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.
NIH ExPorter
NIH ExPorter