Research Project
6852950
T cells perform two basic functions: they regulate the activity of other lymphocytes and they kill abnormal cells such as cancer cells and virally infected cells. In both cases, binding of the T cell to an antigen presenting (APC) induces the polarization of cytoskeletal elements within the T cell. Polarized microtubules form a scaffold along which secretory vesicles containing cytokines or lytic granules containing toxins move, directing their contents at the appropriate APC. Although this process is critical for the fidelity of the immune response, little is known about how it is controlled. This proposal is aimed at understanding how the binding of an appropriate APC triggers the polarization of the microtubule organizing center (MTOC) and subsequent microtubule-dependent secretion. Two distinct, though not mutually exclusive mechanisms that could drive MTOC polarization will be tested, and the role of kinesin and cytoplasmic dynein in lytic granule movement will be evaluated. The studies proposed in Aim 1 test the hypothesis that MTOC polarization is driven by localized microtubule shortening, signaled through phosphorylation of tubulin by ZAP-70 and phosphorylation of MAP4 by MAP-kinases. A panel of Jurkat T cells transfected with mutant and chimeric signaling molecules will be used to ask whether phosphorylation of tubulin and MAP4 correlates with polarization, and the effects of ZAP70 and MAP4 on microtubule dynamics will be investigated in vitro using video microscopy. Aim 2 will test the role of cytoplasmic dynein in MTOC reorientation, by asking whether dynein is recruited to the site of APC binding, and whether transfection with a dynactin subunit then disrupts dynein activity also disrupts MTOC polarization. Aim 3 will probe the contribution of cytoplasmic dynein and kinesin to the microtubule-dependent secretion of lytic granules by killer lymphocytes. CTL and NK cells will be transfected with dominant negative constructs that block the activity of dynein and kinesin, and granule distribution, secretion and cytolytic function will be tested. Changes in motor phosphorylation upon T cell activation will be characterized, to learn how their activity is regulated. The basic mechanisms under investigations are expected to be conserved in T helper cells, virus- and tumor-specific cytotoxic T cells and natural killer cells. Since therapeutic strategies based upon stimulating these cells are currently in clinical trials, gaining a better understanding of these fundamental processes will be critical for their optimal clinical deployment.
