Cell Surface Control of Cellular Physiology

描述（由申请人提供）：本研究的目的是了解vinculin如何对细胞运动施加肿瘤抑制因子样作用。血管蛋白是细胞和组织结构的重要组成部分，介导细胞内细胞骨架和细胞外基质之间的跨膜连接。目前的模型表明，通过在血管蛋白头部区域的talin-integrin复合物和尾部区域的肌动蛋白丝之间的双功能相互作用，血管蛋白刺激黏附并抑制运动，从而加强了这些连接。由于纯化的血管蛋白具有自身抑制作用，因此可以假设，通过调节头尾相互作用（HTI）来暴露或隐藏配体结合位点是血管蛋白调节膜蛋白附着于细胞骨架以控制粘附和运动的机制。这个提议的目标是在活细胞中测试这个模型。我们开发了两种福斯特共振能量转移（FRET）探针，报告了激活和肌动蛋白结合的血管蛋白构象，一系列分子内HTI强度逐渐降低的突变体，以及talin结合突变体。我们建议运用这些工具来实现以下具体目标：1)。使用病毒蛋白FRET探针来测试病毒蛋白组合激活存在冗余机制的假设。Talin和actin丝一起可以激活血管蛋白；我们将测试其他血毒蛋白配体的作用，以及PIP2来定义血毒蛋白激活的信号传导和定位线索。2）. 使用头/尾相互作用突变体和talin结合突变体来验证以下假设：整合素、talin、vinculin和actin之间的相互作用通过激活来调节，从而控制细胞粘附、运动和跨细胞膜的力的转导。3)。使用构象敏感的血管蛋白FRET探针来测试血管蛋白的激活响应活细胞的机械力和收缩性的假设。与Sharon Campbell合作，促进了PIP2在血管蛋白组合激活（Aim1的一部分）中的分析，与Andres Garcia合作，测量细胞中的粘附力，与Susan Gunst合作，测量平滑肌组织（Aim2的一部分）中的张力发展。我们预计这些研究将为蛋白质如何构建结构以跨膜传递力的一般问题提供实质性的新信息，特别是血管素抑制细胞迁移的分子机制。

DESCRIPTION (provided by applicant): The goal of this research is to understand how vinculin exerts a tumor-suppressor-like effect on cell motility. Vinculin is a prominent component of cell and tissue structures that mediate transmembrane connections between the intracellular cytoskeleton and the extracellular matrix. Current models suggest that vinculin stimulates adhesion and inhibits motility by strengthening these connections through bifunctional interactions between talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. Because purified vinculin is autoinhibited, regulation of the head/tail interaction (HTI) to expose or hide ligand binding sites is hypothesized to be the mechanism by which vinculin regulates attachment of membrane proteins to cytoskeleton to control adhesion and motility. A goal of this proposal is to test this model in living cells. We developed two Forster resonance energy transfer (FRET) probes that report on activated and actin-binding conformations of vinculin, a series of mutants having a graded reduction in the strength of the intramolecular HTI, and a talin- binding mutant. We propose to apply these tools to address the following specific aims: 1). Use vinculin FRET probes to test the hypothesis that there are redundant mechanisms for combinatorial activation of vinculin. Talin and actin filaments together can activate vinculin; we will test the roles of other vinculin ligands, as well as PIP2 to define the signaling and localization cues for vinculin activation. 2). Use the head/tail interaction mutants and the talin-binding mutant to test the hypothesis that activation of vinculin regulates interactions between integrin, talin, vinculin, and actin that control cell adhesion, motility, and transduction of force across the cell membrane. 3). Use the conformation-sensitive vinculin FRET probes to test the hypothesis that activation of vinculin responds to mechanical forces and contractility in living cells. Collaborations have been set up with Sharon Campbell to facilitate analyses of PIP2 in combinatorial activation of vinculin (part of Aim1), with Andres Garcia to measure adhesive force in cells, and with Susan Gunst to measure tension development in smooth muscle tissue (parts of Aim2). We anticipate that these studies will provide substantial new information relevant to the general question of how proteins build structures to transmit force across a membrane, and specifically to the molecular mechanism by which vinculin suppresses cell migration. PUBLIC HEALTH REVELANCE: Abnormal cell adhesion and migration are characteristic of cancers that kill people. This project aims to find out how cell migration and adhesion are regulated by a protein called vinculin. By learning how vinculin works, we can better understand how to control the abnormal cell behaviors of cancer.