Cystoskeletal Remodeling During T Cell Activation

T 细胞激活过程中的囊骨骼重塑

• 批准号: 7333282
• 负责人: Janis K. Burkhardt
• 金额: $ 40.4万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333282
• 关键词: Actins; Affect; Agonist; Architecture; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Boxing; Calcium; Cells; Chromosome Pairing; Complex; Condition; Cytoskeleton; Defect; Dose; EEF1A2 gene; Event; Exhibits; Gene Expression; Genetic Polymorphism; Goals; Immune; Immunologic Deficiency Syndromes; In Vitro; Individual; Interleukin-2; Knock-out; Learning; Life; Link; Lupus; Membrane Microdomains; Microfilaments; Microscopy; Molecular; Molecular Conformation; Movement; Mus; Mutation; Pathway interactions; Peptides; Phenotype; Phosphorylation; Physiological; Play; Process; Production; Protein Binding Domain; Proteins; Range; Regulation; Resolution; Role; Signal Transduction; Structure; Synapses; T-Cell Activation; T-Lymphocyte; Testing; Time; Tyrosine Phosphorylation; Variant; YWHAQ gene; adapter protein; cell type; comparative; cytokine; genetic regulatory protein; in vitro Assay; in vivo; mutant; novel strategies; polymerization; research study; response; small hairpin RNA; synaptogenesis

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.

T细胞与APCs的相互作用诱导免疫系统中肌动蛋白细胞骨架的急剧重塑 突触这个过程是T细胞活化和严重免疫缺陷或 自身免疫性疾病由肌动蛋白调节分子如WASP和HS 1的突变引起。本研究将 检验HS 1、WASP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，使得每个组件在调节肌动蛋白中发挥独特的作用 架构，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HS 1置于涉及肌动蛋白，WASP， Itk和Vav。我们将探讨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细胞活化期间进行功能分析。 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 β 1协同驱动肌动蛋白聚合，而HS 1则起稳定肌动蛋白丝的作用。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细胞应答 将被评估。在目标3中，我们将通过在大肠杆菌中表达突变体来进行HS 1的结构-功能分析。 HS 1缺陷型T细胞和测定肌动蛋白反应和T细胞活化的其他方面。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 多态最后，我们将分析HS 1酪氨酸磷酸化，并测试生理效应， 非磷酸化点突变体。总之，这些研究将推动该领域向分子水平发展。 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控制的。 有助于T细胞活化。