Defining STIM1 function at the Immunological Synapse

T细胞的激活是由T细胞和抗原提呈细胞(APC)之间的直接接触启动的，导致 T细胞对APC的极化和免疫突触(IS)的形成。现在一般都是 接受T细胞激活所需的钙信号。此外，钙信号蛋白的重组 在IS形成过程中已经有了广泛的报道，尽管驱动这一事件的潜在机制很少 被描述为这种重组的生理意义。我们小组未发表的观察结果， 揭示了到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止STIM1极化。此外，细胞内钙离子内流是 在表达这些STIM1突变体的细胞中，线粒体Ca~(2+)负荷和MROs的产生都是正常的。此外，Septins是一个已知影响STIM1定位的GTP结合和复杂形成蛋白家族 通过重组ER-PM连接周围的肌醇磷脂，定位于STIM1依赖的IS 举止。基于这些初步发现，我们提出了以下假设：Septin介导的磷脂酰肌醇重组控制STIM1及其相关蛋白对IS的极化，驱动 线粒体钙负荷，代谢重编程和T细胞分化的关键。该提案分为3个目标：目标1：确定STIM1易位在IS形成过程中的机制和作用。 在这里，我们将I.定义STIM1极化在IS形成中的作用，包括检查 针对IS和II的STIM1靶蛋白。确定极化丧失对线粒体功能的影响。目的2：评估STIM1和Septins在IS形成过程中的相互依赖关系。在这里，我们将使用 Septin4/5-KO细胞在钙离子转运和清除及下游信号转导中的作用 T细胞活化。此外，我们还将使用定点突变来消除GTP酶功能，然后在KO细胞中重新表达。STIM1、STIM1突变体及其相关蛋白的定位意义 ORAI1、PMCA4和POST将确定。最后，我们将确定Septins和 STIM1对T细胞活化过程中肌醇磷脂定位的影响目标3：STIM1极化在初级医学中的作用 T细胞活化。在这里，我们将使用过继转移来产生原代T细胞，表达STIM1WT或 STIM1K672M。这将有助于将我们的工作扩展到包括评估STIM1的故障如何移位 IS影响T细胞的分化、细胞因子的产生、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。这项工作的完成可能会有许多应用，特别是考虑到最近 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 对多种自身免疫性疾病和癌症免疫学的治疗具有潜在的意义。

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 Unpublished observations from our group, reveal that translocation to the IS is highly dependent upon a polybasic region at its C-terminus, with neutralization of any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