Cell Surface Controll of Cellular Physiology

描述（由申请人提供）：本研究的长期目标是了解vinculin如何调节细胞粘附和运动。肠菊素对小鼠胚胎发育和调节细胞外基质基质上的粘附和运动是必不可少的。目前的证据支持一个模型，即在局灶黏附中，血管蛋白的作用是在头部区域与talin-integrin复合物和尾部区域与肌动蛋白丝提供双重功能的相互作用。在纯化的血管蛋白中，肌动蛋白和talin结合位点被血管蛋白头部（Vh）和尾部（Vt）结构域的分子内相互作用所掩盖。通过调节头尾相互作用来暴露或隐藏配体结合位点，被认为是血管蛋白调节膜蛋白附着于细胞骨架以控制细胞粘附和运动的机制。目前提议的一个目标是在活细胞中测试这个模型。为此，研究人员开发了两个缺乏分子内头尾相互作用的突变体，并构建了荧光共振能量转移（FRET）探针，该探针可以报告血管蛋白的封闭、开放和肌动蛋白结合构象。当在细胞中表达时，突变体具有定位和动态表型，这与它们稳定局灶粘附斑块蛋白子集的解释一致，这些斑块蛋白代表野生型白细胞素进行的过程中的中间产物。为了阐明这一过程，并测试在病毒蛋白的功能中是否需要调节分子内头尾相互作用，我们提出如下计划。

DESCRIPTION (provided by applicant): The Iong-term goal of this research is to understand how vinculin regulates cell adhesion and motility. inculin is essential for embryonic development in mice and for regulation of adhesion and motility on xtracellular matrix substrates. Current evidence supports a model in which vinculin's role in a focal adhesion is to provide a bifunctional interaction with talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. In purified vinculin the actin and talin binding sites are masked by an intramolecular interaction between the head (Vh) and tail (Vt) domains of vinculin. Regulation of the head/tail interaction to expose or hide the ligand binding sites is hypothesized to be the mechanism by which vinculin regulates the attachment of membrane proteins to cytoskeleton to control adhesion and motility in cells. A goal of the present proposal is to test this model in living cells. For this purpose, two mutants deficient in intramolecular head/tail interaction were developed and a fluorescence resonance energy transfer (FRET) probe that reports on the closed, open, and actin-bound conformations of vinculin have been constructed. When expressed in cells the mutants have a localization and dynamic phenotype consistent with the interpretation that they stabilize a subset of focal adhesion plaque proteins representing an intermediate in a process carried out by wild type vinculin. To elucidate this process and to test the requirement for regulation of the intramolecular head/tail interaction in the function of vinculin, we propose the following plan. Aim 1, Use the loss of function mutants to identify the requirement for regulated head/tail interaction in rescue of adhesion and motility phenotypes in vinculin null cells. Aim 2, test the hypothesis that the phenotype generated by the constitutive talin-binding conformation of vinculin represents a stalled intermediate in endocytosis of the a5bl integrin receptor for FN. Aim 3, use the conformation-sensitive vinculin FRET probe to determine when and where in a cell vinculin undergoes conformational regulation and to test the hypothesis that activation of vinculin responds to changes in mechanical forces and contractility. Aim 4, test the hypothesis that vinculin's effects on cell adhesion and motility are mediated through Rac, RhoA, or Cdc42 pathways.