Cloning and characterization of EXC-1, an IRGP homologue that controls Intracellular Trafficking and the Maintenance of Shape in Small Biological Tubes

Abstract

Biological tubes are ubiquitous structures that carry out vital roles. The formation and maintenance of these tubule structures is a fundamental process in most organisms. The goal of this work is to get a better understanding of the molecules required for these processes. The C. elegans excretory canal provides an ideal model for these studies. The family of EXC proteins is required to maintain the tubule shape of the excretory canal. Mutations in the exc genes (EXcretory Canal abnormal) result in the formation of fluid-filled cysts in the lumen of the canal. exc-1 mutants show cysts in the canals that are often located at the ends of the canals. These cysts vary in size and number; from cysts not much wider than normal lumen up to cysts expanded to the entire diameter of the worm. This dissertation describes the work that I carried out to map, clone, and characterize exc-1. EXC-1 is homologous to the family of Immunity-Related GTPases (IRGP) and contains tandem Ras-homology domains. IRGPs make up a large family of proteins, with many of the members involved in the clearance of infectious pathogens, by assisting in autophagy. EXC-1 contains highest homology to the member of IRGPs, IRGC, of which very little is known. The family of IRGPs, and EXC-1's homology to this family and the Ras superfamily is described here. exc-1 is expressed within the excretory canal and the amphid sheath cells, a glial structure that ensheaths the amphid neuron sensory endings. exc-1 mutants exhibit occasional, abnormal formation of cyst-like structures along the process of the amphid sheath, but does not disrupt the formation of the amphid channel, the passageway for exposure of amphid dendrites to the outside environment. exc-1 interacts genetically with exc-5 (Faciogenital dysplasia protein 4 homologue) and exc-9 (Cysteine Rich Intestinal Protein homologue) in a pathway of exc-9 → exc-1 → exc-5. Binding assays show that EXC-1 and EXC-9 bind when EXC-1 is in its wild-type or GTP-bound, active, form. This work also describes the studies carried out to identify the subcellular role of EXC-1 within the canal. With a group of subcellular markers it has been identified that EXC-1 is required for proper trafficking of material from early endosomes to recycling endosome within the excretory canal, similar to EXC-5. This work indicates that EXC-1 and EXC-9 binding is required for downstream activation of EXC-5 for proper trafficking within the canal and maintenance of tubule shape.