exc-2, an intermediate filament gene, maintains tubular excretory canals in Caenorhabditis elegans along with ifa-4 and other novel genes.

Abstract

The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves. Additionally, a focused reverse genomic screen of genes highly expressed in the canals found 21 new genes that significantly affect outgrowth or diameter of the canals. These genes nearly double the number of candidates that regulate canal size. Two genes act as suppressors on a pathway of conserved genes whose products mediate vesicle movement from early to recycling endosomes. The encoded proteins provide new tools for understanding the process of cellular recycling and its role in maintaining the narrow diameter of single-cell tubulogenesis.