Cystoskeletal Remodeling During T Cell Activation

T 细胞与 APC 的相互作用诱导免疫系统中肌动蛋白细胞骨架的显着重塑 突触。这个过程对于 T 细胞激活、严重免疫缺陷或严重免疫缺陷是绝对必需的。 自身免疫性疾病是由 WASP 和 HS1 等肌动蛋白调节分子突变引起的。这项研究将 检验 HS1、WIP 和 WASP 以协调方式协调肌动蛋白的中心假设 免疫突触的动力学，使得每个成分在调节肌动蛋白中发挥独特的作用 建筑，同时调节其他建筑的功能。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，并分析活 T 细胞中荧光标记蛋白的运动。在目标 2 中，我们将 使用缺乏单独肌动蛋白调节蛋白的细胞在 T 细胞激活过程中进行功能分析。 将进行肌动蛋白动力学和皮质肌动蛋白电镜的视频分析，以检验 WASP 和 WIP 协同驱动肌动蛋白聚合，而 HS1 则起到稳定肌动蛋白丝的作用。此外，肌动蛋白 依赖性 T 细胞反应，范围从免疫突触形成到增殖和细胞因子产生 将被评估。在目标 3 中，我们将通过表达突变体来进行 HS1 的结构功能分析 HS1 缺陷 T 细胞并测定肌动蛋白反应和 T 细胞激活的其他方面。除了 测试结构域和蛋白质-蛋白质相互作用基序，我们将测试狼疮相关插入的功能 多态性。最后，我们将分析HS1酪氨酸磷酸化，并测试其生理效应 不可磷酸化的点突变体。总而言之，这些研究将推动该领域向分子领域迈进。 了解免疫突触的肌动蛋白动力学是如何控制的，以及这个过程是如何进行的 有助于 T 细胞活化。

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.