Mechanistic Analysis of T Cell Polarity by Photoactivation of Single Cells

描述(由申请人提供)：T淋巴细胞在针对感染性因素和癌症的保护性免疫反应中发挥核心作用，但必须严格控制它们的活动，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)细胞骨架的极化实现的。具体地说，在识别抗原提呈细胞(APC)后，T细胞的MT组织中心(MTOC)重新定位到T细胞-APC界面下的位置，也被称为免疫突触(IS)。这使得T细胞能够直接向APC分泌细胞因子或细胞毒因子，从而分别限制了细胞因子介导的通讯或细胞毒杀伤的范围。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极性在体内对T细胞功能的重要性。最近的研究表明，MTOC对APC的极化是由MT马达蛋白Dynein介导的，该蛋白通过二酰甘油(DAG)的局部积累被招募到IS。然而，DAG的积累是如何建立的，以及它是如何与动力蛋白的招募结合在一起的，目前仍不清楚。为了解决这些问题，单细胞成像和功能方法将被用来检验两个相关的假设：第一，二酰甘油激酶(DGK)和新的蛋白激酶C(NPKC)家族的成员对于DAG的稳定积累和随后的MTOC重定位是必需的；第二，nPKC的活性通过MARCKSL1的磷酸化与Dynein偶联，MARCKSL1是一种与调节的dynactin复合体相互作用的PKC底物。我们将致力于以下具体目标：1)确定和表征稳定的DAG积累和MTOC重定向所需的nPKC亚型；2)确定DGK-1和DGK-6在形成极化DAG梯度中所起的作用；以及3)确定MARCKSL1动力学在MTOC重定向过程中的重要性。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亚型(S)参与了偏振响应。对于第二个目的，将使用DGK-1和DGK-6基因敲除小鼠来表征每种DGK亚型在MTOC重定向过程中的特定作用。对于第三个目标，成像研究将与生化方法相结合，以确定MARCKSL1是否通过与动力蛋白复合体的联系来调节极性。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偏振和相关的信号事件进行高分辨率成像分析。这项研究很重要，因为它将确定T细胞中MTOC极化所特别需要的分子和信号事件。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提供基础，也将有助于开发在生理或治疗环境中选择性调节T细胞极性的策略。因此，它与国家卫生研究院的任务相关，因为它将有助于促进基础知识的发展，从而有助于改善人类健康。 公共卫生相关性：T淋巴细胞对有效的免疫反应是至关重要的，以对抗感染性病原体和癌症。它们功能的关键是将自己定位于感染或癌变的靶细胞的能力，这使得它们能够选择性地消除这些细胞，而不会损害健康的旁观者组织。这项建议试图确定这种极化过程是如何在T细胞中发生的，这可能有助于在治疗环境中控制T细胞的极性和功能的策略。

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.