Syntaxin Function in Cell Polarization

突触蛋白在细胞极化中的功能

• 批准号: 6847178
• 负责人: Thomas Weimbs
• 金额: $ 10.64万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847178
• 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，这对于其功能至关重要。 这包括上皮细胞，这些细胞在外界和下面的基底膜和结缔组织之间形成障碍，并构成大多数主要器官。上皮极性的建立和维持取决于使用固醇转运途径的蛋白质对顶端和基底外侧质膜结构域的精确靶向。理解基于两极分化的运输基础的机制是基本的进一步概念性理解极化细胞的功能和功能障碍。 囊泡的传输途径取决于最终膜融合步骤的圈套机械。语法素是最中心的编成分，因为它们几乎与所有其他组件直接相互作用。我们已经发现，语法3和4分别位于顶端和基底外侧结构域，在这些结构域，在这些结构域中，它们控制着传入囊泡的融合。 该提议的中心假设是，语法素定义了囊泡胞吐的位点，而语法3和4的顶端和基底外侧分离对于上皮细胞极性是必需的。为了测试这一点，将确定语法3/4的靶向信号，并将定义它们在到达顶端或基底外侧质膜途中遵循的途径。语法3/4将通过其靶向信号的诱变和产生嵌合分子的诱变重新定位，并将测量对特定膜运输途径和细胞极性的后果。将监控包含GFP标记的顶端和基底外侧标记的高尔基后传输载体的融合位点，我们将询问它们是否专门与预组装的语法3/4簇共定位。 总的来说，该项目旨在为以下基本问题提供答案。 （1）如何在极化MDCK上皮细胞中靶向3/4的语法？ （2）在上皮细胞中极化货物运输所需的语法3/4的相互排斥定位，因此需要形成/维持细胞极性？ （3）在v/t-snare相互作用的特异性中编码了两极分化的运输的保真度？ （4）质膜上是否存在稳定的语法簇，它们是否是载体融合的位置？ （5）语法3/4簇是否专门融合了“他们的”货物载体？ （6）单独的语法素定位是否足以建立融合位点并创建一个表面域？