Tubulogenesis involves the formation of tubule shape and diameter along both apical (lumenal) and basal sides. Once the tubule is formed, this structure needs to be regulated and maintained. The excretory canals provide a simple model to study these processes. The excretory canal cell, located near the terminal bulb of the pharynx, extends two hollow processes to the left and right lateral side of the worm, where they bifurcate and extend anteriorly and posteriorly to form an H-shaped structure. The set of EXC proteins maintain the structure of the apical surface of the canal. Mutations in the exc genes allow formation of fluid-filled cysts in the lumen of the canal. The Exc-1 phenotype exhibits canal cysts of variable size and number that are frequently located at the distal ends of the canals. Cysts can be very small, or as large as the entire diameter of the worm. We have cloned the exc-1 gene via fosmid and gene rescue, RNAi phenocopy, expression pattern, and allele sequencing, and found that this gene encodes a homologue of the family of mammalian Interferon-Inducible GTPases (IIGP), proteins that include a Ras GTPase domain. Overexpression of exc-1 causes the canal cell to form normal-diameter tubules, but with a defective basal surface. This phenotype also occurs through overexpression of other exc genes. exc-1 shows genetic interactions with other members of the exc family; the results imply that EXC-1 acts downstream of the LIM-domain protein EXC-9, but upstream of the guanine exchange factor EXC-5 (See poster by B. Mattingly). We have crossed exc-1 (rh26) mutants to marker strains that show the position of subcellular organelles within the canals. exc-1 mutants appear to have the same effect on subcellular organelles as does loss of EXC-5, a buildup of apical early endosomes with concomitant loss of recycling endosomes, leading to blockage of recycling of material to the apical membrane and presumed weakening of the apical surface. Current work is investigating possible direct protein-protein interactions between EXC-1 and EXC-5, and EXC-1's role in the tubular amphid sheath cells. We are grateful for marker constructs and advice supplied by Barth Grant, John White, Brian Ackley, Erik Lundquist, and Monica Driscoll, as well as support from NINDS R03-NS067323 and the Inez Jay Fund of the University of Kansas.

Affiliation:
- Dept. of Molecular Biosciences, University of Kansas, Lawrence, KS