Role of cell adhesion in organizing membrane growth.

描述（由申请人提供）：胞吐作用的空间调节对于建立和维持细胞表面极性是必不可少的，这是上皮细胞正确功能的重要特性。不能正确地分选和递送特定的质膜蛋白到上皮细胞的正确表面是人类代谢疾病和癌症的共同特征。尽管在理解蛋白质分选的分子机制方面取得了很大进展，但关于高尔基体后转运囊泡如何将顶端和基底侧货物运送到与质膜融合的正确位点的主要问题仍然存在。我们研究的长期目标是了解环境空间线索，如细胞-细胞粘附，如何向细胞骨架和分泌装置发出信号，以组织上皮细胞中的极化运输途径。根据初步研究的结果，提出了一个工作假设。我们的中心假设是，Ral GTP酶被激活后启动E-钙粘蛋白介导的细胞粘附，并在时空的方式调节外囊功能，在形成专门的膜结构域，包括顶端连接复合体，桥粒和初级纤毛。我们的工作模型是Ral GTP酶通过与Sec 5和Exo 84亚基的顺序相互作用来调节外泌囊依赖性运输的不同步骤。这些相互作用反过来又驱动外囊和“辅助因子”之间的缔合/解离循环，所述“辅助因子”将外囊复合物靶向不同的亚细胞位点或以其他方式使它们在这些位点准备特定功能。最后，我们提出，至少有两个不同的外来囊相关的复合物被招募到新生的网站的细胞间接触，随后移动分开，以促进不同类型的细胞间连接的发展。本研究的具体目的是：1）研究Ral-Exocyst复合物在调节上皮极化中的功能; 2）确定Sec 6-Munc 18相互作用如何控制极化胞吐作用; 3）确定Sec 6如何靶向AJC，并确定多种Exocyst相关复合物是否在细胞间连接形成中具有不同的功能。总的来说，我们在本申请中提出的研究试图揭示一种机制的细节，该机制将细胞外事件（即细胞-细胞粘附）与在上皮极性建立期间促进极化膜运输的信号传导途径的激活联系起来。考虑到我们将重点关注的分子-特别是外囊和Ral GTP酶-在多细胞动物中是保守的，我们相信研究它们如何合作建立和维持上皮细胞中不同的连接复合物（例如AJCs和桥粒）将使我们对调节许多其他细胞类型中质膜蛋白复合物组装的机制有重要的了解。因此，这项工作的意义在于，它将指导我们考虑治疗各种人类疾病时的想法，其中细胞内转运和膜蛋白组装受损。 公共卫生相关性：为了使我们的上皮细胞保护我们免受导致疾病的有害物质的侵害，它们必须将质膜的组分组织成结构和功能不同的区域。不这样做会导致毁灭性的人类疾病，包括高胆固醇血症，糖尿病和多囊肾病。这项建议将调查负责指导囊泡携带受体的正确网站在细胞中插入到质膜的分子机制，并扩展我们的理解的基础上异常的膜蛋白组织特征的上皮疾病。

DESCRIPTION (provided by applicant): Spatial regulation of exocytosis is essential for establishing and maintaining cell surface polarity, a property vital to the correct function of epithelia. Failure to properly sort and deliver specific plasma membrane proteins to the correct surface of epithelial cells is a common feature of human metabolic diseases and cancers. Despite much progress in understanding the molecular mechanisms of protein sorting, major questions persist about how post-Golgi transport vesicles ferry apical and basolateral cargo to the correct site of fusion with the plasma membrane. The long-term goal of our research is to understand how environmental spatial cues, such as cell-cell adhesion, signal to the cytoskeleton and secretory apparatus to organize polarized trafficking pathways in epithelial cells. Based upon results of preliminary studies, a working hypothesis has been developed. Our central hypothesis is that Ral GTPases are activated upon initiation of E-cadherin-mediated cell adhesion, and that they regulate Exocyst functions in a spatio-temporal manner during the formation of specialized membrane domains-including the apical junctional complex, desmosomes and primary cilia. Our working model is that Ral GTPases regulate different steps in Exocyst-dependent trafficking, through sequential interactions with the Sec5 and Exo84 subunits. These interactions, in turn, drive an association/dissociation cycle between the Exocyst and "accessory factors" that either target Exocyst complexes to different subcellular sites or otherwise prepare them for specific functions at those sites. Finally, we propose that at least two different Exocyst-related complexes are recruited to nascent sites of intercellular contact, and subsequently move apart to facilitate the development of different types of intercellular junctions. The specific aims of this study are: 1) to investigate the function of Ral-Exocyst complexes in modulating epithelial polarization; 2) to determine how Sec6-Munc18 interactions control polarized exocytosis; and 3) to define how Sec6 is targeted to the AJC and determine whether multiple Exocyst-related complexes have distinct functions in intercellular junction formation. Collectively, the studies we propose in this application seek to uncover details of a mechanism that links an extracellular event, namely cell-cell adhesion, to activation of a signaling pathway that promotes polarized membrane trafficking during the establishment of epithelial polarity. Given that the molecules on which we will focus-specifically the Exocyst and Ral GTPases-are conserved across metazoans, we believe that studying how they collaborate to establish and maintain distinct junctional complexes in epithelial cells (e.g. the AJCs and desmosomes) will give us important insight into mechanisms that regulate the assembly of plasma membrane protein complexes in many other cell types. Thus, the significance of this work is that it will guide our thinking when considering therapies for a variety of human diseases in which intracellular transport and the assembly of membrane proteins is impaired. PUBLIC HEALTH RELEVANCE: In order for our epithelial cells to protect us from harmful agents that cause disease, they must organize components of the plasma membrane into structurally and functionally distinct domains. Failure to do so results in devastating human diseases, including hypercholesterolemia, diabetes, and polycystic kidney disease. This proposal will investigate molecular mechanisms responsible for guiding vesicles carrying receptors to the right site in the cell for insertion into the plasma membrane, and extend our understanding of the basis for abnormalities in membrane protein organization characteristic of epithelial diseases.