Regulation of Integrin Signaling by the Membrane Skeleton

我们在血小板上的研究表明，α-IIb-β3与未受刺激的细胞中的细胞骨架相关联，这种相互作用调节其传递信号的能力。配体结合导致整合素聚集并与细胞骨架蛋白形成复合体：这些复合体被称为焦点复合体和焦点粘连，是激活诱导肌动蛋白细丝聚合和重组的分子所必需的。这些复合体本身是由CdC42、RAC(焦点复合体)和RhoA(焦点粘连)诱导的，可能是通过激活交换因子(如VaV)和效应器(如WASP)。我们工作的总体目标是了解含有β3整合素的整合素与细胞骨架蛋白的结合如何调节CDC42/RAC/Rho激活的配体结合和上游事件。在之前的工作中，我们发现Skelumin是一种细胞骨架蛋白，在完整的细胞中与含有β1和β3的整合素相互作用。我们发现Mu-calain是一种信号分子 未受刺激的血小板中的膜骨架，并表明它是通过在血小板中的α-IIb-β3或在培养细胞中通过含有β1和β3的整合素的信号而被激活的。我们对calain功能的研究发现了一种新型的整合素簇，它是在RAC激活之前由calain诱导的。这些簇不同于焦点复合体和焦点粘连，因为它们含有骨架蛋白、钙蛋白裂解的光影蛋白和β3-整合素，以及活性的粘附素。显性-负性RAC在整合素簇中积累，并阻止进一步扩散。这些发现导致我们提出了一个模型，在该模型中，整合素簇的形成需要骨架蛋白和钙蛋白酶，整合素簇为信号分子的招募提供焦点，信号分子反过来启动CDC42/RAC/RhoA级联激活，从而诱导贴壁细胞中细胞骨架重组的进展。在特定的目标1中，我们将测试这一假设，即整合素亲和力调节、聚集、亲和力调节或导致钙蛋白和CDC42/RAC激活的信号的传输都需要骨架蛋白-整合素相互作用。在特定的目标2中，我们将研究Calain诱导的VaV和WASP切割在启动整合素簇中的CDC42/Rac活性中的作用。在具体目标3中，我们将使用延时视频显微镜来跟踪β3整合素、骨架蛋白、钙蛋白酶、血管紧张素转换酶和WASP的运动。我们将使用FRET技术来确定RAC的激活位置。这些研究将使我们能够直接测试骨架蛋白和钙蛋白酶参与整合素簇形成的想法，整合素簇是RAC激活的部位。他们还将提供不同类型的整合素簇的空间和时间形成的洞察力，以及活细胞中整合素诱导的信号。

Our work on platelets suggests that alpha-IIb-beta3 associates with the cytoskeleton in unstimulated cells and that this interaction regulates its ability to transmit signals. Ligand binding causes integrins to cluster and form complexes with cytoskeletal proteins: these complexes, known as focal complexes and focal adhesions, are necessary for activation of molecules that induce actin filament polymerization and reorganization. The complexes themselves are induced by cdc42, Rac (focal complexes) and RhoA (focal adhesions), presumably by activation of exchange factors such as vav and effectors such as WASP. The overall goal of our work is to understand how association of beta3-containing integrins with cytoskeletal proteins regulates ligand binding and events upstream of activation of cdc42/Rac/Rho. In previous work, we identified skelemin as a cytoskeletal protein that interacts with beta1- and beta3-containing integrins in intact cells. We identified mu-calpain as a signaling molecule associated with the membrane skeleton in unstimulated platelets and showed that it was activated by signaling across alpha-IIb-beta3 in platelets or across beta1- and beta3-containing integrins in cultured cells. Our studies on the function of calpain identified a new type of integrin cluster that is induced by calpain, prior to Rac activation. The clusters are distinct from focal complexes and focal adhesions in that they contain skelemin, calpain-cleaved spectrin and beta3-integrin, and active mu-calpain. Dominant-negative Rac accumulates in the integrin clusters and prevents further spreading. These findings have led us to propose a model in which skelemin and calpain are required for formation of integrin clusters that provide foci for recruitment of signaling molecules that in turn initiate activation of the cdc42/Rac/RhoA cascades that induce progression of cytoskeletal reorganizations in adherent cells. In Specific Aim 1, we will test the hypothesis that skelemin-integrin interactions are required for integrin affinity modulation, clustering, avidity modulation, or transmission of signals leading to calpain and cdc42/Rac activation. In Specific Aim 2, we will investigate the role of calpain-induced vav and WASP cleavage in initiation of cdc42/Rac activity in integrin clusters. In Specific Aim 3, we will use time-lapse video microscopy to track the movement of beta3-integrin, skelemin, calpain, vav, and WASP. We will use FRET technology to identify sites of Rac activation. These studies will allow us to directly test the idea that skelemin and calpain are involved in formation of integrin clusters that are the sites of Rac activation. They will also provide insights into spatial and temporal formation of different types of integrin clusters and integrin-induced signaling in living cells.