Mechanistic Analysis of T Cell Polarity by Photoactivation of Single Cells

描述（由申请人提供）：T淋巴细胞在抵抗感染因子和癌症的保护性免疫反应中起着核心作用，但为了防止自身免疫，它们的活性必须受到严格控制。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）重新定位到T细胞-APC界面下方的位置，该位置也称为免疫突触（is）。这使得T细胞能够向APC定向分泌细胞因子或细胞毒因子，从而分别限制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细胞中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dynein介导的，该蛋白通过二酰基甘油（DAG）的局部积累被募集到is。然而，确切地说，DAG积累是如何建立的，以及它是如何与动力蛋白募集相结合的，目前尚不清楚。为了解决这些问题，单细胞成像和功能方法将用于测试两个相关假设：首先，二酰基甘油激酶（DGKs）和新型蛋白激酶C （nPKC）家族成员是DAG稳定积累和随后MTOC重新定向所必需的；其次，nPKC的活性是通过与调节动力蛋白复合物相互作用的PKC底物Marcksl1的磷酸化与动力蛋白偶联的。将追求以下具体目标：1)识别和表征稳定DAG积累和MTOC重新定向所需的nPKC同工型；2)确定DGK-1和DGK-6在形成极化DAG梯度中的作用；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异构体参与了极化响应。第二个目的是，将使用DGK-1和DGK-6敲除小鼠来表征每种DGK异构体在MTOC重定向过程中的具体作用。对于第三个目标，影像学研究将与生化方法相结合，以确定Marcksl1是否通过与dynactin复合物的关联来调节极性。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极化和相关信号事件进行高分辨率成像分析。这项研究很重要，因为它将确定T细胞中MTOC极化所需的分子和信号事件。这些知识将为未来的研究提供基础，旨在破译T细胞MT极性在体内的作用，它也将有助于在生理或治疗背景下选择性调节T细胞极性的策略的发展。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。

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.