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 Unpublished observations from our group, reveal that translocation to the IS is highly dependent upon a polybasic region at its C-terminus, with neutralization of any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细胞与抗原呈递细胞（APC）的直接接触开始的，导致 T细胞向APC的极化和免疫突触的形成（IS）。现在通常 接受T细胞激活需要Ca2+信号。此外，CA2+信号蛋白的重组 在IS形成期间已广泛报道，尽管驱动此事件的基本机制很差 被描述为这种重组的生理意义。我们小组未发表的观察 揭示向IS的易位高度依赖于其C末端的多重小区域，并具有足以阻止STIM1极化的任何正电荷中和。此外，胞质Ca2+进入 在表达这些STIM1突变体的细胞中，线粒体Ca2+负载和MROS产生的正常状态有缺陷。此外，Septins，一个GTP结合和复杂形成的蛋白质的家族已知会影响STIM1定位 通过对ER-PM连接周围的磷酸肌醇的重组，将其定位于IS依赖于stim1 方式。基于这些初步发现，我们提出了以下假设：磷脂酰肌醇的septin介导的重组刺激性蛋白质和相关蛋白的极化向IS，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。该提议分为3个目标：目标1：定义IS形成过程中STIM1易位的机理和作用。 在这里，我们会的。定义STIM1极化对形成的作用，包括检查的易位 STIM1靶向IS和II。确定损失对线粒体功能的影响。 AIM 2：评估IS形成过程中Stim1和Septins之间的相互依赖性。在这里，我们将使用 septin4/5-ko细胞确定它们对Ca2+进入的贡献，清除和下游信号传导 T细胞激活。此外，我们将使用位置定向的诱变来消除GTPase函数，然后在KO细胞中重新表达。对STIM1，STIM1突变体和STIM1相关蛋白的定位的影响 ORAI1，PMCA4和POST将确定。最后，我们将确定septins和 在T细胞激活过程中，刺激量在磷酸肌醇的定位上。 AIM 3：STIM1极化在主要的作用 T细胞激活。在这里，我们将使用收养转移来生成表达imt1wt或 Stim1k672m。这将有助于扩展我们的工作，以包括评估Stim1失败的方式 IS会影响T细胞分化，细胞因子的产生，膨胀和消除。这些调查会 提供有关调节Stim1定位和功能的细胞机制的新见解，尤其是在 T细胞激活的上下文。这项工作的完成可能会有许多申请，特别是考虑到最近 免疫学进展。因此，IS形成的效率决定免疫的质量和性质 反应，对多种自身免疫性疾病和癌症免疫学的治疗具有潜在的影响。