A spatiotemporal reconstruction of the C. elegans pharyngeal cuticle reveals a structure rich in phase-separating proteins

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. 48% 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.

C. elegansepicuticlins define specific compartments in the apical extracellular matrix and function in wound repair

“…For example EPIC-1 repeats 3-6 are perfect, as are EPIC-2 repeats 2 and 7. Other repetitive intrinsically disordered proteins have been identified in C. elegans aECMs such as the chitinous pharynx cuticle (Kamal et al, 2022) and suggest that aECMs may involve networks of intrinsically disordered proteins. Relative to imperfect repeats, perfect repeats have a higher tendency to be unstructured (Jorda et al, 2010) and are underrepresented in protein structure databases.…”

A Rapidin vivoPipeline to Identify Small Molecule Inhibitors of Amyloid Aggregation

“…Small molecule crystallization is restricted to the pharyngeal cuticle 5 , yet the collagens (which are highly abundant within the body cuticle) are also rich in -bonding capability 9 . Hence, there are likely additional properties of the pharynx cuticle beyond its amyloid-like nature that creates the unique niche that facilitates crystal formation.…”

“…We also constructed a spatiotemporal map for the development of the pharyngeal cuticle of C. elegans 9 . That study revealed the pharynx cuticle to be highly enriched with intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) 9 . Many of these IDP and IDR-rich proteins have amyloidogenic features that include an enrichment in -bonding and hydrogen-bonding capabilities, and have protofilament formation propensity 9 .…”

“…That study revealed the pharynx cuticle to be highly enriched with intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) 9 . Many of these IDP and IDR-rich proteins have amyloidogenic features that include an enrichment in -bonding and hydrogen-bonding capabilities, and have protofilament formation propensity 9 . Furthermore, we and others showed that amyloid-binding dyes specifically stain the pharyngeal cuticle 9,10 .…”

“…Many of these IDP and IDR-rich proteins have amyloidogenic features that include an enrichment in -bonding and hydrogen-bonding capabilities, and have protofilament formation propensity 9 . Furthermore, we and others showed that amyloid-binding dyes specifically stain the pharyngeal cuticle 9,10 . Despite these findings, neither transmission electron microscopy (TEM) nor diagnostic tests provide evidence for extended amyloid fibrils within the pharyngeal cuticle 9,[11][12][13] , which is consistent with the flexible nature of the cuticle 14,15 .…”