Cell Surface Control of Cellular Physiology

这项研究的目的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。 纽蛋白是调节跨膜连接的细胞和组织结构的重要组成部分。 在细胞内细胞骨架和细胞外基质之间。目前的模型表明，纽蛋白 通过双功能相互作用加强这些连接，从而刺激黏附和抑制运动 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。因为 纯化的纽蛋白是自动抑制的，调节头/尾相互作用(HTI)以暴露或隐藏配体结合 位点被认为是纽蛋白调节膜蛋白与 控制黏附和运动的细胞骨架。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，报告了激活和肌动蛋白结合 纽蛋白的构象，一系列分子内强度逐渐降低的突变体 HTI，和一个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探针检验组合存在冗余机制的假设 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PIP2和钙离子来定义纽蛋白的信号和定位信号 激活。2)使用头/尾相互作用突变体和Talin结合突变体来检验假设 纽蛋白的激活调节整合素、talin、vinculin和控制细胞的肌动蛋白之间的相互作用 通过细胞膜提供粘附性、运动性和转导。3)使用构象敏感的 纽蛋白FRET探针测试纽蛋白激活对机械力和 活细胞的收缩能力。已经与莎伦·坎贝尔建立了合作关系，以促进对 PIP2在联合激活纽蛋白(Aim1的一部分)中，与Andres Garcia测量粘附力 并与Susan Gunst一起测量平滑肌组织(AIM2的一部分)的张力发展。我们 预期这些研究将提供与以下一般性问题有关的大量新信息： 蛋白质构建结构，将力传递到膜上。

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 and Ca2+ 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 offeree 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.