Defining STIM1 function at the Immunological Synapse

定义免疫突触处的 STIM1 功能

• 批准号: 10369054
• 负责人: Jonathan A Soboloff
• 金额: $ 39.63万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10369054
• 关键词: Adoptive Transfer; Affect; Antigen-Presenting Cells; Antigens; Autoimmune Diseases; Automobile Driving; Bone Marrow; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Ca(2+)-Transporting ATPase; Cell membrane; Cells; Cellular biology; Charge; Complex; Confocal Microscopy; Effector Cell; Endoplasmic Reticulum; Event; Exhibits; Failure; Family; Fluorescence Microscopy; GTP Binding; Generations; Guanosine Triphosphate Phosphohydrolases; Immune response; Immunology; Investigation; Link; Mediating; Membrane Potentials; Metabolic; Metabolism; Mitochondria; Mutation; Nature; Oxygen Consumption; Phosphatidylinositols; Phospholipase C; Physiological; Process; Production; Proteins; Protocols documentation; Reporting; Rest; Role; STIM1 gene; Series; Signal Transduction; Signaling Protein; Site; Site-Directed Mutagenesis; Superoxides; T cell differentiation; T-Cell Activation; T-Lymphocyte; Translations; Work; adaptive immunity; base; cell transformation; confocal imaging; cytokine; defined contribution; design; effector T cell; experimental study; immunological synapse; immunological synapse formation; insight; mutant; novel; sensor; tool; tumor immunology; ward

T cell activation is initiated by direct contact between T cells and antigen-presenting cells (APCs), leading to polarization of the T cell towards the APC and the formation of the immunological synapse (IS). It is now generally accepted that Ca2+ signals are required for T cell activation. Further, reorganization of Ca2+ signaling proteins during IS formation has been widely reported, although the underlying mechanisms driving this event are poorly described as is the physiological significance of this reorganization. Unpublished observations from our group, reveal that translocation to the IS is highly dependent upon a polybasic region at its C-terminus, with neutralization of any positive charges sufficient to block STIM1 polarization. Further, whereas cytosolic Ca2+ entry was normal in cells expressing these STIM1 mutants, mitochondrial Ca2+ loading and mROS production were defective. Further, Septins, a family of GTP-binding and complex forming proteins known to affect STIM1 localization through reorganization of phosphoinositides around ER-PM junctions, localize to the IS in a STIM1-dependent manner. Based on these preliminary findings, we propose the following hypothesis: Septin-mediated reorganization of Phosphatidylinositides control polarization of STIM1 and associated proteins towards the IS, driving mitochondrial Ca2+ loading, critical for metabolic reprogramming and T cell differentiation. This proposal is organized into 3 aims: Aim 1: Define the mechanisms and role of STIM1 translocation during IS formation. Here, we will i. define the role of STIM1 polarization on IS formation, including examining the translocation of STIM1 target proteins towards the IS and ii. determine the impact of loss of polarization on mitochondrial function. Aim 2: Assess interdependence between STIM1 and septins during IS formation. Here, we will use Septin4/5-KO cells to determine their contribution to Ca2+ entry and clearance and downstream signaling during T cell activation. In addition, we will use site-directed mutagenesis to eliminate GTPase function and then reexpress in KO cells. Implications to the localization of STIM1, STIM1 mutants and STIM1-associated proteins Orai1, PMCA4 and POST will be determined. Finally, we will determine the relationship between Septins and STIM1 on phosphoinositide localization during T cell activation. Aim 3: Role of STIM1 polarization on primary T cell activation. Here, we will use adoptive transfer to generate primary T cells expressing either STIM1WT or STIM1K672M. This will facilitate extending our work to include assessing how the failure of STIM1 to translocate to the IS affects T cell differentiation, cytokine production, expansion and elimination. These investigations will provide new insights into cellular mechanisms regulating STIM1 localization and function, particularly within the context of T cell activation. Completion of this work could have numerous applications, particularly given recent progress in immunology. Hence, the efficiency of IS formation determines the quality and nature of the immune response, with potential implications to the treatment of numerous autoimmune diseases and cancer immunology.

T 细胞激活是通过 T 细胞和抗原呈递细胞 (APC) 之间的直接接触启动的，从而导致 T 细胞向 APC 极化并形成免疫突触 (IS)。现在一般是 公认 Ca2+ 信号是 T 细胞激活所必需的。此外，Ca2+信号蛋白的重组 尽管驱动这一事件的根本机制尚不清楚，但 IS 形成过程中的这一事件已被广泛报道 描述的是这种重组的生理意义。我们小组未发表的观察结果， 研究表明，向 IS 的易位高度依赖于其 C 末端的多碱基区域，中和任何正电荷足以阻止 STIM1 极化。此外，虽然细胞质 Ca2+ 进入是 表达这些 STIM1 突变体的细胞中正常，但线粒体 Ca2+ 负载和 mROS 产生有缺陷。此外，Septins 是已知影响 STIM1 定位的 GTP 结合和复合物形成蛋白家族 通过重组 ER-PM 连接周围的磷酸肌醇，定位到 STIM1 依赖性的 IS 方式。基于这些初步发现，我们提出以下假设：Septin 介导的磷脂酰肌醇重组控制 STIM1 和相关蛋白向 IS 的极化，驱动 线粒体 Ca2+ 负载，对于代谢重编程和 T 细胞分化至关重要。该提案分为 3 个目标： 目标 1：定义 STIM1 易位在 IS 形成过程中的机制和作用。 在这里，我们将.定义 STIM1 极化对 IS 形成的作用，包括检查 STIM1 将蛋白质靶向 IS 和 ii。确定极化丧失对线粒体功能的影响。目标 2：评估 IS 形成过程中 STIM1 和脓毒症之间的相互依赖性。在这里，我们将使用 Septin4/5-KO 细胞以确定其对 Ca2+ 进入和清除以及下游信号传导的贡献 T 细胞激活。此外，我们将使用定点诱变消除GTPase功能，然后在KO细胞中重新表达。对 STIM1、STIM1 突变体和 STIM1 相关蛋白定位的影响 Orai1、PMCA4 和 POST 将被确定。最后，我们将确定 Septins 和 T 细胞激活过程中 STIM1 对磷酸肌醇定位的影响。目标 3：STIM1 极化对初级的作用 T 细胞激活。在这里，我们将使用过继转移来生成表达 STIM1WT 或 STIM1K672M。这将有助于扩展我们的工作，包括评估 STIM1 的易位失败如何 IS 影响 T 细胞分化、细胞因子产生、扩增和消除。这些调查将 为调节 STIM1 定位和功能的细胞机制提供新的见解，特别是在 T 细胞激活的背景。完成这项工作可能会有很多应用，特别是考虑到最近的 免疫学的进展。因此，IS形成的效率决定了免疫的质量和性质。 反应，对许多自身免疫性疾病和癌症免疫学的治疗具有潜在影响。