EXC-5 Controls Intracellular Trafficking in Order to Maintain the Apical Structure of the Caenorhabditis elegans Excretory Canal.

摘要

The goal of this work is to understand how cells form and maintain tubular shapes. The protein EXC-5 is necessary for a small tubular structure in Caenorhabditis elegans to maintain its shape. C. elegans is a small, easily manipulable, genetically tractable, and transparent nematode with a unicellular excretory canal system. The C. elegans excretory canal cell is a long "H"-shaped tubular structure that serves as the osmoregulatory organ for the animal. EXC-5 is a guanine nucleotide exchange factor (GEF). GEF proteins work by binding to GDP-bound small GTPases and facilitating the exchange of GDP with GTP, thus activating the GTPase. The activated GTPases are effectors themselves and interact with numerous other proteins to direct various cellular processes.;In this dissertation I discuss the various methods by which I examined how EXC-5 functions in order to maintain the tubular structure of the excretory canal. I describe an EMS non-complementation screen to identify new alleles of EXC-5, an EMS screen to identify genetic enhancers of EXC-5, methods to create an excretory canal-specific RNAi strain of C. elegans, the genetic interactions between exc-5, cdc-42, mig-2, ced-10 and other genes, the organization of subcellular organelles within the canal cell and in exc-5 mutants, the generation of intein containing proteins to create conditional alleles, and the generation of antibodies that bind to EXC-5.;The data from these studies lead to a model of tubular maintenance where the sorting of material to the apical surface is only required at certain points along the canal where it is undergoing significant restructuring because of growth or physical damage. When exc-5 is mutant, materials are not efficiently sorted to the apical surface where they are needed to maintain the apical surface. Failure of the actin network under osmotic pressure from fluid within the lumen causes cysts to form in the canal cell. When EXC-5 is overexpressed, sorting to the apical surface is enhanced at the expense of sorting to the basal surface. This leads to an inability of the basal surface to adhere to the basement membrane and grow out, causing a convoluted tubule phenotype.