The mechanoreceptor DEG‐1 regulates cold tolerance in Caenorhabditis elegans

Abstract Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH‐1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor‐expressing ASG. Ca2+ imaging revealed that ASG neurons respond to warm temperature via the mechanoreceptor DEG‐1, a degenerin/epithelial Na+ channel (DEG/ENaC), which in turn affects downstream AIN and AVJ circuits. Ectopic expression of DEG‐1 in the ASE gustatory neuron results in the acquisition of warm sensitivity, while electrophysiological analysis revealed that DEG‐1 and human MDEG1 were involved in warm sensation. Taken together, these results suggest that cold tolerance is regulated by mechanoreceptor‐mediated circuit calculation.

Thank you for the submission of your research manuscript to our journal. We have now received the full set of referee reports that is copied below.
As you will see, the referees acknowledge that the findings are potentially interesting but they also have a number of suggestions for how the study should be strengthened, and I think that all of them should be addressed. In particular, the requirement of the Atg2-GABARAP interaction for phagophore closure should be substantiated with additional experimental evidence such as EM. Moreover, the relationship or potential competition between GABARAP and WIPI4 binding should be analysed more rigorously.
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Referee #1:
This manuscript from the McEwan lab addresses the role of ATG2A/B. ATG2 is one of the less well-studied ATG proteins and there is significant interest in this protein, and understanding its function. This manuscript reveals an interaction of ATG2A and B with GABARAPs via a LC3interacting motif (LIR). The LIR motif is close to the interaction motif previously known to modulate interaction between ATG2 and WIPI4. The LIR identified in GABARAP is highly conserved. The authors detail the requirements for recruitment of GABARAP and WIPI4 in a set of well-controlled experiments concluding the ATG2-GABARAP interaction is required for flux, but ATG2-WIPI4 is dispensable. Next, they test the requirements for these interactions for autophagosome closure. The confirmed previous work using the ATG2DKO that there was aberrant formation, and also ATG9 distribution is altered. These phenotypes were rescued by wild-type and YFS ATG2, as was recruitment of Syntaxin 17. The LIR mutant however was unable to rescue any phenotype, and produced protease-sensitive autophagosomes which did not recruit Syntaxin 17. Overall, the data is well presented, convincing and of interest to the field. There are a few points to address: Major points: 1. It would be informative to understand how much GABARAP-ATG2 complex is required to recruit detectable amounts of WIPI4 and to more rigorously test the competition between the LIR and YFS. In Figure 2, IP GFP-GABARAP and probe for WIPI4 with and without ATG2 WT and mLIR by titrating the amount of ATG2. 2. Can the authors IP WIPI4 and detect ATG2A, ATG2B or GABARAP? 3. Figure 2E quantify the pulldowns as the input is uneven, in particular the WIPI4, and the amount of HA-ATG2 mLIR in the IP is less than YFS. Do the authors have evidence that WIPI4 levels are increased in starvation? 4. It would be informative to check the localization of WIPI4 in the ATG2 WT, mLIR and YFS mutants under the conditions used to provide more information about the interaction of the ATG2, GABARAP and WIPI4 complexes. Minor points: Clarify the following statements: -page 6 line 14 "functional consequences and in vivo preferences directed towards GABARAP"what about interaction with LC3A? what functional consequences are shown in Figure 2A that the authors are referring to? -page 7 line 1-2 this sentence is confusing "It remains to be seen....autophagy." Isn't this what the authors are trying to address?

Referee #2:
In this paper, McEwan and colleagues identified a motif in mammalian ATG2 for interaction with GABARAP. This motif is in close proximity to the ATG2-WIPI4 interaction site. They present data indicating that ATG2-GABARAP interaction mutants are unable to close phagophores, resulting in blocked autophagy, similar to ATG2A/B double KO cells. In contrast, the ATG2-WIPI4 interaction mutant restored autophagy flux and appeared to restore what the authors refer to as phagophore closure, similar to wild type ATG2.
This study has been well conducted and contains original and interesting observations. However, there are some suggestions for revisions before publication. Major 1. The authors claim that ATG2-mLIR is critical for phagophore closure. However, far more experimental proof and methods are needed to be really convinced of authors statement and interpretations and their model for closure, as this hypothesis floats up to be the main purpose of this study.
2. In Fig2A, it would be nice to have better blots for myc-ATG2A. It's not easy to see the bands and the loss of interaction in LIR mutant compared to WT. One cannot see any band for GABARAPL1 even if it is visible in EV2A.

In Fig4B
, it is necessary to show LC3/GABARAP blots for the prot K protection assay Minor: 4. Some referencing needs improvement. Examples are Stx17 recruitment, etc.
5. Is it really necessary to introduce multiple terms for LIR? I'd suggest to adhere to Johansen's terminology (see reviews) and refer to these sequences as LIR.

Referee #3:
Bozic et. al identified a new role of ATG2 in phagophore closure mediated by its direct interaction with ATG8s proteins through newly identified LIR domain. This LIR domain, located 30 amino acids upstream of previously characterized WIPI4 interaction motif (YFS), represents a distinct interaction site. The authors claim that Atg2-Atg8 but not Atg2-WIPI4 interaction is needed for phagophore closure and the overall autophagic flux. This research provides an interesting insight into newly identified ATG2 interaction with ATG8s during autophagosome biogenesis. For this study to be fully convincing, the authors' main claim concerning the importance of this interaction for autophagosomal membrane closure requires additional experimental data. Also, the relationship between the two adjacent sites for Atg2 interaction it WIPI4 and Atg8s should be better characterized. It is important for example to determine whether these interactions are mutually exclusive suggesting a competition between these factors during the process of autophagosome biogenesis.
Additional comments Authors conclude that both ATG2A and ATG2B directly interact with GABARAP family of ATG8 proteins, but most of the data are related to ATG2A only (including reconstitution experiments in ATG2A/ATG2B double knockout). The authors emphasis the direct interaction of Atg2 with GABARAP, but most of the experiments are concentrated on LC3B. The reconstitution experiments as well as distinguishing between phagophores and autophagosomes should be based on GABARAP too and not only LC3B. Moreover, in order to conclude that ATG2 interact with GABARAP family, all family members should be tested. Figure 1D and 1E -does GABARAP colocalizes to ATG2A? In panel D the data provided according to GABA-L1 only. In panel E the data present GABARAP only. Does GABA-L1 coimmunoprecipitates with ATG2A? What about GABARAP-L2? Figure 4B -It is unclear why the authors only test protease protection in the presence of BafA but not with other way to block autophagosome-lysosome fusion (for example knockdown of syntaxin 17). Moreover, as indicated above additional tools such as EM and supper-resolution microscopy may help characterize the intermediate membranes obtained when the interaction between Atg2 and Atg8s is disrupted. Figure EV4 -western blots are missing to support the IF data. Authors response to the Referees comments.

Referee #1:
This manuscript from the McEwan lab addresses the role of ATG2A/B. ATG2 is one of the less well-studied ATG proteins and there is significant interest in this protein, and understanding its function. This manuscript reveals an interaction of ATG2A and B with GABARAPs via a LC3interacting motif (LIR). The LIR motif is close to the interaction motif previously known to modulate interaction between ATG2 and WIPI4. The LIR identified in GABARAP is highly conserved. The authors detail the requirements for recruitment of GABARAP and WIPI4 in a set of well-controlled experiments concluding the ATG2-GABARAP interaction is required for flux, but ATG2-WIPI4 is dispensable. Next, they test the requirements for these interactions for autophagosome closure. The confirmed previous work using the ATG2DKO that there was aberrant formation, and also ATG9 distribution is altered. These phenotypes were rescued by wild-type and YFS ATG2, as was recruitment of Syntaxin 17. The LIR mutant however was unable to rescue any phenotype, and produced protease-sensitive autophagosomes which did not recruit Syntaxin 17.
Overall, the data is well presented, convincing and of interest to the field. There are a few points to address: We would like to thank the referee for their constructive comments and believe that the suggested experiments have strengthened the manuscript as a result of their inclusion.
1. It would be informative to understand how much GABARAP-ATG2 complex is required to recruit detectable amounts of WIPI4 and to more rigorously test the competition between the LIR and YFS. In Figure 2, IP GFP-GABARAP and probe for WIPI4 with and without ATG2 WT and mLIR by titrating the amount of ATG2.
As the referee has suggested, we have confirmed the reverse IP experiment that now forms part of Figure 2 ( Figure 2F and quantified in Figure 2G). In summary, endogenous WIPI4 only co-immunoprecipitated with ATG2A-WT and not the mLIR or mYFS (as expected), indicating that WIPI4 does not interact directly with GABARAP but forms a complex of WIPI4-ATG2A-GABARAP.
We also used overexpression of increasing amounts of WIPI4 to assess whether we could out-compete the interaction of GFP-GABARAP with endogenous ATG2A/ATG2B. We show that indeed, having high concentrations of WIPI4 (such as by overexpression) we lose a large proportion of ATG2A and ATG2B binding to GABARAP ( Figure 2H and quantified in Figure 2I). However, the reverse was not true (i.e increasing GABARAP concentration to out compete WIPI4 interaction), indicating that WIPI4 is the more dominant and stable interaction ( Figure EV2J-K). This is also indicated by our ability to immunoprecipiate WIPI4 with endogenous GFP-tagged ATG2A under non-stimulated conditions and that this interaction has been previously shown to be a stable interaction in multiple studies. We believe that upon activation of autophagy, through an as yet unidentified mechanism, stimulates GABARAP recruitment and complex formation between GABARAP-ATG2A-WIPI4 for the efficient formation of autophagosomes.
Due to the limited amounts of anti-WIPI4 antibody (a kind gift from Prof. Sharon Tooze) and its limited use for both immunoprecipitation and immunofluorescence we have been unable to perform these experiments. However, using overexpressed GFP-tagged WIPI4 we could show efficient immunoprecipitation of endogenous ATG2A and ATG2B from cells ( Figure EV2J-K). However, we could not detect endogenous GABARAP/GABARAP-L1. In-light of our results where overexpression of WIPI4 can out compete GABARAP binding, this is not surprising.
We could detect an ATG2-WIPI4 coprecipitation with a limited number of mammalian ATG8s ( Figure EV1D). This also follows on from work by other groups showing both an in vitro and in vivo complex between ATG2s and WIPI4 [1-4].
3. Figure 2E quantify the pulldowns as the input is uneven, in particular the WIPI4, and the amount of HA-ATG2 mLIR in the IP is less than YFS. Do the authors have evidence that WIPI4 levels are increased in starvation?
We have now quantified the co-IPs as suggested and this is now shown as Figure  2D.
As for the WIPI4 levels changing, we believe it is potential stabilisation effect that is also reflected in some of the pull downs. Notably, we see consistent differences between ATG2A/B DKO cells and those reconstituted with ATG2A WT.

It would be informative to check the localization of WIPI4 in the ATG2 WT, mLIR and YFS
mutants under the conditions used to provide more information about the interaction of the ATG2, GABARAP and WIPI4 complexes.
We agree with the referee on this point, however as mentioned above the antibody used to detect WIPI4 by western blot was not suitable for either immunoprecipitation or immunofluorescence as this had been tested extensively in the Tooze lab. Given our results with the overexpression of WIPI4 on GARARAP-ATG2 interaction ( Figure 2H-I), using the GFP-construct to assess this was difficult and provided inconclusive results. However, the referee deems that this is required we are happy to include this in the final version. Currently, we have excluded this due to space constraints for the EMBO reports short report format.
Minor points: Clarify the following statements:  Figure 2A that the authors are referring to?
We apologise for the confusion. We have clarified the sentence to read "..however we have been unable to confirm an endogenous interaction" as we could not detect LC3A in ATG2 IPs. From our results in Figure EV1D, it appears that GABARAP and GABARAP-L1 interact more with ATG2A/B than LC3A and thus was the major focus of this paper. However, we cannot rule out a role for ATG2-LC3A interaction during the autophagy pathway.
-page 7 line 1-2 this sentence is confusing "It remains to be seen....autophagy." Isn't this what the authors are trying to address?
We agree with the referee and have removed this sentence for clarity.
Referee #2: In this paper, McEwan and colleagues identified a motif in mammalian ATG2 for interaction with GABARAP. This motif is in close proximity to the ATG2-WIPI4 interaction site. They present data indicating that ATG2-GABARAP interaction mutants are unable to close phagophores, resulting in blocked autophagy, similar to ATG2A/B double KO cells. In contrast, the ATG2-WIPI4 interaction mutant restored autophagy flux and appeared to restore what the authors refer to as phagophore closure, similar to wild type ATG2.
This study has been well conducted and contains original and interesting observations. However, there are some suggestions for revisions before publication.
We would like to thank the referee for their constructive comments and believe that the suggested experiments have strengthened the manuscript as a result of their inclusion.
1. The authors claim that ATG2-mLIR is critical for phagophore closure. However, far more experimental proof and methods are needed to be really convinced of authors statement and interpretations and their model for closure, as this hypothesis floats up to be the main purpose of this study.
We agree with the referee on this point and would like to thank the referee for the suggestions. We have now included images from both Airyscan super resolution confocal microscopy ( Figure 5 A-C Figure EV4A-B, Movie EV1A and Movie EV1B) and transmission electron microscopy ( Figure 5D) to analyse the effect of the mutants on the formation of autophagosomes. This has led to us observing that the ATG2A-mLIR results in the formation of multiple, immature phagophores that cluster with ER. These remain proteinase K sensitive and are "open" similar to ATG2A/B double knockouts and consistent with observations from other groups [5][6][7]. We have altered our title to reflect these new conclusions as a result of the experiments suggested by the reviewers (see above).

In Fig2A
, it would be nice to have better blots for myc-ATG2A. It's not easy to see the bands and the loss of interaction in LIR mutant compared to WT. One cannot see any band for GABARAPL1 even if it is visible in EV2A.
We agree that this was not clear. This panel has been replaced for both ATG2A and ATG2B and is now Figure EV2H.

In Fig4B, it is necessary to show LC3/GABARAP blots for the prot K protection assay.
We agree that the inclusion of LC3/GABARAP blots would enhance the results and be more complete. However, during the course of the studies, we found that both LC3 and GABARAP are proteinase K resistant, even in the presence of triton x-100 (see below for example (i)). This was also the case for GFP-LC3B (see below, (ii)). This is in spite of the fact that p62/SQSTM1 and HA-ATG2A were efficiently degraded and were model substrates for this assay. We hope the reviewers will take this into account and also the second part of the figure, STX17 localization, to demonstrate that the p62/LC3 structures we observe are not complete autophagosomes, but rather "open" phagophores. Minor: 4. Some referencing needs improvement. Examples are Stx17 recruitment, etc.
We have updated the references for STX17 recruitment accordingly (page 9 of main text).

5.
Is it really necessary to introduce multiple terms for LIR? I'd suggest to adhere to Johansen's terminology (see reviews) and refer to these sequences as LIR.
We agree that multiple names for the same sequence type do not make for easier reading and have simplified the terminology and adhered to only using LIR to describe the sequences as per the referee's suggestion.

Referee #3:
Bozic et. al identified a new role of ATG2 in phagophore closure mediated by its direct interaction with ATG8s proteins through newly identified LIR domain. This LIR domain, located 30 amino acids upstream of previously characterized WIPI4 interaction motif (YFS), represents a distinct interaction site. The authors claim that Atg2-Atg8 but not Atg2-WIPI4 interaction is needed for phagophore closure and the overall autophagic flux. This research provides an interesting insight into newly identified ATG2 interaction with ATG8s during autophagosome biogenesis. For this study to be fully convincing, the authors' main claim concerning the importance of this interaction for autophagosomal membrane closure requires additional experimental data. Also, the relationship between the two adjacent sites for Atg2 interaction it WIPI4 and Atg8s should be better characterized. It is important for example to determine whether these interactions are mutually exclusive suggesting a competition between these factors during the process of autophagosome biogenesis.
We would like to thank the referee for their constructive comments and believe that the suggested experiments have strengthened the manuscript as a result of their inclusion.
Authors conclude that both ATG2A and ATG2B directly interact with GABARAP family of ATG8 proteins, but most of the data are related to ATG2A only (including reconstitution experiments in ATG2A/ATG2B double knockout).

(ii) (i)
The referee is correct to in their observation. We can show that ATG2B interacts with GABARAP/GABARAP-L1 through a conserved LIR sequence that is shared not only with human ATG2A but multiple other species of ATG2s. It was our intention that the manuscript include both human ATG2 isoforms in order to gain a more comprehensive understanding of their biological function. However, we have been unable to reconstitute ATG2A/B double knockout cells with ATG2B WT and mutants to a satisfactory level (either too high or too low) within the timeframe of revision. This is something that we are actively working on to resolve, but unfortunately, we cannot include in this manuscript preparation. We also note that a number of the papers currently published that analyse ATG2 function, are also solely focused on ATG2A and perhaps this is a common problem with ATG2B overexpression. Where possible, we have limited our conclusions to that of the role of ATG2A but have included more in the discussion sections regarding the role of ATG2B in this process. We hope that the referee nonetheless finds our data of importance to the field.
The authors emphasis the direct interaction of Atg2 with GABARAP, but most of the experiments are concentrated on LC3B. The reconstitution experiments as well as distinguishing between phagophores and autophagosomes should be based on GABARAP too and not only LC3B.
We agree and have included GABARAP/GABARAP-L1 or pan GABARAP staining or western blot analysis throughout. These include co-precipitation experiments of GABARAP and WIPI4 with ATG2A WT and mutants ( Figure  Moreover, in order to conclude that ATG2 interact with GABARAP family, all family members should be tested. We agree with the referee that all isoforms should have been tested. We have no included co-precipitation of experiments using GFP-tagged ATG8 isoforms (LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2). From this we can show that both ATG2A and ATG2B co-precipitate with GABARAP and GABARAP-L1 mainly, but also weakly with LC3A ( Figure EV1D). Interestingly, we also found that WIPI4 also co-precipitated with ATG2A/B in these samples. Figure 1D and 1E -does GABARAP colocalizes to ATG2A? In panel D the data provided according to GABA-L1 only.
We have attempted the co-localization experiments with GABARAP and CRISPR tagged GFP-ATG2A. However, the specific GABARAP antibody (Abgent, AP1821a) that has previously been used for IF studies [8] requires cells to be incubated in methanol for 5 minutes (permeabilization) in order to observe GABARAP puncta. This method is unfortunately incompatible with the endogenous ATG2A staining we show in the rest of the figure that used a much less harsh, saponin-based, permeabilization method. However, given the high degree of sequence identity between GABARAP and GABARAP-L1 (86%) and that ATG2A/B seem to interact equally well with both isoforms, we have been unable to precisely define which is the preferred interaction partner. We have updated the text throughout the manuscript to reflect this.
In panel E the data present GABARAP only. Does GABA-L1 co-immunoprecipitates with ATG2A? What about GABARAP-L2?
As per referee 3's earlier suggestion where we tested interaction of ATG2s with all ATG8 family members, we found no or very weak interaction of ATG2A and ATG2B with GABARAP-L2 by co-precipitation from cells ( Figure EV1D). Figure 4B -It is unclear why the authors only test protease protection in the presence of BafA but not with other way to block autophagosome-lysosome fusion (for example knockdown of syntaxin 17).
We apologise for the lack of clarity in this. We used BafA1 in combination with starvation to simply accumulate autophagy vesicles (phagophores/autophagosomes/autolysosomes that would contain (or not in the case of DKO and DKO+2A-mLIR) p62/SQSTM1 that we could test using the limited proteolysis assay. Knockdown of syntaxin17 would also have been an option, however this would have had to also be combined with knockdown of YKT6 to completely block fusion. Using a chemical inhibitor of degradation, we were able simply and reproducibly accumulate the vesicles of interest.
Moreover, as indicated above additional tools such as EM and supper-resolution microscopy may help characterize the intermediate membranes obtained when the interaction between Atg2 and Atg8s is disrupted.
As per referee 3 and also referee 2's suggestion, we have now included images from both Airyscan super resolution confocal microscopy ( Figure 5 A-C Figure  EV4A-B, Movie EV1A and Movie EV1B and transmission electron microscopy ( Figure 5D) to analyse the effect of the mutants on the formation of autophagosomes. This has led to us observing that the ATG2A-mLIR results in the formation of multiple, immature phagophores that cluster with ER. These remain proteinase K sensitive and are "open" similar to ATG2A/B double knockouts and consistent with observations from other groups [5][6][7]. We have altered our title to reflect these new conclusions as a result of the experiments suggested by the reviewers (see above). Figure EV4 -western blots are missing to support the IF data.
As per the reviewer's suggestion, we have included the western blot data to support the IF data ( Figure EV4C). Thank you for your patience while we have reviewed your revised manuscript. Unfortunately, we have not received the report from former referee #2 but since referee #1 and #3 are very positive about the study and support publication in EMBO reports, I have decided to proceed.
I am therefore writing with an 'accept in principle' decision, which means that I will be happy to accept your manuscript for publication once a few minor issues/corrections have been addressed, as follows. 2) Author contributions: please specify the contribution of Beth Milne and Lisa Robertson 3) Movies: please remove their legends from the main manuscript file and supply them as simple README.txt file. Then zip the movie with its legend and upload the .zip file. 4) Please remove the list of abbreviations from the front page. All abbreviations have to be explained in the text.

5)
Please convert the table with plasmids that is currently in Materials and Methods to " Table 2", move it to the end of the manuscript and provide a callout in the text. Table 1 contains red color, which unfortunately cannot be typeset. Please choose another highlight, e.g., underline or bold. 7) Source data: Thank you for providing it. Could you please double-check the data supplied for Figure 2F? You note that the higher exposure for GFP-GABARAP was not used for the publication but it appears to be the scan that was used. Moroever, what is labeled GFP-GABARAP input in the source data file appears as IP in the figure panel. Please double-check this.

8)
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