Cell Surface Control of Cellular Physiology

这项研究的目的是了解纽蛋白如何在细胞上发挥类似肿瘤抑制物的作用。 能动性。纽蛋白是调节跨膜的细胞和组织结构的重要组成部分。 细胞内细胞骨架和细胞外基质之间的连接。当前型号 提示纽蛋白通过加强这些连接来刺激黏附和抑制运动 通过纽蛋白头部结构域的Talin-整合素复合体与肌动蛋白的双功能相互作用 花丝在它的尾域。因为纯化的纽蛋白是自动抑制的，所以头/尾的调节 揭示或隐藏配体结合位点的相互作用(HTI)被认为是 纽蛋白调节膜蛋白与细胞骨架的附着，从而控制黏附和运动。 这项提议的一个目标是在活细胞中测试这一模型。我们发现了两个福斯特共振 能量转移(FRET)探针报告纽蛋白的活化和肌动蛋白结合构象 一系列突变体，分子内HTI的强度逐渐降低，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探针测试组合存在冗余机制的假说 纽蛋白的激活。塔林和肌动蛋白细丝在一起可以激活纽蛋白；我们将测试 其他纽蛋白配体，以及定义纽蛋白信号和定位信号的PIP2 激活。2)。使用头/尾相互作用突变体和Talin结合突变体来测试 纽蛋白激活调节整合素、Talin、纽蛋白和肌动蛋白之间相互作用的假说 它控制着细胞的黏附、运动和跨细胞膜的力的传递。3)。使用 构象敏感的纽蛋白FRET探针验证纽蛋白激活的假说 对活细胞中的机械力和伸缩性做出反应。已经建立了与 Sharon Campbell为了便于分析PIP2在纽蛋白(Aim1的一部分)的组合激活中的作用， 用安德烈斯·加西亚测量细胞粘附力，用苏珊·冈斯特测量张力 在平滑肌组织中的发育(AIM2的部分)。我们预计这些研究将提供 与蛋白质如何构建结构以 通过膜传递力量，特别是到纽蛋白的分子机制 抑制细胞迁移。项目说明： 细胞异常黏附和迁移是致死人的癌症的特征。这个项目的目的是找到 了解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。通过学习纽球蛋白是如何工作的， 我们可以更好地了解如何控制癌症的异常细胞行为。

The goal of this research is to understand how vinculin exerts a tumor-supressor-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 togetther can activate vinculin; we will test the roles of other vinculin ligands, as well as PIP2 to define the signalling 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. Project Narrative: 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.