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的极性是必不可少的，这是一种对上皮细胞的正确功能至关重要的特性。未能正确地将特定的质膜蛋白分选并运送到正确的上皮细胞表面是人类代谢性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，但主要的问题仍然是高尔基体后运输小泡如何将顶端和基底外侧的货物运送到与质膜融合的正确位置。我们研究的长期目标是了解环境空间线索，如细胞-细胞黏附，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，提出了一个可行的假设。我们的中心假设是，在E-钙粘附素介导的细胞黏附开始时，Ral GTP酶被激活，并且它们以时空的方式调节外囊功能，在特殊的膜域的形成过程中--包括顶端连接复合体、桥粒和初生纤毛。我们的工作模型是，Ral GTP酶通过与Sec5和Exo84亚基的顺序相互作用，调节依赖于外囊的运输的不同步骤。这些相互作用反过来推动胞囊和“辅助因子”之间的结合/解离循环，这些辅助因子将胞囊复合体定位于不同的亚细胞位置，或以其他方式使它们为这些位置的特定功能做好准备。最后，我们建议至少有两个不同的外囊相关复合体被招募到细胞间接触的新生部位，然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ERL-胞囊复合体在调节上皮细胞极化中的作用；2)确定Sec6-Munc18相互作用如何控制极化胞吐；3)确定Sec6是如何靶向AJC的，并确定多种胞囊相关复合体是否在细胞间连接形成中具有不同的功能。总而言之，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-细胞黏附，与激活一条信号通路相联系，该信号通路在上皮极性的建立过程中促进极化的膜运输。鉴于我们将关注的分子-特别是外囊和Ral GTP酶-在后生动物中是保守的，我们相信研究它们如何协作在上皮细胞(例如AJCs和桥粒)中建立和维持不同的连接复合体将使我们对许多其他细胞类型中调节质膜蛋白复合体组装的机制有重要的了解。因此，这项工作的意义在于，它将指导我们在考虑治疗各种人类疾病时的思维，在这些疾病中，细胞内运输和膜蛋白组装受到损害。

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.