Tubular Excretory Canal Structure Depends on Intermediate Filaments EXC-2 and IFA-4 in Caenorhabditis elegans

Abstract

The excretory canals of are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in 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 encodes intermediate filament protein IFC-2 EXC-2/IFC-2 protein, fluorescently tagged via clustered regularly interspaced short palindromic repeats/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.

Intestinal intermediate filament polypeptides in C. elegans: Common and isotype-specific contributions to intestinal ultrastructure and function

“…Yet, the data in Fig. 1d together with other recent analyses provide conclusive evidence for expression at other sites [20][21][22] . The situation is further complicated by alternative transcriptional units at the ifc-2 gene locus ( Supplementary Fig.…”

“…Production of the IF-moiety should therefore be efficiently abrogated throughout the mutant worms. As to be expected from results of other laboratories using comparable gene inactivation approaches [20][21][22] , the lumen of the excretory canal was considerably enlarged with alterations of the apical domain in this mutant ( Fig. 6a-c), which was confirmed by light microscopic analyses ( Fig.…”

“…Furthermore, Northern blot experiments using a probe specific for the IF-encoding moiety detected two minor large transcripts and at least one major small transcript, which would account for the alternative transcripts ifc-2a/b and ifc-2d/e from the two different promoters. In accordance, Al-Hashimi et al 21 identified polypeptides corresponding in size to IFC-2a and IFC-2b but not to IFC-2c in anti-GFP immunoblots of knock-in strain BK531 producing an aminoterminally 3xFLAG-and GFP-tagged IFC-2a/b/c reporter. Anti-GFP immunoblotting of our knock-in strain BJ316 encoding carboxyterminally YFP-tagged IFC-2a/e, on the other hand, showed a weak band expected for the IFC-2a::YFP fusion protein and a much stronger band for the expected IFC-2e::YFP fusion protein ( Supplementary Fig.…”

“…They encode 1128 amino acid-long IFC-2a and 1248 amino acid-long IFC-2b differing only at their carboxyterminal 15 and 135 amino acids, respectively. Both isoforms, which have also been referred to as EXC-2a and EXC-2b 21 , consist of an aminoterminal half, that is not related to IFs and a typical IF-moiety at their carboxyterminal half. Isoform IFC-2c (685 amino acids), which has been named ECP-1 by others 20 , corresponds to the aminoterminal half of IFC-2a/b.…”

“…To deduce information on isotype expression in these cells, we side-by-side compared the fluorescence in knock-in strain BJ316 producing carboxyterminally-tagged IFC-2a/e with that in knock-in strain BK531 producing aminoterminally-tagged IFC-2a/b/c ( Supplementary Fig. S3; see also 21 ). As expected, the intestine was fluorescent in BJ316 but not in BK531.…”