Functional analysis of ERM proteins in epithelial morphogenesis

描述（由申请人提供）：形成由独特的跨膜蛋白、相关细胞质蛋白和磷脂组成的专门膜结构域的能力是极化上皮细胞的基本特性。膜结构域，如顶端表面或连接复合体，允许在质膜上的功能空间分离，这是极化上皮细胞所必需的。该过程的核心是在膜的细胞质侧形成蛋白质复合物，其定位跨膜蛋白，调节其信号输出并通过受调节的内吞作用控制其丰度。Ezrin，Radixin，Moesin（ERM）蛋白在这一过程中起着关键作用。 在这个建议中，我们描述的实验设计，以利用两个调查员，安德烈McClatchey（哈佛/MGH）和理查德Fehon（芝加哥大学）的综合专业知识，以扩大我们对ERM功能的理解。研究人员和他们的实验室汇集了两个强大的实验系统，小鼠和果蝇的专业知识。拟议的研究利用多方面的方法，包括遗传学，生物化学，细胞生物学和蛋白质组学，以更好地了解这些高度保守蛋白质的功能。具体而言，我们计划：1）确定将ERM蛋白与发育中的上皮细胞中Rho的活化状态联系起来的分子机制。 2)检查调节ERM活性的分子机制，特别是在ERM活性如何动态调节的背景下。 3)阐明ERM蛋白在细胞中的功能：细胞连接重塑。 4)建立ERM介导的复合物形成的整合模型。这些实验有望为研究ERM蛋白在细胞骨架顶端-基底极性调节、肠腔形成和稳态以及转移等生物学过程中的功能提供新的视角。他们也应该产生一个更好的理解细胞过程中，建立专门的膜结构域极化细胞，并告知我们的理解细胞骨架和连接动力学在形态发生和疾病。

DESCRIPTION (provided by applicant): The ability to form specialized membrane domains composed of unique sets of transmembrane proteins, associated cytoplasmic proteins, and phospholipids, is a fundamental property of polarized epithelial cells. Membrane domains, such as the apical surface or junctional complex, allow spatial segregation of functions at the plasma membrane that are essential for polarized epithelia. Central to this process is the formation of protein complexes on the cytoplasmic side of the membrane that localize transmembrane proteins, regulate their signaling output and control their abundance via regulated endocytosis. The Ezrin, Radixin, Moesin (ERM) proteins organize a key role in this process. In this proposal we describe experiments designed to take advantage of the combined expertise of two investigators, Andrea McClatchey (Harvard/MGH) and Richard Fehon (University of Chicago), to extend our understanding of ERM function. The investigators and their laboratories bring together expertise in two powerful experimental systems, the mouse and Drosophila. The proposed research utilizes a multifaceted approach, including genetics, biochemistry, cell biology and proteomics to better understand the functions of these highly conserved proteins. Specifically, we plan to: 1) Determine the molecular mechanisms that link the ERM proteins to the activation state of Rho in developing epithelial cells. 2) Examine the molecular mechanisms that regulate ERM activity, particularly in the context of how ERM activity is dynamically regulated. 3) Delineate the function of the ERM proteins in cell:cell junction remodeling. 4) To build an integrated model of ERM-mediated complex formation. These experiments are expected to provide novel insight into the functions of ERM proteins in biological processes such as apical-basal polarity cytoskeletal regulation, intestinal lumen formation and homeostasis, and metastasis. They should also yield a better understanding of the cellular processes that establish specialized membrane domains in polarized cells, and inform our understanding of cytoskeletal and junctional dynamics during morphogenesis and in disease.