Mechanisms of vinculin activation and force transmission

 描述(由申请人提供)：本提案的重点是纽蛋白，这是一种细胞骨架蛋白，是局部粘连和粘连连接的重要组成部分。纽蛋白以一种自身抑制的构象存在，激活后作为一种支架来调节细胞活动，导致细胞迁移、细胞存活和胚胎发生。纽蛋白缺失细胞显示出致瘤特性，纽蛋白突变或缺失与心脏病有关。虽然纽蛋白与肌动蛋白和磷脂酰肌醇4，5-二磷酸(PIP2)结合，但我们还不清楚这些相互作用的性质或它们在调节纽蛋白功能中的确切作用。特别是，纽蛋白和肌动蛋白之间的相互作用在连接过程中起着关键作用。 细胞骨架的跨膜受体，这反过来对控制细胞的形态、力传递和运动是重要的。纽蛋白与F-肌动蛋白结合并发生构象变化，导致形成肌动蛋白细丝束所必需的隐蔽二聚体，但发生的构象变化和形成的二聚体尚不清楚。目前还不清楚纽蛋白如何识别PIP2，如何插入膜，以及如何受这种相互作用的调节。我们提出了高度集成的计算和实验方法来生成和测试这些重要相互作用的模型，并评估它们在体外和细胞中对纽蛋白功能的意义。这将通过在体外产生和鉴定在肌动蛋白结合、肌动蛋白诱导的纽蛋白二聚体形成和PIP2结合方面存在特定缺陷的纽蛋白变体，然后在纽蛋白缺失细胞中表达全长野生型蛋白和突变体来实现。这些相互作用在调节纽蛋白的激活状态以及纽蛋白的力响应和传递特性方面的作用将在亚细胞和全细胞水平上进行探讨。

 DESCRIPTION (provided by applicant): This proposal is focused on vinculin, a cytoskeletal protein that is a prominent component of focal adhesions and adherens junctions. Vinculin exists in an autoinhibited conformation and upon activation, functions as a scaffold to regulate cellular events resulting in cell migration, cell survival and embryogenesis. Vinculin null cells display tumorigenic properties and mutation or loss of vinculin is associated with cardiac disease. Although vinculin binds actin and phosphatidylinositol 4,5- bisphosphate (PIP2), we do not understand the nature of these interactions or their precise role in regulating vinculin function. In particular, the interaction between vinculin and actin plays a pivotal role in linking transmembrane receptors to the cytoskeleton, which, in turn, is important for controlling cellular cell morphology, force transmission and motility. Vinculin binds to F-actin and undergoes a conformational change that induces formation of a cryptic dimer necessary for actin filament bundling, but the conformation change that occurs and dimer that is formed is unknown. It is also unclear how vinculin recognizes PIP2, inserts into membranes and is regulated by this interaction. We propose highly integrated computational and experimental approaches to generate and test models for these important interactions and assess their significance in vinculin function both in vitro and in cells. This will be accomplished by generating and characterizing vinculin variants with specific defects in actin binding, actin-induced vinculin dimer formation and PIP2 association in vitro, and then expressing the full length wild type protein and mutants in vinculin null cells. The role of these interactions in regulating the activation state of vinculin as well as vinculin's force response and transmission properties will be probed at both the sub-cellular and whole cell level.