Defining STIM1 function at the Immunological Synapse

T细胞活化通过T细胞和抗原呈递细胞（APC）之间的直接接触而启动，导致T细胞活化。 T细胞向APC的极化和免疫突触（IS）的形成。现在人们普遍 认为Ca 2+信号是T细胞活化所必需的。此外，Ca 2+信号蛋白的重组 在IS形成过程中，尽管驱动这一事件的潜在机制尚不清楚， 这种重组的生理意义。我们小组未发表的观察结果， 揭示了向IS的易位高度依赖于其C末端的多碱基区域，任何正电荷的中和足以阻断STIM 1极化。此外，虽然细胞质Ca 2+内流是 在表达这些STIM 1突变体的细胞中正常，线粒体Ca 2+负载和mROS产生是有缺陷的。此外，已知影响STIM 1定位的GTP结合和复合物形成蛋白质家族Septins 通过ER-PM连接点周围磷酸肌醇的重组，以STIM 1依赖性的方式定位于IS。 方式基于这些初步发现，我们提出以下假设：Septin介导的磷脂酰肌醇的重组控制STIM 1和相关蛋白向IS的极化， 线粒体Ca 2+负载，对代谢重编程和T细胞分化至关重要。本提案分为3个目标：目标1：定义IS形成过程中STIM 1易位的机制和作用。 在这里，我们将我。定义STIM 1极化对IS形成的作用，包括检查 STIM 1靶向IS蛋白，和ii.确定极化丧失对线粒体功能的影响。目的2：评估IS形成过程中STIM 1和septins之间的相互依赖性。在这里，我们将使用 Septin 4/5-KO细胞，以确定它们对Ca 2+进入和清除以及下游信号传导的贡献。 T细胞活化。此外，我们将使用定点突变来消除GTdR功能，然后在KO细胞中重新表达。STIM 1、STIM 1突变体和STIM 1相关蛋白定位的意义 将确定Orai 1、PMCA 4和POST。最后，我们将确定Septins和 STIM 1对T细胞活化期间磷酸肌醇定位的影响。目的3：STIM 1极化对原发性 T细胞活化。在这里，我们将使用过继转移来产生表达STIM 1 WT或STIM 1 WT的原代T细胞。 STIM1K672M。这将有助于扩展我们的工作，包括评估STIM 1易位失败是如何发生的。 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STIM 1定位和功能的细胞机制提供了新的见解，特别是在 T细胞活化的背景。这项工作的完成可能有许多应用，特别是考虑到最近的 免疫学的进步。因此，IS形成的效率决定了免疫的质量和性质。 反应，具有潜在的影响，许多自身免疫性疾病和癌症免疫学的治疗。

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 Unpublished observations from our group, reveal that translocation to the IS is highly dependent upon a polybasic region at its C-terminus, with neutralization of any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.