonal integrity of the PLM mechanosensory neurons in C. elegans. Specifically, epidermal attachment sites are regulated by these genes form the epidermis and compromising these attachment sites results in axonal degeneration. The study provides some evidence that that RAB-35 and AGEF-1 at least partially colocalize in the skin. Finally, the authors provide evidence that the human orthologue BIG2 is capable of functionally replacing AGEF-1a in C. elegans. Overall, the experiments are well designed and the paper is clear and succinct. The conclusions are supported by the findings and provide an important extension of the author's findings a few back, when they identified the role of rab-35 in mediating the epidermal-neuronal attachment sites.

    Major comments:

    1. AGEF-1/BIG2 are known to regulate other GTPases such as ARF-5 or ARF-2. The authors exclude a non-redundant function for ARF-2, but are unable to establish a role for ARF-5 because of the lethality associated with the mutation. Alternative approaches, such as cell specific knock out or knock down experiment. In addition, studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5.__

    We thank the reviewer for this suggestion. We propose addressing these concerns using a tissue-specific degradation for AGEF-1a (please see reviewer #1 major point 2 for details). To establish a role for ARF-5 we propose to do an RNAi mediated knock-down to overcome lethality (please see reviewer #1 major point 3 for details). Finally, we plan to use a split-GFP approach to test the physical interaction between agef-1a and rab-35 in vivo (please see reviewer #1 major point 1 for details)

    __ Phenotypic readout has been limited to only axon breaks. It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch or synaptic integrity could be informative.__

    We have not observed any obvious touch receptor neurons axonal phenotypes other than axonal breaks in these mutants, and we will include a statement that reflects this concept. In relation to the behavior, we have not tested it as the results will be difficult to interpret for two reasons: first, the breaks are not always bilateral and one neuron is sufficient to provide mechanical response; second, the mixed identity of the PLM neurite allows it to retain some function despite being severed. However, if deemed essential, we will perform these experiments.

    __ Overexpression constructs such as SKIN::RAB-35[Q69L], SKIN::BIG2, SKIN::AGEF-1a[E608K] in extrachromosomal transgenes could lead to non-physiological localization or effects. Single copy expression using MosSCI or CRISPR insertions are generally considered better approaches (other than endogenous reporters) to provide accurate insights at the physiological level. While the authors tacitly acknowledge this by conducting the experiments in a rab-35 mutant background and very low transgene concentration, at the very least this caveat regarding the localization should be discussed.__

    This is an important remark, and we appreciate the comment. We acknowledge that experiments using extrachromosomal arrays have inherent caveats, especially for localization studies. To address the RAB-35 localization concern we plan to repeat the localization studies using an endogenously tagged RAB-35 using CRISPR to overcome the possible artifacts caused by extrachromosomal array driven expression (please see reviewer #1 point 1 for more details). For the cell-specific rescues or dominant-negative constructs expression, we believe that using extrachromosomal arrays is sufficient, since this allows us to compare genetically identical transgenic vs non-transgenic siblings of independent lines. Moreover, given these constructs are already driven by a tissue-specific promoter that is inherently stronger than their respective endogenous promoters, even a single-copy insertion would have the same caveats.

    __4. The study does not address clearly whether AGEF-1a acts in parallel to spectrin or upstream/ downstream to it. Epistasis experiments could help to figure out the signaling pathway involved.

    __

    Indeed, this is a concept that we need to communicate more clearly. We have data showing that a mutation in agef-1 does not cause axonal damage on its own, and that it has no effect on the axonal damage caused by *unc-70 *dominant negative mutation alone. We only detect an effect of agef-1 when *tbc-10 *is mutated together with unc-70 (Fig. 1a of manuscript). Together, these data indicate that agef-1 functions upstream of rab-35, thus acting in parallel to unc-70 (see schematic below) to ensure the mechanical stability of neuron epidermal attachment. We plan to include this data and the following schematic as a supplement to better convey the idea and discuss the results appropriately.

    __ The finding that BIG2 rescues the mutant defect is an important finding and rightfully finds its place in the abstract. I wonder whether a reference to the human diseases caused by loss of BIG2 in the abstract and introduction would not increase interest/impact for the study, rather than burying this potentially interesting connection in the discussion.

    __

    We appreciate the reviewer's comment, and welcome the suggestion. We propose to include relevant background about BIG2-related human diseases in the abstract and introduction as suggested and expand the discussion regarding BIG2 mutations.

    __Minor comments:

    1. Some explanation about how mutating the autoinhibitory domain could impact the catalytic activity of a GEF might be helpful.__

    We acknowledge that this notion was not well communicated. We propose to elaborate more about why we think a mutation in the autoinhibitory domain might be affecting the GEF activity and we plan to do further experiments to dissect how this might be happening. Please see reviewer #2 major point 2 for a more detailed explanation.

    __ The paper refers to rme-4(b1001) as a null allele while wormbase refers to the same as a missense allele. It would be more accurate to refer rme-4(b1001) as a strong loss of function or putative null.__

    We agree and will refer to b1001 as a strong loss-of-function.

    __ The paper does not clearly discuss limitations of the hypomorphic agef-1[S784L] and that the observed phenotypes in this hypomorph might underestimate the complete role of AGEF-1a.__

    We thank the reviewer for this suggestion. We propose to elaborate more on these limitations, especially considering the possible new results from the experiments suggested in reply to reviewer #2 major comment point 2.

    __ In figure 1, where there really only one extrachromosomal transgenic line for some of the construct tested? __

    For the *Pdpy-7::AGEF-1a *lines we have scored 3 transgenic lines (data not included) and only one yielded a full rescue. For all extrachromosomal lines presented, we tested 3 independent transgenic lines. For brevity, we only included the result for the positive rescues (1 for BIG2 and 1 for AGEF-1a), except for the Pmec-4 lines, of which none rescued the phenotype (data included in Table S2). We will update Table S2 to include all the lines tested.

    __ The concentrations of transgenes vary in different transgenes. Is there a rationale behind this? __

    Yes, we have attempted multiple concentrations of injections for each transgene and there was some variability for each construct injected, thus we only included the ones where we observed an effect. As mentioned in point 4 above, we will update Table S2 to include details of all lines tested.

    __ In Fig.1e: I may be useful to also show the "WT" phenotype, i.e. the strong defects to get a visual comparison for the degree of rescue. __

    We think this suggestion will help the readers. We will include this as a representative dashed line showing the WT phenotype.

    __Reviewer #4 (Significance (Required)):

    The study has identified AGEF-1a as a regulator of axonal maintenance, functioning to protect neurons against mechanical stress by acting through RAB-35. Additionally, this epidermal GEF, AGEF-1a is functionally conserved as its human orthologue BIG2 can replace AGEF-1a in C. elegans for axonal protection. Important points here are that the findings extend prior work by the authors of non-autonomous mechanism that regulates epidermal-neuronal attachment. In my humble opinion, the human disease connection, in particular with regard to the unexplained neuronal phenotypes in patients could be better developed in the manuscript. It may also increase impact/interest of a wonderful story that right now reads a bit 'wormy'.__

    This is an important remark and we are grateful for the positive comments. The fact that human BIG2 is also conserved in C. elegans points to a fundamental role of this molecule in multicellular life, and it provides a tractable model to investigate the function of this molecule in a physiological context. We welcome the suggestion to elaborate more the connection with the unexplained neuronal phenotypes in patients and use a more accessible language to convey our findings to a wider audience.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    N/A

    4. Description of analyses that authors prefer not to carry out

    __Reviewer #1 __

    "...studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5."

    While pull-down assays, co-IP and FRET would reveal whether AGEF-1a can form a complex with RAB-35, we believe that using a full length AGEF-1a would not only represent a significant technical challenge but will also not prove a direct interaction in a physiological context.

    "...An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081)."

    We think that screening for the relevant rab-35 effector in this context and/or doing effector pull-down/cell based GEF assays would be a significant technical challenge. We propose to address this concern by tempering our claim as suggested by the reviewer.

    "...It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch or synaptic integrity could be informative."

    As indicated above in major point 2 of reviewer 4, these are interesting ideas that might answer how the function of these neurons might be affected. However, in addition to the challenges indicated above, they will not provide further insights into how their integrity is maintained. We believe these will fall outside the scope of the manuscript, but if deemed essential we will perform behavioral analysis.

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    Referee #4

    Evidence, reproducibility and clarity

    Summary:

    In this study Bonacossa-Pereira et al. identify AGEF-1a, an Arf-GEF, as a factor that functions in the epidermis through RAB-35 to regulate axonal integrity of the PLM mechanosensory neurons in C. elegans. Specifically, epidermal attachment sites are regulated by these genes form the epidermis and compromising these attachment sites results in axonal degeneration. The study provides some evidence that that RAB-35 and AGEF-1 at least partially colocalize in the skin. Finally, the authors provide evidence that the human orthologue BIG2 is capable of functionally replacing AGEF-1a in C. elegans. Overall, the experiments are well designed and the paper is clear and succinct. The conclusions are supported by the findings and provide an important extension of the author's findings a few back, when they identified the role of rab-35 in mediating the epidermal-neuronal attachment sites.

    Major comments:

    1. AGEF-1/BIG2 are known to regulate other GTPases such as ARF-5 or ARF-2. The authors exclude a non-redundant function for ARF-2, but are unable to establish a role for ARF-5 because of the lethality associated with the mutation. Alternative approaches, such as cell specific knock out or knock down experiment. In addition, studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5.
    2. Phenotypic readout has been limited to only axon breaks. It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch orsynaptic integrity could be informative.
    3. Overexpression constructs such as SKIN::RAB-35[Q69L], SKIN::BIG2, SKIN::AGEF-1a[E608K] in extrachromosomal transgenes could lead to non-physiological localization or effects. Single copy expression using MosSCI or CRISPR insertions are generally considered better approaches (other than endogenous reporters) to provide accurate insights at the physiological level. While the authors tacitly acknowledge this by conducting the experiments in a rab-35 mutant background and very low transgene concentration, at the very least this caveat regarding the localization should be discussed.
    4. The study does not address clearly whether AGEF-1a acts in parallel to spectrin or upstream/ downstream to it. Epistasis experiments could help to figure out the signaling pathway involved.
    5. The finding that BIG2 rescues the mutant defect is an important finding and rightfully finds its place in the abstract. I wonder whether a reference to the human diseases caused by loss of BIG2 in the abstract and introduction would not increase interest/impact for the study, rather than burying this potentially interesting connection in the discussion.

    Minor comments:

    1. Some explanation about how mutating the autoinhibitory domain could impact the catalytic activity of a GEF might be helpful.
    2. The paper refers to rme-4(b1001) as a null allele while wormbase refers to the same as a missense allele. It would be more accurate to refer rme-4(b1001) as a strong loss of function or putative null.
    3. The paper does not clearly discuss limitations of the hypomorphic agef-1[S784L] and that the observed phenotypes in this hypomorph might underestimate the complete role of AGEF-1a.
    4. In figure 1, where there really only one extrachromosomal transgenic line for some of the construct tested?
    5. The concentrations of transgenes vary in different transgenes. Is there a rationale behind this?
    6. In Fig.1e: I may be useful to also show the "WT" phenotype, i.e. the strong defects to get a visual comparison for the degree of rescue.

    Significance

    The study has identified AGEF-1a as a regulator of axonal maintenance, functioning to protect neurons against mechanical stress by acting through RAB-35. Additionally, this epidermal GEF, AGEF-1a is functionally conserved as its human orthologue BIG2 can replace AGEF-1a in C. elegans for axonal protection. Important points here are that the findings extend prior work by the authors of non-autonomous mechanism that regulates epidermal-neuronal attachment. In my humble opinion, the human disease connection, in particular with regard to the unexplained neuronal phenotypes in patients could be better developed in the manuscript. It may also increase impact/interest of a wonderful story that right now reads a bit 'wormy'.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper investigates the mechanism by which molecular pathways in the skin protect the processes of nerves that innervate them from damage. The authors previously showed that spectrin and the small GTPase RAB-35 act in the epidermis of C. elegans to protect mechanosensory axons from breaking. In this paper they used a suppression screen to identify another gene involved in this process, an ARF-GEF called AGEF-1. Partial loss-of-function mutations in agef-1 suppress the axon-breakage phenotype of spectrin mutations, and genetic experiments by the authors are consistent with the possibility that AGEF-1 could act directly as an exchange factor for RAB-35. Consistent with this model, they show that AGEF-1 and RAB-35 colocalise in the skin.

    Major comments: The experiments in this paper are well-designed and well-controlled, and the interpretations of the results are all reasonable. On the other hand, I don't think the authors' hypothesis that AGEF-1 acts directly as an exchange factor for RAB-35, or that these two proteins directly interact, is definitively proven. This is not an issue of the authors overinterpreting their data--the paper is very carefully and thoughtfully written. However, the most interesting and counterintuitive finding--that an ARF-GEF could also be a RAB-GEF--might be strengthened with more experiments (for example, could they more directly show protein-protein interaction through co-IP or mass spec?).

    Minor comments: There are also two places where the fact that null mutations are lethal (for agef-1 and arf-5) prevented the authors from addressing the effect of agef-1 loss of function in the skin, and addressing whether ARF-5 could be an AGEF-1 target, respectively. In principle, they could have tried to make a CRISPR line in which these genes could be cell-specifically deleted in the skin (using a dpy-7-driven recombinase). I don't think either of these experiments are essential, but if it is feasible to make these lines it would tie up a couple of loose ends.

    Significance

    Overall I think this is an interesting paper on a topic of general interest. The most interesting finding is that an exchange factor for an ARF (a small GRPase involved in vesicle coating/uncoating) could also be an exchange factor for a RAB (a small GTPase involved in vesicle tethering). The evidence presented is suggestive and intriguing, though as noted above not completely definitive. In summary, I think it is an interesting paper in its current form, and anything it could do to more firmly establish a direct interaction between AGEF-1 and RAB-35 would increase its impact and importance.

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    Referee #2

    Evidence, reproducibility and clarity

    This interesting manuscript reports the outcome of a fruitful C. elegans genetic screen with a complex but clever design. Through it, the authors identify AGEF-1 as a GEF that likely regulates the active state of the GTPase RAB-35 in the skin to protect touch receptor axons from mechanical breakage.

    Major points:

    1. Based on localization experiments, the authors claim "AGEF-1a interacts with RAB-35 in the epidermis" (Results heading) and state "these data demonstrate that AGEF-1a interacts with a subset of RAB-35 molecules in the epidermis." In general, localization studies cannot be used to conclude physical interaction (with some exceptions such as single-molecule kinetics). In this case, the data in my view do not even make a compelling argument for co-localization. There is a lot of AGEF-1 and RAB-35 signal everywhere and it may not be meaningful that the signals sometimes overlap. A more quantitative approach with controls would be needed to conclude meaningful co-localization. Importantly, this would still not demonstrate interaction.
    2. The effect of the AGEF-1(S784L) mutation is not clear to me. Naively, as the S784L mutation lies in the auto-inhibitory domain, I would have expected AGEF-1 to become constitutively active, not inactive as the authors seem to suggest. Is the idea that it is constitutively auto-inhibited? The main evidence for a loss of function effect seems to be that a putative dominant negative mutation AGEF-1(E608K) does not further supress axon breakage when co-expressed in trans to AGEF(S784L), but in my view this only shows that, once the defect is suppressed, it cannot be suppressed any further. Defining the nature of the S784L allele is important. Some suggestions, although the authors may come up with different approaches: use of an inducible or cell-specific depletion system like AID/TIR1, Cre/lox, or FLP/FRT to circumvent the lethality of agef-1(0) and reveal what a true loss-of-function looks like; testing if deletion of the auto-inhibitory domain phenocopies S784L to test if this mutation impairs autoinhibition.

    Minor points:

    1. I am not able to see the "vesicle-like structures with a clear luminal space" or RAB-35 being "notably enriched at the membrane near the epidermal furrow" in Fig. 3. The "3D surface rendering" in Fig. 3e is grossly oversampled and should not be included.
    2. As the agef-1a isoform is specifically referenced throughout, please describe the different agef-1 isoforms somewhere to save readers from having to look this up.
    3. The authors include an interesting speculation in the Discussion: "Future investigations of BIG2-associated neurological disorders should consider... hyper-activity of BIG2 as a driver of neuropathology." If the authors have the tools to test the effect of hyperactive BIG2 in this system, it could be an exciting addition.
    4. On a personal note, since GEFs act oppositely to GTPase Activating Proteins (GAPs), I had to stop and re-read carefully whenever the authors referred to a GEF "activating" a GTPase. I understand their meaning (i.e., putting the GTPase in its active GTP-bound state, not activating its GTPase function) but I wanted to point out this potential confusion in case there is a way to better define terms in the Introduction or change word choice. I realize this may be a standard jargon in the field.
    5. Please check the correct nomenclature for CRISPR/Cas9.
    6. p.7 "these molecules act in synergy", consider replacing with "redundantly".

    Significance

    The significance of this story is to show that GEF-GTPases pairing can be highly context-dependent. Previous studies have identified GEFs that pair with RAB-35 and GTPases that pair with AGEF-1, but the authors find that these factors have at best a modest role in the context of skin-axon interactions. Instead, the authors suggest a novel GTPase-GEF pairing of RAB-35 with AGEF-1 and provide evidence that this relationship is conserved in the human homolog of AGEF-1. These results suggest that GTPase-GEF pairings depend not only on chemical affinity but also cellular context.

    The main strength of the study is its clever genetics. For the screen, the authors looked for suppressors of a synthetic defect in axon integrity caused in part by elevated activity of RAB-35 due to loss of its GAP TBC-10. It is satisfying that this screen isolated a mutation in a GEF that in principle could counterbalance the loss of a GAP.

    The main weakness of the study is the lack of direct evidence for an AGEF-1/RAB-35 interaction. While not necessary for publication, the inclusion of biochemical data to support the role of AGEF-1 as a GEF for RAB-35 and the effect of the S784L mutation on this activity would strongly elevate the study. The genetic data for this interaction are consistent with the model but not conclusive, and in my view the colocalization data are not compelling. Nevertheless this is a solid genetic story with a clever screen.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Stability of the PLM axon in C. elegans is maintained through interactions with the epidermis. Previous studies by this group found that loss of the tbc-10 Rab GTPase Activating Protein strongly enhanced the PLM axon break phenotype of unc-70/beta-spectrin mutants. TBC-10 is a GAP for RAB-35 and thus loss of rab-35 suppresses the tbc-10 phenotype. Of the two RAB-35 GEFs, loss of RME-4 partially suppressed the tbc-10 phenotype and FLCN-1 was not involved suggesting that there may be an additional GEF involved. Here Bonacossa-Pereira et al identify a point mutation in agef-1a (vd92) as a suppressor of tbc-10 PLM axon break phenotype (all experiments also have a dominant allele of unc-70) and confirm that point mutation is causative by replicating the mutation via genome editing (vd123). Rescue experiments demonstrate that AGEF-1a is required in the epidermis and not PLM as previous demonstrated with tbc-10 and unc-70. Rescue is dependent on a functional SEC7/GEF activity. AGEF-1a is a functional ortholog to human BIG2/ArfGEF2 as its expression fully rescues tbc-10. AGEF-1a functions upstream of RAB-35 as expression of activated RAB-35 can suppress loss of agef-1. AGEF-1a functions in parallel to RME-4 as the double has stronger suppression of tbc-10. AGEF-1a is an ARF GEF, however it functions independently of ARF-1.2 as loss of arf-1.2 does not suppress tbc-10. They demonstrate that AGEF-1a interacts with RAB-35 through colocalization experiments suggesting that AGEF-1a could directly activate RAB-35. Finally, they demonstrate that AGEF-1a regulates the localization of the LET-805 epidermal attached complex component as it restores localization in a tbc-10 mutant.

    Major comments

    The manuscript is well written and easy to understand.

    The experiments are well done and controlled.

    I enjoyed reading this paper. However...

    Some of the claims are not supported by the data.

    1. The claim that AGEF-1a directly interacts with RAB-35 was not demonstrated. The evidence provided to support a direct interaction are colocalization experiments in Figure 3. AGEF-1a does partially colocalize with RAB-35 in the epidermis. However, colocalization does not indicate a physical interaction direct or indirect. A simple fix would be to change the claim to that they partially colocalize. Optional, a physical interaction could be done with the split-GFP since they already have the AGEF-1 strain or they could perform co-IP experiments, though neither of those are proof of direct interactions.
    2. The claim that AGEF-1a facilitates RAB-35 activation is not supported. While it is likely that AGEF-1a facilitates RAB-35 activation based on the epistasis experiments as well as studies in mammalian cells there were no experiments to demonstrate that modulating AGEF-1a activity resulted in a change in RAB-35 activity. I would suggest tempering this claim to something along the line that the data are consistent with AGEF-1a regulating RAB-35 activity as shown in mammalian cells. An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081).
    3. The claim that AGEF-1a functions independently of ARF-1.2 is not well supported. The fact that the ARF-1.2 mutant does not suppress tbc-10 suggests that ARF-1.2 may not be involved but does not eliminate the possibility that ARF-1.2 functions redundantly with ARF-5 or WARF-1/ARF-1.1. This can be resolved by toning down the claim. Alternatively, this can be tested by RNAi of arf-5 and warf-1 in tbc-10 and arf-1.2; tbc-10 mutants.

    Minor comments

    Figure 1C the CRISPR generated allele (vd123) is referred to as [S784L] and then in 1E vd92 is referred to as [S784L]. Perhaps it would be clearer if the allele name was used instead of the amino acid change.

    Page 6 "We reasoned that if the S784L mutation we isolated causes a similar loss of the GTPase activation function, then SKIN::AGEF-1a[E608K] would not have the capacity to restore the rate of PLM axon breaks to background levels in agef-1[S784L]; tbc-10; vdSi2 animals." It was unclear to me whether you were testing if the S784L mutation could be disrupting a GEF independent function or might disrupt the nucleotide exchange activity as might be tested in a biochemical assay. There are many reasons this change could cause a loss of function phenotype (ie. Improper folding, mislocalization, etc.). The most clear explanation would be that you were testing if GEF function was required for rescue rather than testing if the S784L mutation disrupted GEF activity.

    Page 13. It was unclear how testing if AGEF-1, RME-4, ARF-5 and RAB-35 form complexes in vivo (I assume you are suggesting colocalize based on figure 3 interpretation) would resolve how AGEF-1 was regulating RAB-35.

    Cross-commenting

    I agree with the comments made by the other reviewers and I stand by my own as well. I will echo that it is important to know the nature of their agef-1 allele.

    Significance

    Bonacossa-Pereira et al identify AGEF-1 as a regulator of axon integrity that functions in a pathway with RAB-35 in the epidermis is an exciting finding. As pointed out in the discussion, mutations in the human ortholog cause neurodevelopmental defects which leads to obvious characterization of BIG2/ArfGEF2 in neurons while this study indicates that this protein can have cell non-autonomous roles in regulating neurons. These findings could have important implications for understanding the etiology of these defects that would be of interest to neurobiologists and clinical researchers.

    The finding of this paper would also be of interest to cell biologists and particularly those studying the roles of Rab and Arf GTPases in membrane trafficking, such as myself. The idea that AGEF-1 might function as a Rab35 GEF is provocative and would generate a lot of interest and skepticism from the field. However, there is no data to support that AGEF-1 would be a direct regulator of Rab35 over the previously demonstrated cross regulation of Rab35 by Arf GTPases. Therefore, it would be fine to speculate in the discussion a direct interaction, but I would refrain from suggesting this as a model and elsewhere in the manuscript.

  6. Version published to 10.1101/2025.06.03.657768 on bioRxiv