Topogenesis of NA/K-ATPASE in Polarized Epithelial Cells

描述（由申请人提供）：极化转运上皮由紧密粘附的细胞单层组成，它们在体内不同的生物区室之间形成选择性渗透性屏障，并通过这些区室之间离子和溶质的载体运输来调节离子稳态。上皮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钙粘蛋白介导的细胞间接触形成单层，并将蛋白质定位到功能不同的质膜结构域。我们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，蛋白质复合物在细胞中的组装和功能的生化分析，以及蛋白质功能的体外重构。在之前的资助期间，我们定义了细胞-细胞粘附的阶段，并获得了相关机制的初步证据，确定了几种质膜蛋白如何靶向和组织在特定的膜结构域，并开发了新的体外方法来解剖细胞-细胞接触质膜上的蛋白质相互作用和功能。我们将这些结果和结论转化为肾移植患者缺血性损伤的研究。我们的工作假设是，细胞-细胞粘附为蛋白质复合物的局部组装建立了空间线索，以连接信号Nexus （Par复合体）控制细胞-细胞粘附（钙粘蛋白/连环蛋白）、高尔基转运囊泡（Sec6/8复合体）的递送、有丝分裂纺锤体取向和紧密连接功能。我们将通过以下方法来检验这个假设：1)。明确钙粘蛋白介导的细胞-细胞粘附的分子机制，2)。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/融合到钙粘蛋白基质膜结构域的机制；分析Par复合体和连接信号Nexus的组装/功能。这些研究的意义在于，研究结果将明确钙粘蛋白为基础的细胞-细胞粘附形成膜结构域的分子联系和机制，并为理解不同疾病状态下上皮细胞功能异常提供基线。

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.