Meisosomes, folded membrane microdomains between the apical extracellular matrix and epidermis
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eLife assessment
This study thoroughly characterizes the morphology of an interesting folded membrane structure that links the epidermis to the cuticle in C. elegans. This structure, here named the meiosome, has been noted by several previous researchers. The study would be strengthened by providing additional support to the notion that the VHA-5::GFP transgenic reporter, used by the authors, faithfully labels the meisosome, and by stronger evidence that meiosomes indeed serve as attachment platforms between the cuticle and the epidermis.
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Abstract
Apical extracellular matrices (aECMs) form a physical barrier to the environment. In Caenorhabditis elegans , the epidermal aECM, the cuticle, is composed mainly of different types of collagen, associated in circumferential ridges separated by furrows. Here, we show that in mutants lacking furrows, the normal intimate connection between the epidermis and the cuticle is lost, specifically at the lateral epidermis, where, in contrast to the dorsal and ventral epidermis, there are no hemidesmosomes. At the ultrastructural level, there is a profound alteration of structures that we term ‘meisosomes,’ in reference to eisosomes in yeast. We show that meisosomes are composed of stacked parallel folds of the epidermal plasma membrane, alternately filled with cuticle. We propose that just as hemidesmosomes connect the dorsal and ventral epidermis, above the muscles, to the cuticle, meisosomes connect the lateral epidermis to it. Moreover, furrow mutants present marked modifications of the biomechanical properties of their skin and exhibit a constitutive damage response in the epidermis. As meisosomes co-localise to macrodomains enriched in phosphatidylinositol (4,5) bisphosphate, they could conceivably act, like eisosomes, as signalling platforms, to relay tensile information from the aECM to the underlying epidermis, as part of an integrated stress response to damage.
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Public Review
Reviewer #1 (Public Review):
- “In fact, it is not surprising that the collagen mutants display a detached cuticle, because the extracellular domains of MUP-4 and MUA-3 (the transmembrane receptors of apical hemidesmosomes that are primarily responsible for tethering the epidermis to the cuticle) both contain vWFA collagen-binding domain (Hong et al., JCB 2001; Bersher et al., JCB 2001). Hence loss of certain collagens in the cuticle directly affects cuticle-epidermis attachment due to defective ligand-receptor interactions is a much more plausible explanation.”
We agree with the reviewer that a specific molecular interaction likely mediates the attachment of the cuticle to the epidermis, not only in the area above the hemidesmosomes, but also in the area of the meisosomes. The collagens that potentially associate with …
Public Review
Reviewer #1 (Public Review):
- “In fact, it is not surprising that the collagen mutants display a detached cuticle, because the extracellular domains of MUP-4 and MUA-3 (the transmembrane receptors of apical hemidesmosomes that are primarily responsible for tethering the epidermis to the cuticle) both contain vWFA collagen-binding domain (Hong et al., JCB 2001; Bersher et al., JCB 2001). Hence loss of certain collagens in the cuticle directly affects cuticle-epidermis attachment due to defective ligand-receptor interactions is a much more plausible explanation.”
We agree with the reviewer that a specific molecular interaction likely mediates the attachment of the cuticle to the epidermis, not only in the area above the hemidesmosomes, but also in the area of the meisosomes. The collagens that potentially associate with MUP-4 and/or MUA-3 in the muscle regions have not been identified, nor in the main epidermal region, where the putative receptor is not known. We have modified the text accordingly.
“Likewise, it is more resonable to propose that lack of certain collagens in the cuticle directly affects cuticle stiffness, rather than working indirectly through epidermal meisosomes.”
We agree with the reviewer that the loss of specific structural components of the cuticle could well affect stiffness directly, especially if the furrows are affected; non-furrow collagen mutants do not show this phenotype. An analogy might be the increased stiffness that corrugation provides. We have modified the text accordingly. Our future research aims precisely at modelling these physical aspects.
- “VHA-5::GFP does not co-localize with fluorescent markers for MVB, recycling endosomes and autophagolysosomes. By claiming this, the authors made a huge assumption that the overexpressed VHA-5::GFP fusion protein can only possibly associate with four types of organelles (meisosomes, MVB, recycling endosomes and autophagolysosomes) but not any other known or to-be-identified subcellular structures. In addition, a previous study did report that VHA-5 is localized in several other places besides the apical membrane stacks (Liegeois et al., JCB 2006).”
The reviewer cites the Liegeois paper that we mention above, which, in our opinion, and that of reviewer 2 (“VHA-5 is well known to localise to the apical membrane stacks (Liegeois 2006) and could be served as marker of apical membrane structure”), provides extremely strong support for our position. In Liegeois et al., 2006, there is a quantification of immunogold staining that shows that >85% of VHA-5 is found in meisosomes (Fig S5D). By providing the results of co-localisation analyses with 3 cytoplasmic vesicular markers, we simply wanted to illustrate the specificity of the signal to the non-initiated. Importantly, we now provide strong evidence that VHA-5::GFP marker co-localises with apical plasma membrane macrodomains revealed by both a PH domain of PLCδ and a CAAX marker. As our ultrastructural analyses demonstrate that meisosomes are composed by apical membrane folds, this again is wholly consistent with VHA-5 being a bonafide marker of meisosomes.
Reviewer #2 (Public Review):
The reviewer questioned the need to give another name to the “apical membrane stacks”. We made this proposition after consultation with a broad community of researchers in the field. We believe that this simpler name provides a link to an analogous structure in yeast, the eisosome, also at the interface between the aECM and the cell.
The reviewer wrote, “The major problem of this paper is that there is not much new information”, that it was known, for example, that “"furrowless" dpy mutants result in complete disorganization of the epidermis”. In addition to demonstrating that the furrowless Dpy mutants have very particular and specific phenotypes, without affecting the presence of hemidesmosomes (PMID: 33033182), nor different vesicular markers (FIgure 6S2), we would like to point out that reviewer #1 commented, “the work presented by Aggad et al. is rich in novelty”, and Reviewer #3, “The major strengths of the paper are the novelty”. We have re-written and reorganised the text and hope Reviewer #2 appreciates the novelty more in the revised version.
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eLife assessment
This study thoroughly characterizes the morphology of an interesting folded membrane structure that links the epidermis to the cuticle in C. elegans. This structure, here named the meiosome, has been noted by several previous researchers. The study would be strengthened by providing additional support to the notion that the VHA-5::GFP transgenic reporter, used by the authors, faithfully labels the meisosome, and by stronger evidence that meiosomes indeed serve as attachment platforms between the cuticle and the epidermis.
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Reviewer #1 (Public Review):
In this work, Aggad et al. focused on the multi-folded membrane structure (termed meisosomes) located between the apical extracellular matrix and the epidermal cells of the C. elegans. The authors performed detailed analysis on the morphology and 3D distribution of the meisosomes at different developmental stages of the C. elegans skin. They also investigated factors affecting the biogenesis and reorganization of the meisosomes, as well as the involvement of meisosomes in cuticle synthesis and maintenance. The meisosomes are particularly intriguing membrane structures connecting the epidermis to the extracellular matrix, which potentially have vital functions but were given very little attention before this study. Therefore, the work presented by Aggad et al. is rich in novelty and may greatly benefit the …
Reviewer #1 (Public Review):
In this work, Aggad et al. focused on the multi-folded membrane structure (termed meisosomes) located between the apical extracellular matrix and the epidermal cells of the C. elegans. The authors performed detailed analysis on the morphology and 3D distribution of the meisosomes at different developmental stages of the C. elegans skin. They also investigated factors affecting the biogenesis and reorganization of the meisosomes, as well as the involvement of meisosomes in cuticle synthesis and maintenance. The meisosomes are particularly intriguing membrane structures connecting the epidermis to the extracellular matrix, which potentially have vital functions but were given very little attention before this study. Therefore, the work presented by Aggad et al. is rich in novelty and may greatly benefit the related fields if the main conclusions stand. However, the authors' claims are not very well-supported by the data due to improper use of reporters and mutants, as well as some flaws in experimental design.
1. One major problem with this manuscript is the investigation about meisosome functions. Instead of generating knockdown animals or mutants that directly and specifically disrupt meisosome structures, the authors used several cuticular collagen mutants, which harbor multiple complex cuticular and epidermal defects. Therefore, the main conclusions drawn from the analysis using collagen mutants, such as "meisosomes may play an important role in attaching the cuticle to the underlying epidermal cell" or "furrow collagens are required for stiffness potentially as they are essential for the presence of normal meisosomes" do not stand well. In fact, it is not surprising that the collagen mutants display a detached cuticle, because the extracellular domains of MUP-4 and MUA-3 (the transmembrane receptors of apical hemidesmosomes that are primarily responsible for tethering the epidermis to the cuticle) both contain vWFA collagen-binding domain (Hong et al., JCB 2001; Bersher et al., JCB 2001). Hence loss of certain collagens in the cuticle directly affects cuticle-epidermis attachment due to defective ligand-receptor interactions is a much more plausible explanation. Likewise, it is more resonable to propose that lack of certain collagens in the cuticle directly affects cuticle stiffness, rather than working indirectly through epidermal meisosomes. In a word, this study did not answer the long-standing question since the 1980s: what are the primary functions of the apical membrane stacks (AKA meisosomes) in the C. elegans epidermis?
2. Another problem with this manuscript is the representation of meisosome structures by VHA-5::GFP reporter alone from Figure 3 to Figure 7. The authors claim that VHA-5::GFP is a meisosome-specific marker, but only provided indirect and superficial evidence to support this claim: 1) VHA-5::GFP signal is distributed in the same general epidermal area as the majority of meisosomes (so are many other membrane organelles in the C. elegans epidermis);2. VHA-5::GFP does not co-localize with fluorescent markers for MVB, recycling endosomes and autophagolysosomes. By claiming this, the authors made a huge assumption that the overexpressed VHA-5::GFP fusion protein can only possibly associate with four types of organelles (meisosomes, MVB, recycling endosomes and autophagolysosomes) but not any other known or to-be-identified subcellular structures. In addition, a previous study did report that VHA-5 is localized in several other places besides the apical membrane stacks (Liegeois et al., JCB 2006). In a word, there is no solid, direct evidence showing that VHA-5::GFP can specifically represent meisosomes and faithfully visualize meisosome morphology in the C. elegans epidermis. There are also no alternative approaches for meisosome morphological analysis to back up the results obtained from VHA-5::GFP reporter. Therefore, most of the data from Figure 3-7 can only be interpreted as the influence of various factors on the distribution patterns of VHA-5::GFP, not just meisosomes.
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Reviewer #2 (Public Review):
The manuscript by Aggad et al., describes an interesting folded structure that links the epidermis to the cuticle in C. elegans. They analyzed the structure by TEM and tomography and found groups of parallel folds in both L4 and adult animals. They show VHA-5 localizes to this structure and have used VHA-5::GFP transgenic reporter to investigate differently cuticle furrow-related genes by RNAi. It is an important step to describe the character of this structure, which the authors named "meisosomes". However, the structure has been reported and well defined as "apical membrane stacks" in previous studies and reviewed by a few articles (Liegeois et al., 2006, Hyenne et al., 2015, Chisholm and Xu, 2012, Cohen and Sundaram 2020). It is very confusing that the authors want to change the name of this structure.
The…
Reviewer #2 (Public Review):
The manuscript by Aggad et al., describes an interesting folded structure that links the epidermis to the cuticle in C. elegans. They analyzed the structure by TEM and tomography and found groups of parallel folds in both L4 and adult animals. They show VHA-5 localizes to this structure and have used VHA-5::GFP transgenic reporter to investigate differently cuticle furrow-related genes by RNAi. It is an important step to describe the character of this structure, which the authors named "meisosomes". However, the structure has been reported and well defined as "apical membrane stacks" in previous studies and reviewed by a few articles (Liegeois et al., 2006, Hyenne et al., 2015, Chisholm and Xu, 2012, Cohen and Sundaram 2020). It is very confusing that the authors want to change the name of this structure.
The major problem of this paper is that there is not much new information. It is already known that these stacks exist, VHA-5 localizes to the stacks, cuticle damage induces AMPs, "furrowless" dpy mutants result in complete disorganization of the epidermis, defective cuticle structure causes abnormalities via gene expression, etc. The function of these stacks remains unknown. Another issue is the transgenic reporter of VHA-5::GFP, which is not endogenously expressed, and its puncta intensity only reflects the protein distribution but not the stack structure.
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Reviewer #3 (Public Review):
This study by Aggad, Pujol, and colleagues provides some exciting new insights into a largely overlooked organelle/structure present in C. elegans epidermial cells, the "meiosome". Although noted by several previous researchers, this folded-membrane structure was never fully characterized. In particular, the authors provide an important and thorough characterization of meiosome morphology during development. The authors also provide data suggesting that meiosomes may function to provide attachment points between the epidermis and overlying cuticle, although this portion was less clear cut. In addition, the authors show that certain cuticle collagens can affect the morphology and position of meiosomes in addition to the formation of molting-associated actin cables. Some of these latter results, which suggest …
Reviewer #3 (Public Review):
This study by Aggad, Pujol, and colleagues provides some exciting new insights into a largely overlooked organelle/structure present in C. elegans epidermial cells, the "meiosome". Although noted by several previous researchers, this folded-membrane structure was never fully characterized. In particular, the authors provide an important and thorough characterization of meiosome morphology during development. The authors also provide data suggesting that meiosomes may function to provide attachment points between the epidermis and overlying cuticle, although this portion was less clear cut. In addition, the authors show that certain cuticle collagens can affect the morphology and position of meiosomes in addition to the formation of molting-associated actin cables. Some of these latter results, which suggest an 'outside-in' type of patterning regulation, run counter to certain previous models.
The major strengths of the paper are the novelty of describing a 'new organelle' and the thoroughness and clarity of the morphological analysis. The various EM studies were particularly well done and likely required a good deal of technical development, which may be of use to others in the field. One clear weakness is that it's not currently clear if the reported cuticle detachment defect is due to altered meiosomes, to the altered cuticle composition, or perhaps both, and thus the exact function(s) of meiosomes is left open. Other concerns include the use of extrachromosomally expressed VHA-5::GFP as a meiosome-specific marker. Although this could certainly be the case, it wasn't proven.
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