Role of Cell Adhesion in Organizing Membrane Growth

描述（由申请人提供）：本提案的长期目标是了解调节胞吐的分子机制，以便理解和开发用于胞吐缺陷的破坏性人类疾病的治疗方法，如糖尿病和多囊肾病。这项工作的总体目标是在分子水平上剖析上皮细胞极性发育过程中胞吐的时空调节。该提案侧重于运输机制的三个组成部分：Exocyst（一种假定的囊泡捆绑因子），Munc18c（捆绑/融合机制的调节剂）和Syntaxin 4（融合机制的一个组成部分）。根据初步研究的结果，提出了一个关于基底-外侧膜极化运输是如何建立的工作假设。细胞-细胞粘附促进胞囊与e -钙粘蛋白相关蛋白复合物定义的细胞间接触位点的组装、运动和关联；转运囊泡与这些位点的关联与胞囊亚基（Sec6）和Munc18c之间的相互作用相吻合，这反过来调节Syntaxin 4的活性，导致膜融合。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the molecular mechanisms that regulate exocytosis in order to comprehend and develop therapies for devastating human diseases in which exocytosis is defective, such as diabetes and polycystic kidney disease. The overall objective of this work is to dissect the spatio-temporal regulation of exocytosis at a molecular level during development of epithelial cell polarity. This proposal focuses on three components of the trafficking machinery: the Exocyst (a putative vesicle tethering factor), Munc18c (a regulator of tethering/fusion machinery), and Syntaxin 4 (a component of the fusion machinery). Based upon results of preliminary studies, a working hypothesis has been developed for how polarized trafficking to the basal-lateral membrane is established. Cell-cell adhesion promotes assembly, movement and association of Exocyst with intercellular contact sites defined by E-cadherin-associated protein complexes; association of transport vesicles with these sites coincides with an interaction between an Exocyst subunit (Sec6) and Munc18c, and this in turn regulates the activity of Syntaxin 4, leading to membrane fusion. The specific aims of this study are to: 1) identify mechanisms that specify targeting patch assembly at sites of cell-cell adhesion; 2) determine the functional significance of Sec6 binding to Munc18c in basal-lateral membrane trafficking; and 3) determine whether conformational changes in Sec6 regulate its association with Munc18c. The research plan to achieve these aims involves a combination of cell biology, biochemistry and molecular biology approaches. The significance of these studies is that they will define signaling pathways and specific interactions that couple an extracellular spatial cue (cell adhesion) to organization of components involved in membrane trafficking (vesicle docking/fusion machinery) leading to establishment of cell polarity. In the long term these studies will provide new insights into understanding the basis for abnormalities in membrane protein organizations characteristic of epithelial diseases. Also, because this proposal focuses on how information is transferred from vesicle to plasma membrane to initiate exocytosis, it has broad implications for many processes including antigen presentation, neurotransmission and hormone secretion.