Topogenesis of NA/K-ATPASE in Polarized Epithelial Cells

我们工作的长期目标是了解上皮细胞是如何组织成单层的， 专门的钙粘蛋白介导的细胞-细胞接触，并将蛋白质定位于功能不同的血浆 膜结构域我们整合创新的实验方法来解决这些问题： 蛋白质和蛋白质复合物的分析，蛋白质和生物传感器的高分辨率活细胞成像， 细胞中蛋白质复合物组装和功能的生化分析以及新型体外重建 测定。在此资助期间，我们定义了细胞-细胞粘附的阶段和相关机制， 确定了质膜蛋白如何靶向和组织在形成基底外侧 膜结构域对细胞-细胞粘附的影响。我们将在这些结果的基础上进行拟议的研究。我们将 定义α-连环蛋白和p120在调节细胞-细胞粘附和膜动力学中的作用。我们将 检测内源性α-连环蛋白和p120对质膜的隔离作用 独立于E-钙粘蛋白，并使用细胞迁移的位置特异性标签，肌动蛋白 使用活细胞成像、FRET、生物传感器和小分子抑制剂研究细胞动力学和细胞-细胞粘附。我们 将使用亲和纯化和MALDI/TOF质谱来确定调节α-连环蛋白的蛋白质 以及p120功能和与肌动蛋白细胞骨架的关联。我们将剖析调节 基底外侧质膜靶向贴片处囊泡的分选。我们将使用创新的重组技术 对与E-钙粘蛋白底物结合的膜贴片进行测定，以观察转运囊泡如何 传递到目标补丁，以及目标补丁的每个组件如何起作用。这些 研究将扩展到Lgl、Discs Large、Scribble和Par蛋白在细胞发育中的功能。 表面极性我们将研究微管和隔蛋白在细胞间粘附中的作用 以及囊泡递送至基底外侧质膜靶向贴片。我们将定义septins的作用， 细胞-细胞粘附，并在指定Glu-MT组织和囊泡运输到血浆 膜，并研究septins/MT和靶向补丁之间的蛋白质-蛋白质相互作用。 总之，这些研究将提供信号和结构蛋白的全面分子图像 参与细胞-细胞粘附和细胞表面极性发展的网络。

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 integrate innovative experimental approaches to address these problems: structural analysis of proteins and protein complexes, high resolution live cell imaging of proteins and biosensors, biochemical analysis of protein complex assembly and function in cells, and novel in vitro reconstitution assays. During this funding period, we defined stages in cell-cell adhesion and the mechanism involved, and determined how plasma membrane proteins are targeted to and organized in the forming basolateral membrane domain upon cell-cell adhesion. We will build upon these results in proposed studies. We will define roles of alpha-catenin and p120 in regulating cell-cell adhesion and membrane dynamics. We will examine the effects of sequestering endogenous alpha-catenin and p120 to the plasma membrane independently of E-cadherin, and to mitochondria using location-specific tags on cell migration, actin dynamics and cell-cell adhesion using live cell imaging, FRET, biosensors and small molecule inhibitors. We will use affinity purification and MALDI/TOF Mass Spectrometry to define proteins that regulate alpha-catenin and p120 functions and association with the actin cytoskeleton. We will dissect mechanisms regulating sorting of vesicles at the basolateral plasma membrane targeting patch. We will use innovative reconstitution assays on membrane patches bound to an E-cadherin substrate to visualize how transport vesicles are delivered to the targeting patch, and how each of the componentsof the targetign patch function. These studies will be extended to functions of Lgl, Discs Large, Scribble and Par proteins in the development of cell surface polarity. We will investigate the role of microtubule (MT) and septin cytoskeleton in cell-cell adhesion and vesicle delivery to the basolateral plasma membrane targeting patch. We will define roles of septins in cell-cell adhesion and in specifying Glu-MT organization and vesicle trafficking towards the plasma membrane, and investigate protein-protein interactions between septins/MTs and the targeting patch. Together, these studies will provide a comprehensive molecular picture of the signaling and structural protein networks involved in cell-cell adhesion and the development of cell surface polarity.