A spatiotemporal reconstruction of the C. elegans pharyngeal cuticle reveals a structure rich in phase-separating proteins
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Evaluation Summary:
Cuticles are specialized extracellular matrices that cover the bodies of ecdysozoans, which make up 85% of all animals. How cuticles are formed is very poorly understood, in particular in light of the fact that cuticles are shed and regrown as animals grow. The authors present a comprehensively and carefully curated resource of the components of the pharyngeal cuticle of C. elegans and provide a spatio-temporal framework to understand cuticle assembly. In doing so, the authors propose a function for a large class of intrinsically disordered proteins (IDPs). The significance of this work is high because our understanding of both cuticle formation and of IDPs is poor.
(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. The reviewers remained anonymous to the authors.)
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Abstract
How the cuticles of the roughly 4.5 million species of ecdysozoan animals are constructed is not well understood. Here, we systematically mine gene expression datasets to uncover the spatiotemporal blueprint for how the chitin-based pharyngeal cuticle of the nematode Caenorhabditis elegans is built. We demonstrate that the blueprint correctly predicts expression patterns and functional relevance to cuticle development. We find that as larvae prepare to molt, catabolic enzymes are upregulated and the genes that encode chitin synthase, chitin cross-linkers, and homologs of amyloid regulators subsequently peak in expression. Forty-eight percent of the gene products secreted during the molt are predicted to be intrinsically disordered proteins (IDPs), many of which belong to four distinct families whose transcripts are expressed in overlapping waves. These include the IDPAs, IDPBs, and IDPCs, which are introduced for the first time here. All four families have sequence properties that drive phase separation and we demonstrate phase separation for one exemplar in vitro. This systematic analysis represents the first blueprint for cuticle construction and highlights the massive contribution that phase-separating materials make to the structure.
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Evaluation Summary:
Cuticles are specialized extracellular matrices that cover the bodies of ecdysozoans, which make up 85% of all animals. How cuticles are formed is very poorly understood, in particular in light of the fact that cuticles are shed and regrown as animals grow. The authors present a comprehensively and carefully curated resource of the components of the pharyngeal cuticle of C. elegans and provide a spatio-temporal framework to understand cuticle assembly. In doing so, the authors propose a function for a large class of intrinsically disordered proteins (IDPs). The significance of this work is high because our understanding of both cuticle formation and of IDPs is poor.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
Cuticles are specialized extracellular matrices that cover the bodies of ecdysozoans, which make up 85% of all animals. How cuticles are formed is very poorly understood, in particular in light of the fact that cuticles are shed and regrown as animals grow. The authors present a comprehensively and carefully curated resource of the components of the pharyngeal cuticle of C. elegans and provide a spatio-temporal framework to understand cuticle assembly. In doing so, the authors propose a function for a large class of intrinsically disordered proteins (IDPs). The significance of this work is high because our understanding of both cuticle formation and of IDPs is poor.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This manuscript explores the genetic makeup of the C. elegans pharyngeal cuticle. By taking advantage of multiple gene expression datasets, the authors extract a high confidence set of genes that are expressed by pharyngeal cells as they go through the molting process. The majority of these genes encode proteins that are predicted to be secreted, consistent with them becoming part of the cuticle. Among these secreted proteins, the authors identify a series of intrinsically disordered proteins that, together with a previously described family of secreted IDPs, may form a kind of aggregate that promotes flexibility of the pharyngeal cuticle.
Overall, the analyses are accurately described, including possible shortcomings that are carefully taken into account in the interpretations provided. The manuscript is …
Reviewer #1 (Public Review):
This manuscript explores the genetic makeup of the C. elegans pharyngeal cuticle. By taking advantage of multiple gene expression datasets, the authors extract a high confidence set of genes that are expressed by pharyngeal cells as they go through the molting process. The majority of these genes encode proteins that are predicted to be secreted, consistent with them becoming part of the cuticle. Among these secreted proteins, the authors identify a series of intrinsically disordered proteins that, together with a previously described family of secreted IDPs, may form a kind of aggregate that promotes flexibility of the pharyngeal cuticle.
Overall, the analyses are accurately described, including possible shortcomings that are carefully taken into account in the interpretations provided. The manuscript is well written, and the implications of the findings are insightfully discussed.
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Reviewer #2 (Public Review):
Kamal and colleagues study the mechanism of chitinous cuticle formation in ecdysozoa using the C. elegans pharyngeal cuticle as a model. They make extensive use of published material including spatio-temporal transcriptomics, electron micrographs, protein-protein interaction databases, and others. They combine this meta-analysis with a few focused yet highly informative experiments to provide a synthesized version of the knowledge. They use this synthesis to identify new families of secreted proteins that are predicted to be intrinsically disordered proteins (IDPs) and show that one such IDP can undergo phase separation in vitro.
The paper is well-written, the analysis is rigorous, and the discussion is insightful. I anticipate this will be a widely cited paper, as it leads to several interesting hypotheses …
Reviewer #2 (Public Review):
Kamal and colleagues study the mechanism of chitinous cuticle formation in ecdysozoa using the C. elegans pharyngeal cuticle as a model. They make extensive use of published material including spatio-temporal transcriptomics, electron micrographs, protein-protein interaction databases, and others. They combine this meta-analysis with a few focused yet highly informative experiments to provide a synthesized version of the knowledge. They use this synthesis to identify new families of secreted proteins that are predicted to be intrinsically disordered proteins (IDPs) and show that one such IDP can undergo phase separation in vitro.
The paper is well-written, the analysis is rigorous, and the discussion is insightful. I anticipate this will be a widely cited paper, as it leads to several interesting hypotheses to pursue.
I could identify no major concerns in their data and claims.
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Reviewer #3 (Public Review):
In this study, Kamal et al. use canonical amyloid and chitin-bindings dyes and available gene expression datasets to develop a spatiotemporal blueprint for the C. elegans pharyngeal cuticle. They observe three known and three novel families of low complexity protein families expressed in successive waves to detach and reconstruct pharyngeal cuticles during molts. They predict that these IDR-rich proteins promote phase separation and malleability of the pharyngeal cuticle.
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