Cytoskeletal Remodeling During T Cell Activation

描述（由申请人提供）：T细胞与APC的相互作用诱导免疫突触处肌动蛋白细胞骨架的显著重塑。这个过程是T细胞活化所必需的，严重的免疫缺陷或自身免疫性疾病是由肌动蛋白调节分子如WASP和HS 1突变引起的。这项研究将测试的中心假设，HS 1，ESTA，和WASP的功能协调的方式，以协调肌动蛋白的动态在免疫突触，这样每个组件在调节肌动蛋白的结构中发挥着独特的作用，同时调制其他的功能。在目标1中，我们将询问这些蛋白质直接相互作用的程度，并试图将HS 1置于涉及WASP，WASP，Itk和Vav的肌动蛋白调节复合物的背景下。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，靶向和构象，并分析活T细胞中荧光标记蛋白质的运动。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在T细胞活化过程中进行功能分析。将进行肌动蛋白动力学和皮层肌动蛋白EM的视频分析，以测试WASP和WASP合作驱动肌动蛋白聚合，而HS 1起稳定肌动蛋白丝的作用。此外，将评估从免疫突触形成到增殖和细胞因子产生的肌动蛋白依赖性T细胞应答。在目标3中，我们将通过在HS 1缺陷的T细胞中表达突变体并测定肌动蛋白反应和T细胞活化的其他方面来进行HS 1的结构-功能分析。除了测试结构域和蛋白质-蛋白质相互作用基序，我们将测试狼疮连锁插入多态性的功能。最后，我们将分析HS 1酪氨酸磷酸化，并测试非磷酸化点突变体的生理效应。总之，这些研究将推动该领域的分子理解如何控制免疫突触的肌动蛋白动力学，以及这个过程如何有助于T细胞活化。

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.