Syntaxin Function in Cell Polarization

描述(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，这对它们的功能是必不可少的。这包括在外部世界和底层基底膜和结缔组织之间形成屏障的上皮细胞，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运输途径将蛋白质精确定位到顶端和基底侧质膜域。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是非常重要的。 囊泡运输途径依赖于膜融合的最后一步的诱捕机制。Synaxins是最核心的陷阱成分，因为它们与几乎所有其他成分直接相互作用。我们发现，Synaxins 3和4分别特异性地定位于顶端和基底外侧区域，在那里它们控制着传入运输小泡的融合。 这一建议的中心假设是，合成素定义了囊泡胞吐的位置，并且合成素3和4的顶端和基侧分离对于上皮细胞的极性是必要的。为了测试这一点，将识别Synaxins 3/4的靶向信号，并定义它们在到达顶端或基底侧质膜的路径。Synaxins 3/4将通过其靶向信号的突变和嵌合分子的产生而重新定位，并将测量其对特定膜运输途径和细胞极性的影响。我们将监测含有GFP标记的尖端和基侧标记的高尔基体后运输载体的融合部位，并询问它们是否与预先组装的Synaxin 3/4簇特异性地共定位。 总体而言，该项目旨在为以下基本问题提供答案。(1)Synaxins 3/4在极化的MDCK上皮细胞中是如何靶向的？(2)Synaxins 3/4的互斥定位是否是极化上皮细胞中极化货物运输所必需的，从而是细胞极性形成/维持所必需的？(3)极化运输的保真度是否编码于v/t-SNARE相互作用的特异性？(4)细胞膜上是否存在稳定的Synaxin簇，它们是载体融合的位点吗？(5)Synaxin 3/4簇是否仅专门融合‘他们的’货物载体？(6)单靠Synaxin的定位就足以建立一个融合位点并创建一个表面域吗？

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?