A polarity pathway for exocyst-dependent intracellular tube extension

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   Nov 25, 2020

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

   The manuscript by Abrams and Nance describes how the polarity proteins PAR-6 and PKC-3/aPKC promote lumen extension of the unicellular excretory canal in C. elegans. Using tissue-specific depletion methods they find that CDC-42 and the RhoGEF EXC-5/FGD are required for luminal localization of PAR-6, which recruits the exocyst complex required for lumen extension. Interestingly, they show that the ortholog of the mammalian exocyst receptor, PAR-3, is dispensable for …

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

   The manuscript by Abrams and Nance describes how the polarity proteins PAR-6 and PKC-3/aPKC promote lumen extension of the unicellular excretory canal in C. elegans. Using tissue-specific depletion methods they find that CDC-42 and the RhoGEF EXC-5/FGD are required for luminal localization of PAR-6, which recruits the exocyst complex required for lumen extension. Interestingly, they show that the ortholog of the mammalian exocyst receptor, PAR-3, is dispensable for luminal membrane extension. Overall, this is a well-written and interesting manuscript.*

   1.Because depletion of PAR-3 in the canal causes milder defects than PAR-6 or CDC-42 the authors suggest that they cannot rule out the possibility that an alternative isoform of PAR-3 is expressed and buffering the defect. They should perform canal-specific RNAi-mediated depletion of the entire PAR-3 gene to determine if this is true.

   We agree with Reviewer 1 that it would be useful to provide additional evidence that an alternative isoform of PAR-3 lacking the ZF1 degron is not expressed. While tissue-specific RNAi could be used, we have not been successful obtaining complete knockdown in previous tissue-specific RNAi experiments. Moreover, this approach does not target any maternal PAR-3 protein that may be inherited by the excretory cell. As an alternative approach to address this point, we will analyze par-3::zf1::yfp/par-3(null) worms following excretory-cell-specific expression of zif-1, and compare to par-3::zf1::yfp/par-3::zf1::yfp siblings. We would expect the excretory cell phenotype to become more severe if additional, ‘phenotype-buffering’ forms of PAR-3 were present, or if there was incomplete degradation of PAR-3::ZF1::YFP in our previous experiments.

   2.The authors suggest that GTP-loaded (activated) CDC-42 recruits PAR-6 to the luminal membrane. It would be nice if they could use a biosensor, such as the GBD-WSP-1 reagent from Buechner's lab to confirm that EXC-5 depletion also reduces activated CDC-42, as would be expected. This should be achievable since there is strong CDC-42 signal, even in the cytoplasm.

   This is an excellent suggestion. We will utilize a CDC-42 biosensor – an integrated cdc42p::gfp::wsp-1(gbd) strain created in our lab and previously validated and characterized (Zilberman et al. 2017). We have confirmed that the biosensor is detected in the excretory canal and appears enriched at or near the lumenal membrane. We will cross the biosensor into the *exc-5::zf1::mScarlet *background. This will allow us to assess lumenal enrichment, and using heat shock inducible ZIF-1, determine if there is a reduction in biosensor lumenal enrichment when EXC-5::ZF1::mScarlet is acutely degraded.

   If the biosensor is difficult to measure at the canal lumen, an alternative approach would be to use an available *exc-5 *null allele to examine genetically if cdc-42 and exc-5 are acting in the same pathway. We could cross CDC-42exc(-) larvae into *exc-5(rh232) *and quantify excretory canal phenotypes. If CDC-42 and EXC-5 are indeed functioning in the same pathway we would expect no enhancement of the CDC-42exc(-) phenotype.

   3.Related to point 2, (i) does mutation of the CRIB domain of PAR-6 impair its recruitment to the luminal membrane, and (ii) does this mutant exacerbate canal defects when PAR-3 is depleted?

   (i) Our lab has previously generated and characterized a transgenic par6P::par-6(*CRIB)::gfp *strain (Zilberman et al., 2017). We will examine this strain to determine if expression is detectable in the excretory canal, and if so, we will compare lumenal enrichment of PAR-6(CRIB)::GFP to control worms expressing wild-type PAR-6::GFP.

   (ii) This is a very interesting experiment, as it would help address if the mild phenotype observed in PAR-3 depleted animals is due to the remaining PAR-6 that is recruited by CDC-42. Our lab has previously shown that par6P::par-6(*CRIB)::gfp *cannot rescue the embryonic lethality of a *par-6 *mutant, in contrast to *par-6::gfp *(Zilberman et al. 2017). This indicates that the CRIB domain is needed for PAR-6 function during embryogenesis and suggests that CRIB domain mutations introduced by CRISPR would almost certainly be lethal, precluding analysis of the excretory cell.

   As an alternative experiment, we would determine if PAR-3 localizes to the lumenal membrane independently of CDC-42; such a finding would imply that PAR-3 and CDC-42 likely have independent contributions to PAR-6 localization (rather than CDC-42 promoting PAR-6 localization by localizing PAR-3). To do this, we will degrade ZF1::YFP::CDC-42 in the excretory cell and examine the localization of PAR-3::mCherry compared to controls. We have all of the strains needed for this experiment.

   4.The authors hypothesize that partial recruitment of PAR-6 by CDC-42 is sufficient for luminal membrane extension to explain the mild defects caused by PAR-3 depletion. Since depletion of PAR-6 and CDC-42 alone causes milder canal truncations the authors should co-deplete these proteins (as well as PAR-3 and CDC-42) to determine if there is an additive effect.

   This is an excellent suggestion in principal. However, it is not possible to know in any given degradation experiment whether the targeted protein is completely degraded; we can only say it is no longer detectable by fluorescence. Thus, any degron allele (in the presence of ZIF-1) could behave like a strong hypomorph rather than a null. It would not be possible to interpret double degradation experiments in such a case, as a more severe phenotype in the double could simply be a result of combining two hypomorphic alleles, further reducing pathway activity even if the genes function together in the same pathway. To interpret this experiment properly, a null allele of at least one of the genes would have to be used. This is not possible since par and cdc-42 null mutants are lethal and there is also maternal contribution. As an alternative to these double depletion experiments, we will deplete PAR-6::ZF1::YFP or PAR-3::ZF1::YFP in exc-5 null mutant larvae, as unlike cdc-42, exc-5 is not an essential gene.

   *5.In figure 2, the authors show that depletion of PKC-3 causes more severe canal truncations than PAR-6. Since these proteins function in the same complex what do they think is the reason for this difference? This point could be discussed more in the manuscript. *

   As described in the previous point, incomplete degradation could produce modestly different phenotypes even for genes that act in the same pathway. Therefore, it is not possible to determine whether PAR-6 and PKC-3 have different roles using this approach. We will add text to the discussion bringing up this point.

   6.Related to point 5, more experiments with PKC-3 should be done to determine if, for example, localization of SEC-10 is similarly affected as ablation of PAR-3, PAR-6 and CDC-42.

   We agree, and will address this point by acutely degrading ZF1::GFP::PKC-3 and examining transgenic SEC-10::mCherry, as we have done for other par genes.

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

   The manuscript by Abrams & Nance describes a precise investigation of the role of PAR proteins in the recruitment of the exocyst during and after the extension of the C. elegans excretory canal. State-of-the-art genetic techniques are used to acutely deplete proteins only in the targeted cell, and examine the localization of endogenously expressed markers. Experiments are well described and carefully quantified, with systematic statistical analysis. The manuscript is easy to follow and the bibliography is very good. Most conclusions are well supported.*

   1) I am not entirely convinced by the presence of CDC-42 at the lumenal membrane (Fig3G); it seems to be more sub-lumenal that really lumenal. It peaks well before PAR-6 (Fig3H) which itself seem slightly less apical that PAR-3 (Fig3F). Could you use super-resolution microscopy (compatible with endogenous expression levels) to more precisely localize CDC-42? Similar point for PAR-3 and PAR-6 which do not seem to colocalize completely - a longitudinal line scan along the lumenal membrane might provide the answer even without super-resolution; this could help explain why these two proteins do not have the same function. These suggestions are easy to do provided the authors can have access to super-resolution (Airyscan to name it; although other methods will be perfectly acceptable I believe it is the most simple one).

   We agree that the CDC-42 localization peak does not precisely match the PAR-6 peak. As the reviewer notes, resolving the subcellular localization of these two proteins will not be feasible using standard confocal microscopy. We will image the ZF1::YFP::CDC-42; PAR-6:mKate strain using a Zeiss LSM 880 with Airyscan to determine if their subcellular localization patterns are distinct.

   To examine PAR-3 and PAR-6 colocalization at the lumen, we will acquire additional confocal images of the PAR-6-ZF1-YFP; PAR-3-mCherry strain and examine colocalization of the clusters along the lumenal membrane. As a positive control for two proteins that should co-localize, we will image ZF1::GFP::PKC-3; PAR-6-mKate; these two proteins bind directly and co-localize in nearly all cells in which they have been examined.

   2) The same group has described a CDC-42 biosensor to detect its active form. It could be used here to precisely pinpoint where active CDC-42 is required: in the cytoplasm? At the lumenal membrane? colocalizing with what other protein? This will require the expression of a transgene under an excretory cell specific promotor and a simple injection strategy while helping to strengthen the description of the CDC-42 role.

   See Reviewer 1 point #2.

   3) As the authors certainly know, there is a PAR-6 mutation which prevents its binding to CDC-42. They could express this construct in the excretory canal a simple extrachromosomal array should be sufficient) to validate the direct interaction between these proteins in this cell.

   See Reviewer 1 point #3.

   4) What is the lethality of ZIF-1-mediated depletion of the various factors under the exc promoter? Can homozygous strains be maintained? Authors just have to add a sentence in the Mat&Met section.

   All of the strains with excretory cell-specific degradation we have examined are viable when grown on NGM plates. We will add this point to the materials and methods.

   Provided that the authors have access to an Airyscan, all the questions asked here can be answered in two months (one month for constructs, one month for injection and data analysis) at a very minor cost.

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

   Strengths of this manuscript include the use of endogenously tagged proteins (rather than over-expressed transgenes) for high resolution imaging and a cell-type specific acute depletion strategy that avoids complicating pleiotropies and allows tests of molecular epistasis. While some results were fairly expected based on prior studies of Cdc42, PAR proteins, and the exocyst in other tissues or systems, differences in the requirements for par-6 and pkc-3 vs. par-3 strongly suggest that the former genes play more important roles in exocyst recruitment. I was also excited to see a connection made between EXC-5 and PKC-3 localization.

   1.Lumen formation vs. lumen extension. The abstract and introduction use these two terms almost interchangeably, but they are not the same and more care should be taken to avoid the former term. The data here do not demonstrate any roles for par or other genes in lumen formation, but do demonstrate roles in lumen extension and organization/shaping.

   We agree and will correct wording to indicate that lumen extension is affected.

   2.Related to the above, mutant phenotypes here are surprisingly mild and variable. The authors discuss possible reasons for the particularly mild phenotype of par-3 mutants, but don't specifically address the mild phenotypes of the others. Clearly quite a bit of polarization and apical membrane addition occurs in ALL of the mutants. Is this because those early steps use other/redundant molecular players, or is depletion too late or incomplete to reveal an early role?

   We agree with Reviewer 3 and will bring up these points in the discussion. Degradation of proteins strongly predicted to function together (RAL-1 and SEC-5; PAR-6 and PKC-3) produce similar although not identical phenotypes; as discussed above we consider it likely that these differences reflect minor differences in degradation efficiency below our ability to detect by fluorescence. As Reviewer 3 points out, the excretory-specific driver we use to express ZIF-1 may not be active at the very earliest stages of lumen formation, and degradation could take 45 minutes or more after the promoter becomes active (Armenti et al, 2014). Thus, we agree that phenotypes could be more severe if it were possible to completely deplete each tagged protein prior to the onset of lumen formation. However, this caveat does not change the interpretations of our experiments since all proteins are degraded with the same driver. We have avoided mentioning that the phenotypes we observe reflect the ‘null’ phenotype for these reasons. We will emphasize these points in the discussion.

   *The authors introduce a new reagent, "excP" (the promoter for T28H11.8), which they use to drive canal cell expression of ZIF-1 for their degron experiments. Please provide more information about when in embryogenesis this promoter becomes active, how that compares to when the par genes, sec-5, ral-1 and cdc-42 are first expressed, and what canal length is at that time. It would also be helpful to show the timeframe for degron-based depletion using this reagent (Figure 1C shows only depletion at L4, days later). *

   Publicly available single cell RNA seq data (https://pubmed.ncbi.nlm.nih.gov/31488706/ and https://cello.shinyapps.io/celegans_explorer/) suggest that canal expression of the endogenous T28H11.8 gene doesn't really ramp up until the 580-650 minute timepoint, which is several hours after par gene canal expression (270-390 minutes) and the initiation of canal lumen formation (bean stage, 400-450 minutes). These data suggest that excP might come on too late to test requirements in lumen formation and early stages of extension. This caveat should be at least mentioned.

   See point #2 above. We agree that providing more information on expression from the T28H11.8 promoter would be important for interpreting the severity of phenotypes. We will raise this point in the discussion, and include existing published expression data and a more detailed analysis of the excP::mCherry transgene.

   3.There are two major aspects to the mutant phenotypes observed here: short lumens and cystic lumens. A short lumen makes sense intuitively, but the cysts could use a little more explanation. (What are cysts? What is thought to be the basis of their formation?). It is intriguing that cysts in sec-5 vs. ral-1 mutants (Figure 1) and par-6 vs. pkc-3 mutants (Figure 4) seem to have a very different size and overall appearance. Are these consistent differences, and if so, what could be the explanation for them?

   This is an interesting point. Since it is not practical to perform time-lapse imaging to watch canal cysts form, we analyzed only L1 and L4 larvae. We believe from our imaging that these are discontinuous regions of the lumen. One explanation for the expansion and dilation of the cystic lumens by L4 stage could be that the canal lumen has been expanded by fluid buildup resulting from a defect in canal function in osmoregulation, but we have not tested this directly. The reviewer also raises an interesting point regarding different appearances of cysts in SEC-5 and RAL-1 depleted larvae compared to PAR-6 and PKC-3. It is possible that these differences arise because SEC-5 and RAL-1 might direct whether vesicles will fuse at all, whereas PAR proteins direct where they will fuse in the cell (i.e. there could be fusion at basal surfaces, or just reduced apical fusion). We will bring up these points in the discussion.

   4.The authors did not test if PKC-3, like PAR-6, is required to recruit exocyst to the canal cell apical membrane, but their prior studies in the embryo suggested that it is (Armenti et al 2014). They also did not test if EXC-5 is required to recruit PAR-6 and the exocyst (along with PKC-3), or if CDC-42 is required to recruit PKC-3 (along with PAR-6). There seems to be an assumption that PAR-6 and PKC-3 are regulated and function in a common manner (as is often the case), but that has not been demonstrated here specifically. The basis for this assumption and alternatives to the linear model should be acknowledged.

   As discussed above (Reviewer 1 point #6), we will directly test whether PKC-3 is required to recruit SEC-10::mCherry to the lumenal membrane. We agree with Reviewer 3 that we have not shown that PAR-6 and PKC-3 always function similarly, although this is expected based on their similar phenotypes and co-dependent functions in other cells. We will mention this caveat in the discussion.

   *5.EXC-5 is presumed to act upstream of CDC-42 based on shared phenotypes and the known Rho GEF activity of its mammalian homologs. However, direct evidence for this is currently lacking. In future, the authors might test if depleting EXC-5 affects CDC-42 activation/GTP-loading by using CDC-42 biosensors that have been reported in the literature (e.g. Lazetic et al 2018). *

   See Reviewer 1 point #2.

   ***Minor comments:**

   Figure 1, Figure 4, Figure S3, Figure S4 Blue color/CFP indicates the apical/luminal membrane or the apical region of the canal cytoplasm, not the actual lumen as the labels suggest. The lumen is a hollow cavity on the opposite side of the plasma membrane from these markers, and it is shown as white in the Figure 1A upper right cartoon.*

   Thank you for pointing this out. We will correct the figure labelling.

   Figure 2, Figure S2 I'm not confident in the statistical analysis used here (Fisher's Exact test on two bins, 50% canal length), given that four length bins (not two) were defined. I recommend consulting a statistician.

   Our rationale for using two bins for the statistical analysis was because control larvae nearly all have a similar canal length (L1 stage: 99% of larvae have canal length that is 51-75% of body length; L4 stage: 98% of larvae have canal length that is 76-100% of body length), making it straightforward to ask if mutants are shorter. We chose not to make more granular phenotypic comparisons, as we cannot rule out that subtle differences in degradation efficiency, rather than differences in biological function, underlie any differences in canal length of the degron mutants. We will consult with a statistician to determine if this is an acceptable way to statistically compare controls and mutants.

   p.3 "Born during late embryogenesis..." Actually, the canal cell is born at ~270 minutes after first cleavage, which is in the first half of embryogenesis, not what I would call "late".

   We agree and will correct the wording.

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   The C. elegans excretory canal cell is a classic model for studying single cell tubulogenesis, where a cell establishes an intracellular apical domain that extends to form a lumen. Prior studies in this system have identified a set of gene products that localize to the growing apical domain and/or are important for its organization and size, but the molecular pathways through which these various gene products act remain poorly understood. Here, Abrams and Nance are able to connect the dots among several of these to flesh out a pathway for apical membrane addition. Specifically, they demonstrate that CDC-42 is needed to …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   The C. elegans excretory canal cell is a classic model for studying single cell tubulogenesis, where a cell establishes an intracellular apical domain that extends to form a lumen. Prior studies in this system have identified a set of gene products that localize to the growing apical domain and/or are important for its organization and size, but the molecular pathways through which these various gene products act remain poorly understood. Here, Abrams and Nance are able to connect the dots among several of these to flesh out a pathway for apical membrane addition. Specifically, they demonstrate that CDC-42 is needed to recruit PAR-6, and that PAR-6 is needed to recruit the exocyst to the apical membrane and to promote proper apical membrane growth and organization. EXC-5, a candidate GEF for CDC-42, also appears to act in this pathway.

   Strengths of this manuscript include the use of endogenously tagged proteins (rather than over-expressed transgenes) for high resolution imaging and a cell-type specific acute depletion strategy that avoids complicating pleiotropies and allows tests of molecular epistasis. While some results were fairly expected based on prior studies of Cdc42, PAR proteins, and the exocyst in other tissues or systems, differences in the requirements for par-6 and pkc-3 vs. par-3 strongly suggest that the former genes play more important roles in exocyst recruitment. I was also excited to see a connection made between EXC-5 and PKC-3 localization.

   Major comments:

   1.Lumen formation vs. lumen extension. The abstract and introduction use these two terms almost interchangeably, but they are not the same and more care should be taken to avoid the former term. The data here do not demonstrate any roles for par or other genes in lumen formation, but do demonstrate roles in lumen extension and organization/shaping.

   2.Related to the above, mutant phenotypes here are surprisingly mild and variable. The authors discuss possible reasons for the particularly mild phenotype of par-3 mutants, but don't specifically address the mild phenotypes of the others. Clearly quite a bit of polarization and apical membrane addition occurs in ALL of the mutants. Is this because those early steps use other/redundant molecular players, or is depletion too late or incomplete to reveal an early role?

   The authors introduce a new reagent, "excP" (the promoter for T28H11.8), which they use to drive canal cell expression of ZIF-1 for their degron experiments. Please provide more information about when in embryogenesis this promoter becomes active, how that compares to when the par genes, sec-5, ral-1 and cdc-42 are first expressed, and what canal length is at that time. It would also be helpful to show the timeframe for degron-based depletion using this reagent (Figure 1C shows only depletion at L4, days later).

   Publicly available single cell RNA seq data (https://pubmed.ncbi.nlm.nih.gov/31488706/ and https://cello.shinyapps.io/celegans_explorer/) suggest that canal expression of the endogenous T28H11.8 gene doesn't really ramp up until the 580-650 minute timepoint, which is several hours after par gene canal expression (270-390 minutes) and the initiation of canal lumen formation (bean stage, 400-450 minutes). These data suggest that excP might come on too late to test requirements in lumen formation and early stages of extension. This caveat should be at least mentioned.

   3.There are two major aspects to the mutant phenotypes observed here: short lumens and cystic lumens. A short lumen makes sense intuitively, but the cysts could use a little more explanation. (What are cysts? What is thought to be the basis of their formation?). It is intriguing that cysts in sec-5 vs. ral-1 mutants (Figure 1) and par-6 vs. pkc-3 mutants (Figure 4) seem to have a very different size and overall appearance. Are these consistent differences, and if so, what could be the explanation for them?

   4.The authors did not test if PKC-3, like PAR-6, is required to recruit exocyst to the canal cell apical membrane, but their prior studies in the embryo suggested that it is (Armenti et al 2014). They also did not test if EXC-5 is required to recruit PAR-6 and the exocyst (along with PKC-3), or if CDC-42 is required to recruit PKC-3 (along with PAR-6). There seems to be an assumption that PAR-6 and PKC-3 are regulated and function in a common manner (as is often the case), but that has not been demonstrated here specifically. The basis for this assumption and alternatives to the linear model should be acknowledged.

   5.EXC-5 is presumed to act upstream of CDC-42 based on shared phenotypes and the known Rho GEF activity of its mammalian homologs. However, direct evidence for this is currently lacking. In future, the authors might test if depleting EXC-5 affects CDC-42 activation/GTP-loading by using CDC-42 biosensors that have been reported in the literature (e.g. Lazetic et al 2018).

   Minor comments:

   Figure 1, Figure 4, Figure S3, Figure S4 Blue color/CFP indicates the apical/luminal membrane or the apical region of the canal cytoplasm, not the actual lumen as the labels suggest. The lumen is a hollow cavity on the opposite side of the plasma membrane from these markers, and it is shown as white in the Figure 1A upper right cartoon.

   Figure 2, Figure S2 I'm not confident in the statistical analysis used here (Fisher's Exact test on two bins, <50% and >50% canal length), given that four length bins (not two) were defined. I recommend consulting a statistician.

   p.3 "Born during late embryogenesis..." Actually, the canal cell is born at ~270 minutes after first cleavage, which is in the first half of embryogenesis, not what I would call "late".

   Significance

   Polarized plasma membrane addition is critical for the development of epithelial tissues, so understanding the mechanisms that control this is of broad interest to many cell and developmental biologists. This study will be of particularly high interest to researchers working on PAR proteins, the exocyst, or single cell tube development.

   The results here add to the existing body of evidence for PAR-dependent recruitment of exocyst to expanding apical/luminal surfaces (e.g. Bryant et al 2010; Jones et al 2011, 2014; Armenti et al 2014) and to evidence for key functional distinctions between PAR-6 & PKC-3 vs. PAR-3 (e.g. Achilleos et al 2010; Jones et al 2014). The results here are more robust than in those prior studies and more clearly illustrate directionality due to the authors' acute depletion strategy, which avoids major tissue disruptions that could secondarily affect protein localization.

   expertise keywords: C. elegans, epithelia, tubulogenesis

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   Referee #2

   Evidence, reproducibility and clarity

   The manuscript by Abrams & Nance describes a precise investigation of the role of PAR proteins in the recruitment of the exocyst during and after the extension of the C. elegans excretory canal. State-of-the-art genetic techniques are used to acutely deplete proteins only in the targeted cell, and examine the localization of endogenously expressed markers. Experiments are well described and carefully quantified, with systematic statistical analysis. The manuscript is easy to follow and the bibliography is very good. Most conclusions are well supported.

   I only have a few minor questions or remarks:

   1. I am not entirely convinced by the …

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   Referee #2

   Evidence, reproducibility and clarity

   The manuscript by Abrams & Nance describes a precise investigation of the role of PAR proteins in the recruitment of the exocyst during and after the extension of the C. elegans excretory canal. State-of-the-art genetic techniques are used to acutely deplete proteins only in the targeted cell, and examine the localization of endogenously expressed markers. Experiments are well described and carefully quantified, with systematic statistical analysis. The manuscript is easy to follow and the bibliography is very good. Most conclusions are well supported.

   I only have a few minor questions or remarks:

   1. I am not entirely convinced by the presence of CDC-42 at the lumenal membrane (Fig3G); it seems to be more sub-lumenal that really lumenal. It peaks well before PAR-6 (Fig3H) which itself seem slightly less apical that PAR-3 (Fig3F). Could you use super-resolution microscopy (compatible with endogenous expression levels) to more precisely localize CDC-42? Similar point for PAR-3 and PAR-6 which do not seem to colocalize completely - a longitudinal line scan along the lumenal membrane might provide the answer even without super-resolution; this could help explain why these two proteins do not have the same function. These suggestions are easy to do provided the authors can have access to super-resolution (Airyscan to name it; although other methods will be perfectly acceptable I believe it is the most simple one).

   2. The same group has described a CDC-42 biosensor to detect its active form. It could be used here to precisely pinpoint where active CDC-42 is required: in the cytoplasm? At the lumenal membrane? colocalizing with what other protein? This will require the expression of a transgene under an excretory cell specific promotor and a simple injection strategy while helping to strengthen the description of the CDC-42 role.

   3. As the authors certainly know, there is a PAR-6 mutation which prevents its binding to CDC-42. They could express this construct in the excretory canal a simple extrachromosomal array should be sufficient) to validate the direct interaction between these proteins in this cell.

   4. What is the lethality of ZIF-1-mediated depletion of the various factors under the exc promoter? Can homozygous strains be maintained? Authors just have to add a sentence in the Mat&Met section.

   Provided that the authors have access to an Airyscan, all the questions asked here can be answered in two months (one month for constructs, one month for injection and data analysis) at a very minor cost.

   Significance

   The reviewer has an expertise in cell polarity and membrane trafficking, using C. elegans as a model.

   The manuscript by Abrams & Nance describes a precise investigation of the role of PAR proteins in the recruitment of the exocyst during and after the extension of the C. elegans excretory canal. The interactions between these factors have already been examined in a number of models and contexts. In particular it follows a previous study from the same group (Armenti et al, Dev Biol, 2014) which established that the exocyst and RAL-1 controls polarized secretion in this model, and that PAR proteins are required for the polarized localization of the exocyst, but using the early embryo. This new manuscript is entirely focused on the excretory canal and 1) confirms the previous results, and 2) significantly extends them by precisely dissecting the role of CDC-42 and the apical PAR proteins. It will be of interest to researchers investigating the links between polarity and membrane trafficking with the description of a molecular cascade required for membrane trafficking in the context of a single-cell tube.

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   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Abrams and Nance describes how the polarity proteins PAR-6 and PKC-3/aPKC promote lumen extension of the unicellular excretory canal in C. elegans. Using tissue-specific depletion methods they find that CDC-42 and the RhoGEF EXC-5/FGD are required for luminal localization of PAR-6, which recruits the exocyst complex required for lumen extension. Interestingly, they show that the ortholog of the mammalian exocyst receptor, PAR-3, is dispensable for luminal membrane extension. Overall, this is a well-written and interesting manuscript.

   Major comments

   1.Because depletion of PAR-3 in the canal causes milder defects …

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   Referee #1

   Evidence, reproducibility and clarity

   The manuscript by Abrams and Nance describes how the polarity proteins PAR-6 and PKC-3/aPKC promote lumen extension of the unicellular excretory canal in C. elegans. Using tissue-specific depletion methods they find that CDC-42 and the RhoGEF EXC-5/FGD are required for luminal localization of PAR-6, which recruits the exocyst complex required for lumen extension. Interestingly, they show that the ortholog of the mammalian exocyst receptor, PAR-3, is dispensable for luminal membrane extension. Overall, this is a well-written and interesting manuscript.

   Major comments

   1.Because depletion of PAR-3 in the canal causes milder defects than PAR-6 or CDC-42 the authors suggest that they cannot rule out the possibility that an alternative isoform of PAR-3 is expressed and buffering the defect. They should perform canal-specific RNAi-mediated depletion of the entire PAR-3 gene to determine if this is true.

   2.The authors suggest that GTP-loaded (activated) CDC-42 recruits PAR-6 to the luminal membrane. It would be nice if they could use a biosensor, such as the GBD-WSP-1 reagent from Buechner's lab to confirm that EXC-5 depletion also reduces activated CDC-42, as would be expected. This should be achievable since there is strong CDC-42 signal, even in the cytoplasm.

   3.Related to point 2, (i) does mutation of the CRIB domain of PAR-6 impair its recruitment to the luminal membrane, and (ii) does this mutant exacerbate canal defects when PAR-3 is depleted?

   4.The authors hypothesize that partial recruitment of PAR-6 by CDC-42 is sufficient for luminal membrane extension to explain the mild defects caused by PAR-3 depletion. Since depletion of PAR-6 and CDC-42 alone causes milder canal truncations the authors should co-deplete these proteins (as well as PAR-3 and CDC-42) to determine if there is an additive effect.

   5.In figure 2, the authors show that depletion of PKC-3 causes more severe canal truncations than PAR-6. Since these proteins function in the same complex what do they think is the reason for this difference? This point could be discussed more in the manuscript.

   6.Related to point 5, more experiments with PKC-3 should be done to determine if, for example, localization of SEC-10 is similarly affected as ablation of PAR-3, PAR-6 and CDC-42.

   Significance

   This manuscript builds off their previous work on the role of the exocyst in excretory canal extension and in our view represents an important advance that is relevant to biological tube development across phyla. Therefore, this work should be of interest to biologists studying tubulogenesis in many different model systems.

   My areas of expertise include model organism genetics, biological tube development, and biochemistry.

   Read the original source
5. 

   Version published to 10.1101/2020.10.05.327247 on bioRxiv

   Oct 5, 2020