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通过 专门的钙粘附素介导的细胞-细胞接触，并将蛋白质定位到不同功能的血浆 膜结构域。我们整合了创新的实验方法来解决这些问题：结构 蛋白质和蛋白质复合体的分析，蛋白质和生物传感器的高分辨率活细胞成像， 蛋白质复合体在细胞内组装和功能的生化分析及新的体外重组 化验。在这一资助期间，我们定义了细胞-细胞黏附的阶段和涉及的机制，以及 确定质膜蛋白是如何定位和组织在形成基侧的 膜结构域对细胞-细胞的黏附。我们将在拟议的研究中以这些结果为基础。我们会 明确α-连环蛋白和p120在调节细胞-细胞黏附和膜动力学中的作用。我们会 检测内源性α-连环蛋白和p120对质膜的影响 不依赖于E-钙粘蛋白，并使用细胞迁移的特定位置标记与线粒体结合，肌动蛋白 使用活细胞成像、FRET、生物传感器和小分子抑制剂的动力学和细胞-细胞黏附。我们 将使用亲和纯化和MALDI/TOF质谱学来定义调节α-连环蛋白的蛋白质 P120的功能及其与肌动蛋白细胞骨架的关系。我们将剖析调控机制 基侧质膜靶向贴片上囊泡的分选。我们将使用创新的重组 结合在E-钙粘附素底物上的膜片上的分析以可视化运输小泡是如何 递送到目标贴片，以及目标贴片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Septin细胞骨架在细胞-细胞黏附中的作用 并将囊泡输送到基侧质膜靶向贴片。我们将定义Septins在 细胞-细胞黏附与谷氨酸-金属硫蛋白组织和囊泡向血浆的转运 并研究Septins/MTS与靶向贴片之间的蛋白质-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景。 网络参与了细胞间的黏附和细胞表面极性的发展。

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.