Topogenesis of NA/K-ATPASE in Polarized Epithelial Cells

描述（由申请人提供）：极化转运上皮细胞包括紧密粘附的细胞单层，其在体内不同生物区室之间形成选择性渗透屏障，并通过离子和溶质在这些区室之间的矢量转运来调节离子稳态。上皮细胞的结构和功能异常是许多人类疾病的特征。我们工作的长期目标是了解上皮细胞如何通过专门的钙粘蛋白介导的细胞-细胞接触组织成单层，并将蛋白质定位于功能不同的质膜结构域。我们已经整合了不同的实验方法来解决这些问题：蛋白质和蛋白质复合物的结构分析，高分辨率活细胞成像，蛋白质复合物组装和功能在细胞中的生化分析，以及蛋白质功能的体外重建。在之前的资助期间，我们定义了细胞间粘附的阶段，并获得了所涉及机制的初步证据，确定了几种质膜蛋白如何靶向并组织在特定的膜结构域中，并开发了新的体外方法来解剖细胞间接触质膜的蛋白质相互作用和功能。我们将这些结果和结论应用于肾移植患者缺血性损伤的研究。我们的工作假设是，细胞-细胞粘附建立了空间线索的蛋白质复合物的本地化组装到一个连接信号连接（Par复合物），控制细胞-细胞粘附（钙粘蛋白/连环蛋白），后高尔基体运输囊泡（Sec 6/8复合物），有丝分裂纺锤体的方向，和紧密连接功能。我们将通过以下方式来检验这个假设：1）。明确钙粘蛋白介导的细胞间粘附的分子机制。体外重建钙粘蛋白/连环蛋白-肌动蛋白细胞骨架相互作用，3）。囊泡对接/融合到基于钙粘蛋白的质膜结构域的机制，以及4）。分析Par复合体和连接信号连接的组装/功能。这些研究的意义是，结果将定义分子连接和机制控制膜结构域形成的钙粘蛋白为基础的细胞-细胞粘附，并提供了一个基线，了解异常的上皮细胞功能在不同的疾病状态。

DESCRIPTION (provided by applicant): Polarized transporting epithelia comprise tightly adherent cell monolayers that form selective permeability barriers between different biological compartments in the body and regulate ionic homeostasis by vectorial transport of ions and solutes between those compartments. Structural and functional abnormalities of epithelia are characteristic of many human diseases. The long-term goals of our work are to understand how epithelial cells organize into monolayers through specialized cadherin-mediated cell-cell contacts, and localize proteins to functionally different plasma membrane domains. We have integrated different experimental approaches to address these problems: structural analysis of proteins and protein complexes, high-resolution live cell imaging, biochemical analysis of protein complex assembly and function in cells, and in vitro reconstitution of protein functions. During the previous funding period, we defined stages in cell-cell adhesion and obtained preliminary evidence of a mechanism involved, determined how several plasma membrane proteins are targeted to and organized in specific membrane domains, and developed new in vitro methods to dissect protein interactions and functions at cell-cell contact plasma membranes. We translated these results and conclusions to studies of ischemic damage in kidney transplant patients. Our working hypothesis is that cell-cell adhesion establishes spatial cues for localized assembly of protein complexes to a Junctional Signaling Nexus (Par complex) that controls cell-cell adhesion (cadherin/catenins), delivery of post-Golgi transport vesicles (Sec6/8 complex), mitotic spindle orientation, and tight junction function. We will test this hypothesis by: 1). Define molecular mechanisms of cadherin-mediated cell-cell adhesion, 2). In vitro reconstitute cadherin/catenin-actin cytoskeleton interactions, 3). Mechanism of vesicle docking/fusion to cadherin-based plasma membrane domains, and 4). Analyze the Par complex, and assembly/function of a Junctional Signaling Nexus. The significance of these studies is that results will define molecular linkages and mechanisms controlling membrane domain formation by cadherinbased cell-cell adhesions, and provide a base line for understanding abnormalities in epithelial cell functions in different disease states.