Mechanistic Analysis of T Cell Polarity by Photoactivation of Single Cells

描述（由申请人提供）：T 淋巴细胞在针对传染源和癌症的保护性免疫反应中发挥着核心作用，但必须严格控制其活性以防止自身免疫。这种控制被认为部分是通过 T 细胞微管 (MT) 细胞骨架的极化来实现的。具体来说，在识别抗原呈递细胞 (APC) 后，T 细胞的 MT 组织中心 (MTOC) 重新定向到 T 细胞-APC 界面正下方的位置，该位置也称为免疫突触 (IS)。这使得T细胞能够定向向APC分泌细胞因子或细胞毒性因子，从而分别限制细胞因子介导的通讯或细胞毒性杀伤的范围。我们工作的长期目标是确定控制 T 细胞 MT 极性的分子机制，并确定 MT 极性对体内 T 细胞功能的重要性。最近的研究表明，MTOC 向 APC 的极化是由 MT 运动蛋白动力蛋白介导的，该蛋白通过二酰基甘油 (DAG) 的局部积累而被招募到 IS。然而，DAG 积累到底是如何建立的以及它如何与动力蛋白招募结合仍不清楚。为了解决这些问题，将使用单细胞成像和功能方法来测试两个相关的假设：首先，二酰甘油激酶（DGK）和新型蛋白激酶C（nPKC）家族的成员是DAG的稳定积累和随后的MTOC重新定向所必需的；其次，nPKC 活性通过 Marcksl1 的磷酸化与动力蛋白偶联，Marcksl1 是一种与调节性动力蛋白复合物相互作用的 PKC 底物。将追求以下具体目标： 1) 识别和表征稳定 DAG 积累和 MTOC 重新定位所需的 nPKC 同工型； 2)确定DGK-1和DGK-6在塑造偏振DAG梯度中所起的作用； 3) 确定 MTOC 重新定向期间 Marcksl1 动态的重要性。对于第一个目标，功能丧失实验将与荧光成像相结合，以确定哪些 nPKC 同种型参与偏振响应。对于第二个目标，DGK-1 和 DGK-6 敲除小鼠将用于表征每种 DGK 同工型在 MTOC 重新定向过程中的具体作用。对于第三个目标，成像研究将与生化方法相结合，以确定 Marcksl1 是否通过与 dynactin 复合物结合来调节极性。这项工作将依赖于一种创新的光激活系统，该系统能够对 MTOC 偏振和相关信号事件进行高分辨率成像分析。这项研究很重要，因为它将识别 T 细胞中 MTOC 极化所需的分子和信号事件。这些知识将为未来旨在破译 T 细胞 MT 极性在体内的作用的研究奠定基础，并且还将有助于开发在生理或治疗背景下选择性调节 T 细胞极性的策略。因此，它与 NIH 的使命相关，因为它将有助于提高有助于改善人类健康的基础知识。 公共卫生相关性：T 淋巴细胞对于针对传染源和癌症的有效免疫反应至关重要。它们功能的关键是能够将自身定位于受感染或癌变的靶细胞，这使得它们能够选择性地消除这些细胞，而不伤害健康的旁观者组织。该提案旨在确定 T 细胞中这种极化过程如何发生，这可能有助于制定治疗环境中控制 T 细胞极性和功能的策略。

DESCRIPTION (provided by applicant): T lymphocytes play a central role in protective immune responses against infectious agents and cancer, but their activity must be tightly controlled in order to prevent autoimmunity. This control is thought to be achieved, in part, by the polarization of the T cell's microtubule (MT) cytoskeleton. Specifically, upon recognition of an antigen-presenting cell (APC), the MT organizing center (MTOC) of the T cell reorients to a position just beneath T cell-APC interface, which is also called the immunological synapse (IS). This enables the T cell to secrete cytokines or cytotoxic factors directionally toward the APC, thereby limiting the scope of cytokine- mediated communication or cytotoxic killing, respectively. The long-term goal of our work is to identify the molecular mechanisms that control MT polarity in T cells and to determine the importance of MT polarity for T cell function in vivo. Recent studies have shown that MTOC polarization toward the APC is mediated by the MT motor protein dynein, which is recruited to the IS by the localized accumulation of diacylglycerol (DAG). Precisely how DAG accumulation is established and how it is coupled to dynein recruitment, however, remain unclear. To address these issues, single cell imaging and functional approaches will be used to test two related hypotheses: first, that diacylglycerol kinases (DGKs) and members of the novel protein kinase C (nPKC) family are required for the stable accumulation of DAG and for subsequent MTOC reorientation; and second, that nPKC activity is coupled to dynein by phosphorylation of Marcksl1, a PKC substrate that interacts with the regulatory dynactin complex. The following specific aims will be pursued: 1) Identify and characterize the nPKC isoforms that are required for stable DAG accumulation and reorientation of the MTOC; 2) Determine the roles played by DGK-1 and DGK-6 in shaping the polarizing DAG gradient; and 3) Determine the importance of Marcksl1 dynamics during MTOC reorientation. For the first aim, loss-of-function experiments will be combined with fluorescence imaging to determine which nPKC isoform(s) are involved in the polarization response. For the second aim, DGK-1 and DGK-6 knockout mice will be used to characterize the specific role of each DGK isoform during MTOC reorientation. For the third aim, imaging studies will be combined with biochemical approaches to determine whether Marcksl1 regulates polarity through association with the dynactin complex. This work will rely upon an innovative photoactivation system that enables high- resolution imaging analysis of MTOC polarization and associated signaling events. This research is important because it will identify molecules and signaling events that are required specifically for MTOC polarization in T cells. This knowledge will provide a foundation for future studies aimed at deciphering the role of T cell MT polarity in vivo, and it will also contribute to the development of strategies to modulate T cell polarity selectively in physiological or therapeutic contexts. Hence, it is relevant to the NIH mission in that it will contribute to the advancement of basic knowledge that could aid in the improvement of human health. PUBLIC HEALTH RELEVANCE: T lymphocytes are crucial for effective immune responses against infectious agents and cancer. Key to their function is the ability to orient themselves toward infected or cancerous target cells, which allows them to selectively eliminate these cells without harming healthy bystander tissue. This proposal seeks to determine how this polarization process takes place in T cells, which could contribute to strategies for controlling T cell polarity and function in therapeutic contexts.