Intermediate filaments EXC-2 and IFA-4 Maintain Luminal Structure of the Tubular Excretory Canals inCaenorhabditis elegans

Abstract

C. elegans excretory canals form a useful model for understanding formation of narrow 33 tubes. exc-2 mutants start to form normal canals that then swell into fluid-filled cysts. We show 34 that exc-2 encodes a large intermediate filament (IF) protein previously not thought to be located 35 in the canals. EXC-2 is located at the apical (luminal) membrane, binds to another IF protein, 36 and appears to be one of three IF proteins that form a flexible meshwork to maintain the thin 37 canal diameter. This work provides a genetically useful model for understanding the interactions 38 of IF proteins with other cytoskeletal elements to regulate tube size and growth. 39 3 ABSTRACT 40 The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance 41 of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that 42 mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts 43 that lack a complete terminal web at the apical surface, and accumulate filamentous material in 44 the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes 45 intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via 46 CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate 47 filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are 48 not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament 49 protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either 50 protein results in narrow but shortened canals. These results are consistent with a model 51 whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling 52 of narrow tubules in concert with actin filaments that guide the extension and direction of tubule 53 outgrowth, while allowing the tube to bend as the animal moves. 54 93 While most of the original exc genes have been cloned (GRUSSENDORF et al. 2016; 94 SUNDARAM and BUECHNER 2016), mutations in the exc-2 gene cause particularly severe canal 95 defects. In these mutants, the canal length is shortened by over half, the animals accumulate 96 multiple cysts in the canals, and are sensitive to growth at low osmolarity (BUECHNER et al. 97 1999). Four alleles of this gene were discovered in the original screen, which suggested that it 98 encodes a large protein. Here we report that exc-2 encodes the intermediate filament IFC-2, and 99 additionally found that mutations in the ifa-4 intermediate filament gene also cause cystic canal 100 6 defects similar to those of exc-2 mutants. Overexpression of either exc-2 or ifa-4 results in 101 shortened canals with small or no cysts. EXC-2 and IFA-4 proteins bind to each other and are 102 located at the apical membrane of the canals. The position of EXC-2 at the apical membrane 103 occurs independently of IFB-1 and IFA-4 function in the canals. These result...

Bubble guts–Controlling apical membrane morphology in the C. elegans intestine

“…In the marginal cells of the pharynx, we observed a pattern of short radial filaments, reminiscent of the IF containing tonofilaments (Karabinos et al, 2003) , 2004). We also observed expression of BBLN-1 in the uterine seam and vulva, as previously reported for IFA-1, MUA-6/IFA-2, IFB-1 and EXC-2/IFC-2 (Al-Hashimi et al, 2018;Hapiak et al, 2003;Karabinos et al, 2003;Williams et al, 2015;Woo et al, 2004). To investigate the localization of BBLN-1 relative to IFs, we engineered an endogenous IFB-1::mCherry fusion.…”

“…In bbln-1(null) animals, the lumen of the canal greatly increased in width and seemed divided into circular confinements with little or no luminal continuation between them (Figure S2C). These canal deformations were visibly distinct from the phenotypes caused by ERM-1 tagging, but are unlikely the result of the lack of BBLN-1 alone, since loss of bbln-1 does not induce excretory canal deformation as shown by expression of IFC-2 (Figure S2D), one of the main intermediate filaments in the excretory canal (Al-Hashimi et al, 2018). In bbln-1(null) animals, ERM-1 retained its apical localization, but VHA-9 appeared to additionally localize to the cytoplasm.…”

“…Therefore, the strong reduction in apical BBLN-1 likely reflects the loss of all IFs from the apical domain. We also examined the localization of BBLN-1 in the excretory canal of ifc-2(RNAi) animals, as IFC-2 has been shown to be important for the structure of this tissue (Al-Hashimi et al, 2018). We observed a marked relocalization of BBLN-1 from the apical cortex to the cytoplasm and basal membrane, which suggests that dependency on IFs is a general aspect of BBLN-1 localization (Figure 3C).…”