The excretory canals of C. elegans represent an attractive model for studying maintenance of seamless, narrow, single-cell biological tubes. Many genes have been identified that play a role in the maintenance of canal diameter.Mutation of the
exc-2 gene leads to short (30-40% of wild-type length) canals containing multiple fluid-filled cysts. In order to identify the
exc-2 gene, we carried out whole-genome sequencing for the allele
rh90. The results showed a frameshift insertion in candidate gene
ifa-4, which encodes intermediate filament A4. RNAi against
ifa-4 in N2 worms causes formation of short, cystic canals similar to those of
exc-2 mutant worms. In order to confirm the identity of the
exc-2 gene, we performed a rescue assay and a complementation test between
exc-2 and
ifa-4. Surprisingly, injection of the
ifa-4 sequence into
exc-2 worms did not rescue the phenotype, and the two genes complemented each other, which indicates that
exc-2 and
ifa-4 are different genes. Sequencing of three additional alleles of
exc-2 showed the same frameshift, as did sequencing of our wild-type strains! Galaxy Cloudmapping uses an older version of the genome (WS220); the latest version of Wormbase (WS247) has corrected this sequence error. We have now performed whole-genome sequencing of all 4 alleles of
exc-2, two of which were outcrossed to Hawaiian wild-type worms to allow identification of the
exc-2 mutation. We are now screening candidate genes via confirmation of mutations via Sanger sequencing, RNAi, and fosmid rescue in order to identify the
exc-2 gene, which should have mutations in all 4 alleles.Our lab's previous work suggests that
exc-2 acts genetically upstream of
exc-9,
exc-1, and
exc-5. A FRAP analysis of mutants labeled with endosomal tags suggests that, while most endosomal compartments are unaffected, the turnover of RAB-11 on recycling endosomes requires wild-type EXC-2 function. EXC-2 may therefore participate in endosomal recycling.