Role of cell adhesion in organizing membrane growth.

描述（申请人提供）：胞吐的空间调节对于建立和维持细胞表面极性是必不可少的，这是上皮正常功能的重要性质。不能正确地分类和传递特定的质膜蛋白到上皮细胞的正确表面是人类代谢性疾病和癌症的共同特征。尽管在了解蛋白质分选的分子机制方面取得了很大进展，但关于后高尔基转运囊泡如何将根尖和基底外侧的货物运送到与质膜融合的正确位置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，以组织上皮细胞的极化运输途径。根据初步研究的结果，提出了一个可行的假设。我们的中心假设是，在e -钙粘蛋白介导的细胞粘附开始时，Ral gtpase被激活，并且在形成特殊膜域（包括顶端连接复合体、桥粒和初级纤毛）的过程中，它们以时空方式调节囊泡功能。我们的工作模型是，通过与Sec5和Exo84亚基的连续相互作用，Ral GTPases调节了胞囊依赖性运输的不同步骤。这些相互作用，反过来，驱动胞囊和“辅助因子”之间的关联/解离循环，这些“辅助因子”将胞囊复合物靶向到不同的亚细胞位点，或者以其他方式为这些位点的特定功能做准备。最后，我们提出至少有两种不同的囊泡相关复合物被招募到细胞间接触的新生位点，并随后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作用；2)确定Sec6-Munc18相互作用如何控制极化胞吐；3)明确Sec6如何靶向AJC，确定多种囊泡相关复合物在细胞间连接形成中是否具有不同的功能。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）与在上皮极性建立过程中促进极化膜运输的信号通路的激活联系起来。鉴于我们将重点关注的分子-特别是外囊和Ral gtpass -在后生动物中是保守的，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）将使我们对许多其他细胞类型中调节质膜蛋白复合物组装的机制有重要的见解。因此，这项工作的意义在于，它将指导我们在考虑治疗各种细胞内运输和膜蛋白组装受损的人类疾病时的思考。

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.