Cystoskeletal Remodeling During T Cell Activation

T细胞与APC的相互作用诱导免疫过程中肌动蛋白细胞骨架的戏剧性重构 Synapse。这一过程对于T细胞的激活是绝对必要的，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HS1突变引起的。这项研究将 测试以下中心假设：HS1、WIP和WASP协同工作以协调肌动蛋白 免疫突触的动力学，因此每个成分在调节肌动蛋白方面扮演着不同的角色 建筑，同时调节其他人的功能。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.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协同推动肌动蛋白聚合，而HS1则稳定肌动蛋白细丝。此外，肌动蛋白- 从免疫突触形成到增殖和细胞因子产生的依赖性T细胞反应 将会被评估。在目标3中，我们将通过表达突变体来分析HS1的结构和功能。 HS1缺陷的T细胞和检测肌动蛋白反应和其他方面-T细胞的激活。除了……之外 测试结构域和蛋白质相互作用基序，我们将测试狼疮相关插入的功能 多态。最后，我们将分析HS1酪氨酸的磷酸化，并测试其生理效应 非磷酸化的点突变体。综上所述，这些研究将把这一领域推向分子 了解免疫突触的肌动蛋白动力学是如何控制的，以及这一过程是如何进行的 有助于T细胞的激活。

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.