Syntaxin Function in Cell Polarization

突触蛋白在细胞极化中的功能

• 批准号: 6696359
• 负责人: Thomas Weimbs
• 金额: $ 27.23万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6696359
• 关键词: Golgi apparatus; MDCK cell; SDS polyacrylamide gel electrophoresis; apical membrane; basolateral membrane; biological signal transduction; cell fusion; cell membrane; cellular polarity; crosslink; epithelium; exocytosis; fluorescence microscopy; green fluorescent proteins; intracellular transport; membrane fusion; membrane proteins; mutant; protein localization; protein structure function; syntaxin; vesicle /vacuole

DESCRIPTION (provided by applicant): The majority of human cell types are polarized, i.e. they exhibit asymmetry, which is essential to their function. This includes epithelial cells that form barriers between the outside world and the underlying basement membrane and connective tissue, and make up most major organs. Establishment and maintenance of epithelialcell polarity depends on the precise targeting of proteins to the apical and basolateral plasma membrane domains usingvesicular transport pathways. Understanding the mechanismsthat underlie polarized trafficking is of fundamental importanceto understand functionand dysfunction of polarized cells. Vesicle transport pathways depend on the SNARE machinery for the final membrane fusion step. Syntaxins are the most central SNARE component as they directly interact with almost all other components. We have found that syntaxins 3 and 4 are specifically localized at the apical and basolateral domain, respectively, where they govern the fusion of incoming transport vesicles. The central hypothesis of this proposal is that syntaxins define the sites of vesicle exocytosis and that the apical and basolateral separation of syntaxins 3 and 4 is necessary for epithelial cell polarity. To test this, the targeting signals of syntaxins 3/4 will be identified, and the pathways that they follow en route to the apical or basolateral plasma membrane will be defined. Syntaxins 3/4 will be re-localized by mutagenesis of their targeting signals and by generation of chimeric molecules, and the consequences on specific membrane trafficking pathways and on cell polarity will be measured. The fusion sites of post-Golgi transport carriers containing GFP-tagged apical and basolateral markers will be monitored and we will ask whether they specifically co-localize with pre-assembled syntaxin 3/4 clusters. Collectively, this project is designed to provide answers to the following fundamental questions. (1) How are syntaxins 3/4 targeted in polarized MDCK epithelial cells? (2) Is the mutually exclusive localization of syntaxins 3/4 required for polarized cargo transport in epithelial cells and hence for the formation/maintenance of cell polarity? (3) Is the fidelity of polarized trafficking encoded in the specificity of v/t-SNARE interactions? (4) Do stable syntaxin clusters exist on the plasma membrane, and are they the sites of carrier fusion? (5) Do syntaxin 3/4 clusters specifically fuse only 'their' cargo carriers? (6) Is the localization of a syntaxin alone enough to establish a fusion site and create a surface domain?

描述（由申请人提供）：大多数人类细胞类型都是极化的，即它们表现出不对称性，这对其功能至关重要。 这包括上皮细胞，它们在外界与下面的基底膜和结缔组织之间形成屏障，并构成大多数主要器官。上皮细胞极性的建立和维持取决于利用囊泡运输途径将蛋白质精确靶向顶端和基底外侧质膜结构域。了解极化运输的机制对于了解极化细胞的功能和功能障碍至关重要。 囊泡运输途径取决于最终膜融合步骤的 SNARE 机器。突触蛋白是最核心的 SNARE 组件，因为它们直接与几乎所有其他组件交互。我们发现突触融合蛋白 3 和 4 分别特异性定位于顶端和基底外侧区域，在那里它们控制传入运输囊泡的融合。 该提议的中心假设是突触融合蛋白定义了囊泡胞吐作用的位点，并且突触融合蛋白 3 和 4 的顶端和基底外侧分离对于上皮细胞极性是必要的。为了测试这一点，将识别突触融合蛋白 3/4 的靶向信号，并定义它们到达顶端或基底外侧质膜的路径。突触融合蛋白 3/4 将通过其靶向信号的诱变和嵌合分子的生成而重新定位，并且将测量对特定膜运输途径和细胞极性的影响。将监测含有 GFP 标记的顶端和基底外侧标记的后高尔基体运输载体的融合位点，我们将询问它们是否与预组装的突触蛋白 3/4 簇特异性共定位。 总的来说，该项目旨在为以下基本问题提供答案。 (1) 突触融合蛋白 3/4 如何靶向极化 MDCK 上皮细胞？ (2) 上皮细胞中极化货物运输以及细胞极性的形成/维持是否需要突触融合蛋白 3/4 的相互排斥定位？ (3) 极化贩运的保真度是否编码在 v/t-SNARE 相互作用的特异性中？ (4)质膜上是否存在稳定的突触融合蛋白簇，它们是载体融合的位点吗？ (5) Syntaxin 3/4 簇是否专门只融合“它们的”货运载体？ (6) 仅突触融合蛋白的定位是否足以建立融合位点并创建表面结构域？