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

目前，已知有几种蛋白质在发育过程中影响上皮细胞的极性。然而，它们的信号通路和机制仍然难以捉摸。这在一定程度上是由于适配器分子的丰富，这些分子介导了极性信号与执行细胞骨架的相互作用。本项目的目的是研究Ezrin-Radisin-moesin(Ezrin-Radisin-moesin)蛋白在线虫中的功能，该蛋白被定义为膜-细胞骨架连接物。我们已经开始深入分析ERM-1，它在线虫肠道上皮细胞连接复合体的发育过程中具有重要的功能。这项研究的具体目标如下：1)利用？RNA介导的干扰(RNAi)消除所有19个Band-4.1/ERM蛋白的功能，并在组合亚群中消除它们。表型分析将集中在我们已经获得或鉴定了一系列标志性抗体的上皮细胞中微丝、质膜亚区和连接的排列。2)在资助期间，我们将致力于产生针对最有希望的候选Band-4.1/ERM蛋白的抗体，以分析它们在固定样本中的时空分布。使用抗ERM-1的抗体，到目前为止，我们已经鉴定出1个Band-4.1/ERM蛋白，它表达在肠上皮的顶膜区。3)鉴定和研究与ERM-1相互作用的蛋白的功能，从而在上皮分化中发挥重要作用。这将通过酵母双杂交分析来鉴定ERM-1相互作用蛋白。一旦确定，将使用RNAi方法和免疫定位来研究这些蛋白质的功能。

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.