Ectocytosis prevents accumulation of ciliary cargo in C. elegans sensory neurons

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1. 

   Version published to 10.7554/elife.67670 on eLife

   Sep 17, 2021
2. 

   eLife

   Jul 22, 2021

   Author Response

   Reviewer #1 (Public Review):

   [...] One intriguing aspect is that ectosomes were not detected in supporting glia by transmission electron microscopy and by light microscopy analysis of PKD-2::GFP and CIL-7::GFP conducted by Blacque and Barr (see for instance 10.7554/eLife.50580). The authors discuss this point and rationalize the discrepancy by stating that the fluorescent protein that they are using is brighter at the low pH of endosomes and lysosomes that the FPs previously used. Considering that the FP is fused to the intracellular domain of the membrane protein, the FP will not be exposed to the pH of the endosomes in the target cell. The authors' explanation is not valid and the basis for the discrepant results remains unresolved.

   Reviewer#1 is correct: the C-terminal FP would not be exposed to acidic pH in …

   Author Response

   Reviewer #1 (Public Review):

   [...] One intriguing aspect is that ectosomes were not detected in supporting glia by transmission electron microscopy and by light microscopy analysis of PKD-2::GFP and CIL-7::GFP conducted by Blacque and Barr (see for instance 10.7554/eLife.50580). The authors discuss this point and rationalize the discrepancy by stating that the fluorescent protein that they are using is brighter at the low pH of endosomes and lysosomes that the FPs previously used. Considering that the FP is fused to the intracellular domain of the membrane protein, the FP will not be exposed to the pH of the endosomes in the target cell. The authors' explanation is not valid and the basis for the discrepant results remains unresolved.

   Reviewer#1 is correct: the C-terminal FP would not be exposed to acidic pH in endosomes. We tested whether increasing the pH of endo-lysosomal compartment would increase the fluorescence or the number of EVs within AMsh cytoplasm by exposing the animals to NH4Cl (Fazeli et al., 2016). We did not observe significant differences induced by NH4Cl. Therefore, quenching plays no role. We understand the concerns raised and removed all sentences suggesting quenching of FP may play a role. Still, EV produced from PCMC are captured by the supporting glia and - at least for GCY-22-wrmScarlet - this occurs through phagocytosis of basal ectosomes budding from ASER PCMC rather than by fusion of EV with AMsh plasma membrane. Therefore, the EVs within AMsh are likely traveling through endolysosomal pathway.

   As Reviewer#1, we were intrigued other labs did not report our observations of EV cargo transfer to glia. How can we explain PKD-2::GFP and CIL-7::GFP overexpressed in CEM were not observed in the associated glia? We observed export of TSP-6 to AMsh by Amphid neurons is severely reduced in TSP-6-wrmScarlet knock-in strain compared to TSP-6-wrmScarlet overexpression strains. Therefore, the absence of PKD-2::GFP and CIL-7::GFP export to the associated glia could be explained by differences in expression levels of the cargos, although -as noticed by Reviewer#1- PKD-2::GFP and CIL-7::GFP are also overexpressed in (Wang et al., 2014). Alternatively, each neuron/ cargo can have specific properties preventing or promoting export of cargo from PCMC to glia. For example, CEM neurons releasing PKD-2::GFP and CIL-7::GFP have a specific trafficking machinery (including expression of the kinesin KLP-6 and the tubulin TBA-6 (Akella and Barr, 2021) that can contribute to potentiate PKD-2/CIL-7 ectocytosis from cilia tip. Strong ectocytosis from CEM cilia tip might secondarily prevent PKD-2/CIL-7 export from PCMC to glia. If true, we would predict trafficking mutants preventing PKD-2::GFP and CIL-7::GFP entry in CEM cilia should promote their export to CEPsh. We could not test this. Finally, the morphology of CEM-CEPsh glia ensemble might matter. Particularly the extent to which CEM PCMC is embedded by glia might contribute to the export properties. We are missing this EM information to elaborate further.

   As correctly alluded to in the discussion, primary cilia regulate their protein composition by shedding ectosomes and overexpression of ciliary proteins may lead to increased ciliary ectocytosis. Therefore, it is also conceivable that the extracellularly shed material the authors observe is a non-physiological consequence of their experimental design rather than a manifestation of physiological ectocytosis. In all fairness to the authors, all published studies on PKD1 and PKD2 ectocytosis by the Barr lab have used overexpression systems. And the discussion clearly spells out the possibility that the observed transfer of ciliary material from ciliated neurons to glial cells may be caused by overexpression of fusion proteins. Nonetheless, the abstract and result sections do not mention the possibility that the observed results are caused by overexpression. It would be of great help to the community to clearly indicate from the introduction onward that the shedding of material by ciliated neurons may be a result of overexpression, in this study and in past publications.

   Thank you for mentioning this. As stated previously, we performed new experiments with the same cilia proteins expressed at endogenous levels to avoid any overexpression artefacts. Our new results prove ectocytosis from the cilia tip do take place in physiological condition for GCY-22-GFP and TSP6-wrmScarlet and that export to glia still occurs for TSP-6-wrmScarlet in physiological conditions. As suggested by Reviewer #1, we now state the overexpression concerns in the discussion.

   To firmly determine the physiological extend of ciliary signaling receptor transfer from ciliated neuron to glial cells, the authors are encouraged to consider using an endogenously tagged protein instead of an overexpression system. For the GCY-22 receptor, the knock-in animals have already been developed and published by Gert Jansen's group (doi: 10.1016/j.cub.2020.08.032). A comparison of the localization between the overexpression strains and the endogenous expression strains of GCY-22::FP will be valuable to the paper and to the general discussion of ectocytosis. The Jansen lab has generated mutants of GCY-22 that no longer localize to cilia; studying whether such mutants still end up in glial cells would help clarify the route taken by ciliary material that ends up in glial cells.

   As described above, osm-3 kinesin-II anterograde IFT motor and che-3 dynein retrograde motor mutants show increased PCMC accumulation and increased transfer to AMsh glia, suggesting that accumulation of cargoes in PCMC likely drives their export to AMsh. Similarly, overexpression of GCY-22-wrmScarlet in ASER induced its accumulation PCMC and its export to AMsh. We used a mutants for AP-1 μ1 clathrin adaptor unc-101(m1) mutants to prevent GCY-22-wrmScarlet sorting and trafficking to cilia. In unc-101(m1), GCY-22-wrmScarlet was not enriched in cilia and did not export to AMsh. Therefore, the sorting and trafficking machinery mediating ciliary cargoes to accumulate in cilia is required for GCY-22-wrmScarlet export to AMsh (Figure 4).

   The authors point out in the discussion that the DiI dye transfer experiment rules out issues related to overexpression. It is however unclear whether the route taken by DiI from the environment to the support cell is the same as the route taken by receptors overexpressed in ciliated neurons. Can the authors conduct co-localization studies with DiI and one of the overexpressed FP-tagged ciliary membrane protein?

   We tried the suggested experiment and observed ~ 40% of the DiO vesicles in AMsh also carrying TSP-6-wrmScarlet in all amphid neurons. However, we are sceptical about the approach taken in this experiment. The donor cells are not the same; TSP-6-wrmScarlet is exported from all amphid neurons (under the arrestin-4 promoter, driving the expression in most ciliated neurons); DiO is exported from a subset of these amphid neurons ASK, ADL, ASI, AWB, ASH and ASJ. Also, we expect endosomes to fuse along the endolysosomal pathway leading to an increased colocalization towards the cell body, independently of the original EV content.

   Reviewer #2 (Public Review):

   [...] 1. The overexpression of fluorescently tagged transmembrane proteins may be a concern, because it often leads to aberrant neurite morphology. For example, the ciliary base in Fig. 4A seems abnormally swollen. This could confound the authors' ability to faithfully measure EV dynamics in vivo.

   As stated by Reviewer#1, all previously published studies on ectocytosis in C. elegans used overexpression and we were aware of this limitation. We provide new results using endogenously tagged GCY-22 and TSP-6 EV cargos. We obtained much cleaner results regarding the effect of cilia trafficking mutants in these knock-in strains. We highlight overexpression concerns in the discussion. We agree with Reviewer#2: measuring PCMC deformation in presence of overexpressed GCY-22-wrmScarlet is prone to artefacts. Instead, we explored all PCMC shape using mKate expression in Figure 7.

   1. Other activities of glia that are important for shaping cilia may also be impaired by the use of a dominant negative dynamin to block endocytosis. By comparison, the use of a glial-specific dominant negative RAB-28 to block exocytosis also causes severe defects in cilia morphology (Singhvi et al. 2016). Thus, this experiment does not directly demonstrate a requirement for glial EV pruning in maintaining cilia shape.

   As stated by Reviewer#1, all previously published studies on ectocytosis in C. elegans used overexpression and we were aware of this limitation. We provide new results using endogenously tagged GCY-22 and TSP-6 EV cargos. We obtained much cleaner results regarding the effect of cilia trafficking mutants in these knock-in strains. We highlight overexpression concerns in the discussion. We agree with Reviewer#2: measuring PCMC deformation in presence of overexpressed GCY-22-wrmScarlet is prone to artefacts. Instead, we explored all PCMC shape using mKate expression in Figure 7.Ablation of AMsh, AMsh exocytosis defect in AMsh::RAB1(DN) or secretome defect in pros-1 were previously shown to cause severe truncation of AWC and AFD NREs as well as defects in the associated sensory functions probably because of changes in the microenvironment of these embedded cilia. In animals expressing DYN-1(K46A), we observed severe truncation of ~10% of AFD NRE but none for AWC cilia (Figure 7). We did not observe thermotaxis defect nor chemotaxis defect to IAA, suggesting AFD and AWC sensory responses are maintained. Therefore, our results contrast with the effects of AMsh exocytosis block.

   Nevertheless, we agree with Reviewer#2 that we cannot exclude DYN-1(K46A) could indirectly affect AMsh function, leading to cilia shape changes independently of the EV capture defects caused by DYN-1(K46A). We highlight this possibility in the discussion.

   1. The distinction between puncta brightness, size, and number is unclear. For example, in Fig. 7A, glial puncta in ttx-1 mutants seem to be approximately as numerous as in wild-type animals but much less bright. The authors interpret this as export being "strongly reduced" - but why does this affect brightness rather than number? In most figures, the results are either not quantified or are summarized as a ratio of overall glia/neuron fluorescence intensity. More precise quantification of puncta brightness, size, and number would improve the manuscript.

   We agree that glia/neuron fluorescence intensity was not appropriate. We improved ttx-1 analysis and we now provide puncta number and intensity (Figure 6B and Figure 6- Supplement 1A, 1B). Their number in AMsh is heavily reduced in ttx-1 as well as their fluorescence intensity. However, as we stated, export from AFD to AMsh is maintained in ttx-1 in absence of microvilli. More quantifications have been done for puncta number, intensity and size in other experiments. These are now presented in Figure 5-Supplement 1C, 1D and Figure 3 - Supplement 1B.

   Read the original source
3. 

   eLife

   Jul 22, 2021

   Evaluation Summary:

   Razzauti and Laurent investigate the formation of extracellular vesicles (EVs) by the cilia of C. elegans sensory neurons and the potential functions of this process. Consistent with previous findings, they show that EVs can be released from two distinct sites of the cilium; further, they show that several different classes of sensory neurons can produce EVs, that these can be taken up by a neighboring glial cell, and that this process may be important for the morphology and function of ciliated sensory neurons. However, it remains unclear whether these phenomena may be a consequence of the experimental system (ciliary protein overexpression); additionally, the link between EV uptake by glia and maintenance of neuronal structure and function is not convincingly established.

   (This preprint has been reviewed by eLife. …

   Evaluation Summary:

   Razzauti and Laurent investigate the formation of extracellular vesicles (EVs) by the cilia of C. elegans sensory neurons and the potential functions of this process. Consistent with previous findings, they show that EVs can be released from two distinct sites of the cilium; further, they show that several different classes of sensory neurons can produce EVs, that these can be taken up by a neighboring glial cell, and that this process may be important for the morphology and function of ciliated sensory neurons. However, it remains unclear whether these phenomena may be a consequence of the experimental system (ciliary protein overexpression); additionally, the link between EV uptake by glia and maintenance of neuronal structure and function is not convincingly established.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Jul 22, 2021

   Reviewer #1 (Public Review):

   Razzauti and Laurent investigate extracellular vesicle formation in ciliated neurons from the amphid sensory compartment, using overexpression of ciliary membrane proteins fused to fluorescent proteins and live imaging. Consistent with past studies from Maureen Barr's lab in male cephalic neurons, they find that ciliary membrane proteins are shed from two distinct sites in amphid sensory neurons, namely the tip of cilia and a pre-ciliary zone previously named the periciliary membrane compartment (PCMC). One novel conclusion reached by the current study is that the secretion of ciliary material via extracellular vesicles is not limited to male cephalic neurons but is likely a general phenomenon of all ciliated neurons.

   The most exciting and novel finding of the paper is that puncta of ciliary material …

   Reviewer #1 (Public Review):

   Razzauti and Laurent investigate extracellular vesicle formation in ciliated neurons from the amphid sensory compartment, using overexpression of ciliary membrane proteins fused to fluorescent proteins and live imaging. Consistent with past studies from Maureen Barr's lab in male cephalic neurons, they find that ciliary membrane proteins are shed from two distinct sites in amphid sensory neurons, namely the tip of cilia and a pre-ciliary zone previously named the periciliary membrane compartment (PCMC). One novel conclusion reached by the current study is that the secretion of ciliary material via extracellular vesicles is not limited to male cephalic neurons but is likely a general phenomenon of all ciliated neurons.

   The most exciting and novel finding of the paper is that puncta of ciliary material originating from the ciliated neurons are found in the cytoplasm of the supporting glia. These findings suggest that ectosome shed from the PCMC of neurons are phagocytosed by the supporting glia. As discussed in the manuscript, the phagocytosis of ciliary material by support cells has long been documented in the highly specialized setting of photoreceptors. The present study suggests that this process of ciliary material transfer between neurons and glial cells may be widely prevalent in the nervous system.

   The paper is well written and the experimental quality is high. The diagrams are clear and to the point.

   One intriguing aspect is that ectosomes were not detected in supporting glia by transmission electron microscopy and by light microscopy analysis of PKD-2::GFP and CIL-7::GFP conducted by Blacque and Barr (see for instance 10.7554/eLife.50580). The authors discuss this point and rationalize the discrepancy by stating that the fluorescent protein that they are using is brighter at the low pH of endosomes and lysosomes that the FPs previously used. Considering that the FP is fused to the intracellular domain of the membrane protein, the FP will not be exposed to the pH of the endosomes in the target cell. The authors' explanation is not valid and the basis for the discrepant results remains unresolved.

   As correctly alluded to in the discussion, primary cilia regulate their protein composition by shedding ectosomes and overexpression of ciliary proteins may lead to increased ciliary ectocytosis. Therefore, it is also conceivable that the extracellularly shed material the authors observe is a non-physiological consequence of their experimental design rather than a manifestation of physiological ectocytosis. In all fairness to the authors, all published studies on PKD1 and PKD2 ectocytosis by the Barr lab have used overexpression systems. And the discussion clearly spells out the possibility that the observed transfer of ciliary material from ciliated neurons to glial cells may be caused by overexpression of fusion proteins. Nonetheless, the abstract and result sections do not mention the possibility that the observed results are caused by overexpression. It would be of great help to the community to clearly indicate from the introduction onward that the shedding of material by ciliated neurons may be a result of overexpression, in this study and in past publications.

   To firmly determine the physiological extend of ciliary signaling receptor transfer from ciliated neuron to glial cells, the authors are encouraged to consider using an endogenously tagged protein instead of an overexpression system. For the GCY-22 receptor, the knock-in animals have already been developed and published by Gert Jansen's group (doi: 10.1016/j.cub.2020.08.032). A comparison of the localization between the overexpression strains and the endogenous expression strains of GCY-22::FP will be valuable to the paper and to the general discussion of ectocytosis. The Jansen lab has generated mutants of GCY-22 that no longer localize to cilia; studying whether such mutants still end up in glial cells would help clarify the route taken by ciliary material that ends up in glial cells.

   The authors point out in the discussion that the DiI dye transfer experiment rules out issues related to overexpression. It is however unclear whether the route taken by DiI from the environment to the support cell is the same as the route taken by receptors overexpressed in ciliated neurons. Can the authors conduct co-localization studies with DiI and one of the overexpressed FP-tagged ciliary membrane protein?

   Read the original source
5. 

   eLife

   Jul 22, 2021

   Reviewer #2 (Public Review):

   The manuscript uses lipid dyes and genetically encoded reporters to show that material is transferred from C. elegans ciliated sensory neurons to their associated glia. The presence of punctate signal in the glia and observations from time-lapse imaging suggest that transfer is mediated by extracellular vesicles that bud from the ciliary base. Distinct pools of vesicles bud from the ciliary tip. While ciliary EV release has been demonstrated for IL2 neurons and male-specific neurons (Wang et al., Curr. Biol. 2014), the authors extend this to include at least ASE, AFD, and either ASH, ASI, or both. The mechanism of EV release is not determined, although consistent with previous work EV release is found to modestly increase when ciliary transport is disrupted, for example through mutations in the BBSome …

   Reviewer #2 (Public Review):

   The manuscript uses lipid dyes and genetically encoded reporters to show that material is transferred from C. elegans ciliated sensory neurons to their associated glia. The presence of punctate signal in the glia and observations from time-lapse imaging suggest that transfer is mediated by extracellular vesicles that bud from the ciliary base. Distinct pools of vesicles bud from the ciliary tip. While ciliary EV release has been demonstrated for IL2 neurons and male-specific neurons (Wang et al., Curr. Biol. 2014), the authors extend this to include at least ASE, AFD, and either ASH, ASI, or both. The mechanism of EV release is not determined, although consistent with previous work EV release is found to modestly increase when ciliary transport is disrupted, for example through mutations in the BBSome (Akella et al., eLife 2020). Genetic disruption of glial phagocytosis alters cilia morphology, opening the possibility that glia maintain cilia shape by pruning EVs, although a direct link to EVs is not made. In animals with ablated glia, EVs are still released and are taken up by other cell types, similar to what has been shown for exophers in other sensory neurons (Melentijevic et al., Nature 2017). Overall, this paper offers an important contribution by extending the phenomenon of EV release to additional classes of neurons, defining new markers with which to study EVs, and providing intriguing time-lapse images of their production. However, it falls short of advancing our understanding of how EVs are released from cilia or what their function is.

   1. The overexpression of fluorescently tagged transmembrane proteins may be a concern, because it often leads to aberrant neurite morphology. For example, the ciliary base in Fig. 4A seems abnormally swollen. This could confound the authors' ability to faithfully measure EV dynamics in vivo.

   2. Other activities of glia that are important for shaping cilia may also be impaired by the use of a dominant negative dynamin to block endocytosis. By comparison, the use of a glial-specific dominant negative RAB-28 to block exocytosis also causes severe defects in cilia morphology (Singhvi et al. 2016). Thus, this experiment does not directly demonstrate a requirement for glial EV pruning in maintaining cilia shape.

   3. The distinction between puncta brightness, size, and number is unclear. For example, in Fig. 7A, glial puncta in ttx-1 mutants seem to be approximately as numerous as in wild-type animals but much less bright. The authors interpret this as export being "strongly reduced" - but why does this affect brightness rather than number? In most figures, the results are either not quantified or are summarized as a ratio of overall glia/neuron fluorescence intensity. More precise quantification of puncta brightness, size, and number would improve the manuscript.

   Read the original source
6. 

   Version published to 10.1101/2021.02.14.430969v1 on bioRxiv

   Feb 15, 2021