Cell Surface Control of Cellular Physiology

描述(申请人提供)：这项研究的目标是了解纽蛋白如何在细胞运动中发挥肿瘤抑制因子样的作用。纽蛋白是细胞和组织结构中的重要组成部分，它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，纽蛋白通过在纽蛋白头部区域的talin-整合素复合体和其尾部区域的肌动蛋白细丝之间的双功能相互作用来加强这些连接，从而刺激黏附和抑制运动。由于纯化的纽蛋白是自身抑制的，调节头部/尾巴相互作用(HTI)以暴露或隐藏配体结合部位被认为是纽蛋白调节膜蛋白与细胞骨架的附着以控制黏附和运动的机制。这项提议的一个目标是在活细胞中测试这一模型。我们开发了两个Forster共振能量转移(FRET)探针，报告了vinculin的激活和肌动蛋白结合构象，一系列分子内HTI强度逐渐降低的突变体，以及一个talin结合突变体。我们建议应用这些工具来解决以下具体目标：1)。使用纽蛋白FRET探针来检验纽蛋白组合激活存在冗余机制的假设。Talin和肌动蛋白细丝一起可以激活vinculin；我们将测试其他vinculin配体以及PIP2的作用，以确定vinculin激活的信号和定位信号。2)。使用头/尾相互作用突变体和talin结合突变体来检验这一假设，即vinculin的激活调节整合素、talin、vinculin和肌动蛋白之间的相互作用，从而控制细胞的黏附、运动和跨细胞膜的力的传递。3)。使用构象敏感的纽蛋白FRET探针来测试纽蛋白的激活对活细胞中的机械力和收缩能力做出反应的假设。已经与Sharon Campbell建立了合作关系，以促进PIP2在vinculin(Aim1的一部分)的组合激活中的分析，与Andres Garcia合作来测量细胞中的粘附力，并与Susan Gunst合作来测量平滑肌组织(AIM2的一部分)的张力发展。我们预计，这些研究将提供大量新的信息，涉及蛋白质如何构建结构以跨膜传递力的一般问题，特别是纽蛋白抑制细胞迁移的分子机制。 公共卫生评论：细胞异常黏附和迁移是致死人的癌症的特征。该项目旨在找出细胞迁移和黏附是如何由一种名为纽蛋白的蛋白质调节的。通过了解纽蛋白的工作原理，我们可以更好地了解如何控制癌症的异常细胞行为。

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.