Syntaxin Function in Cell Polarization

描述（由申请人提供）：大多数人类细胞类型是极化的，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，它们形成外部世界与底层基底膜和结缔组织之间的屏障，并构成大多数主要器官。上皮细胞极性的建立和维持依赖于通过囊泡转运途径将蛋白质精确靶向至顶端和基底侧质膜结构域。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具有重要意义。 囊泡转运途径依赖于SNARE机制进行最后的膜融合步骤。Syntaxins是SNARE最核心的组成部分，因为它们直接与几乎所有其他组成部分相互作用。我们已经发现，突触融合蛋白3和4是专门定位在顶端和基底外侧域，分别，在那里他们管理传入的运输囊泡的融合。 这个建议的中心假设是，突触融合蛋白定义的网站囊泡胞吐和突触融合蛋白3和4的顶端和基底侧分离是必要的上皮细胞极性。为了测试这一点，将识别突触融合蛋白3/4的靶向信号，并定义它们在到达顶端或基底侧质膜的途中所遵循的途径。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产生而重新定位，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标记的顶侧和基底侧标记物的后高尔基体转运载体的融合位点，并且我们将询问它们是否与预组装的突触融合蛋白3/4簇特异性共定位。 总的来说，这个项目旨在为以下基本问题提供答案。(1)突触融合蛋白3/4如何靶向极化的MDCK上皮细胞？(2)突触融合蛋白3/4的互斥定位是否是上皮细胞中极化货物运输所必需的，从而是细胞极性的形成/维持所必需的？(3)极化运输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，它们是否是载体融合的位点？(5)syntaxin 3/4簇是否仅特异性融合“它们的”货物载体？(6)单独定位一个突触融合蛋白是否足以建立一个融合位点并产生一个表面结构域？

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?