Cytoskeletal Remodeling During T Cell Activation

描述(由申请人提供)：T细胞与APC的相互作用导致免疫突触肌动蛋白细胞骨架的戏剧性重塑。这一过程是T细胞激活所必需的，严重的免疫缺陷或自身免疫性疾病是由于肌动蛋白调节分子如WASP和HS1的突变造成的。这项研究将检验这一中心假设，即HS1、WIP和WASP以一种协调的方式发挥作用，以协调免疫突触的肌动蛋白动力学，从而使每个组件在调节肌动蛋白结构方面发挥不同的作用，同时调节其他组件的功能。在目标1中，我们将询问这些蛋白质在多大程度上直接相互作用，并寻求将HS1置于涉及WIP、WASP、ITK和Vav的肌动蛋白调控复合体的背景下。我们将询问这些肌动蛋白调节分子如何影响彼此的稳定性、靶向性和构象，并分析荧光标记蛋白在活T细胞中的运动。在目标2中，我们将使用缺乏单个肌动蛋白调节蛋白的细胞进行T细胞激活过程中的功能分析。将进行肌动蛋白动力学和皮质肌动蛋白EM的视频分析，以测试WASP和WIP合作推动肌动蛋白聚合，而HS1作用于稳定肌动蛋白细丝的想法。此外，还将评估肌动蛋白依赖的T细胞反应，范围从免疫突触形成到增殖和细胞因子产生。在目标3中，我们将通过在HS1缺失的T细胞中表达突变体并检测肌动蛋白反应等方面来分析HS1的结构和功能。除了测试结构域和蛋白质相互作用基序外，我们还将测试狼疮连锁插入多态的功能。最后，我们将分析HS1酪氨酸的磷酸化，并测试非磷酸化点突变的生理效应。综上所述，这些研究将推动该领域朝着分子理解的方向发展，即免疫突触的肌动蛋白动力学是如何控制的，以及这个过程如何有助于T细胞的激活。

DESCRIPTION (provided by applicant): Interaction of T cells with APCs induces dramatic remodeling of the actin cytoskeleton at the immune synapse. This process is absolutely required for T cell activation, and severe immunodeficiency or autoimmune disease result from mutations in actin regulatory molecules like WASP and HS1. This study will test the central hypothesis that HS1, WIP, and WASP function in a coordinate fashion to orchestrate actin dynamics at the immune synapse, such that each component plays a distinct role in regulating actin architecture, while modulating the function of the others. In Aim 1, we will ask to what extent these proteins interact directly, and seek to place HS1 in the context of the actin regulatory complex involving WIP, WASP, Itk and Vav. We will ask how these actin regulatory molecules affect one another's stability, targeting and conformation, and analyze movements of fluorescently tagged proteins in living T cells. In Aim 2, we will conduct functional analyses during T cell activation using cells lacking individual actin regulatory proteins. Video analysis of actin dynamics and EM of cortical actin will be performed to test the idea that WASP and WIP collaborate to drive actin polymerization, while HS1 acts to stabilize actin filaments. In addition, actin- dependent T cell responses ranging from immune synapse formation to proliferation and cytokine production will be assessed. In Aim 3, we will conduct structure-function analysis of HS1 by expressing mutants in HS1-deficient T cells and assaying actin responses and other aspects-of T cell activation. In addition to testing domains and protein-protein interaction motifs, we will test the function of a lupus-linked insertion polymorphism. Finally, we will analyze HS1 tyrosine phosphorylation, and test the physiological effects of non-phosphorylatable point mutants. Taken together, these studies will advance the field toward a molecular understanding of how actin dynamics at the immune synapse are controlled, and how this process contributes to T cell activation.