Analysis of ERM-1 (ezrin-radixin-moesin) function during epithelial development in C. elegans.

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At present, several proteins are known to affect epithelial cell polarity during development. However, their signalling pathways and mechanisms are still elusive. This is in part due to the abundance of adaptor molecules which mediate the interaction of polarity cues with the executive cytoskeleton. The aim of this project is to investigate the function of band-4.1/ERM (ezrin-radixin-moesin) proteins in C.elegans, a protein family that is defined as membrane-cytoskeleton linkers. We have started in-depth analysis of erm-1 that has an essential function during development of the junctional complex in the gut epithelium of C.elegans. The specific goals of the proposed research are as follows: 1) Use of ìRNA mediated interference (RNAi)î to eliminate the function of all 19 band-4.1/ERM proteins individually and in combinatorial subgroups. The phenotypic analysis will focus on the arrangements of microfilaments, plasmamembrane subdomains and junctions in epithelia for which we have obtained or identified a series of marker antibodies. 2) Over the course of the funding period, we will work to generate antibodies against most promising candidate band-4.1/ERM proteins to analyse their spatio-temporal distribution in fixed specimens. Using antibodies against ERM-1, to date we have identified 1 band-4.1/ERM protein that is expressed on the apical membrane domain of the gut epithelium. 3) Identify and study the functions of proteins that interact with ERM-1 and thus could play important roles in epithelia differentiation. This will be done by yeast 2-hybrid analysis for identification of ERM-1 interacting proteins. Once identified, the functions of those proteins will be studied with the RNAi approach and immunolocalization.