Mechanisms of vinculin activation and force transmission

 描述（由申请方提供）：该提案重点关注黏着斑蛋白，黏着斑蛋白是一种细胞骨架蛋白，是局灶性粘连和粘连连接的重要组成部分。白藜芦醇以自抑制构象存在，并且在活化时，作为支架发挥作用以调节细胞事件，导致细胞迁移、细胞存活和胚胎发生。黏着斑蛋白缺失细胞显示出致瘤特性，黏着斑蛋白的突变或缺失与心脏疾病相关。虽然黏着斑蛋白结合肌动蛋白和磷脂酰肌醇4，5-二磷酸（PIP 2），我们不了解这些相互作用的性质或它们在调节黏着斑蛋白功能的确切作用。特别是黏着斑蛋白和肌动蛋白之间的相互作用在连接 细胞骨架的跨膜受体，这反过来又是重要的控制细胞的细胞形态，力传递和运动。纤维蛋白与F-肌动蛋白结合，并发生构象变化，诱导形成肌动蛋白丝成束所需的隐蔽二聚体，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PIP 2、插入细胞膜并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的相互作用的模型，并评估其在体外和细胞中粘着斑蛋白功能的意义。这将通过在体外产生和表征在肌动蛋白结合、肌动蛋白诱导的粘着斑蛋白二聚体形成和PIP 2缔合中具有特异性缺陷的粘着斑蛋白变体，然后在粘着斑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黏着斑蛋白的激活状态以及黏着斑蛋白的力响应和传输特性的作用将在亚细胞和全细胞水平上进行探索。

 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.