Multiple pools of dynein and their roles in T cell effector functions (P1028)

Abstract

Dramatic cytoskeletal activities and protein movements are seen when T cells engage cognate targets or antigen presenting cells. These include formation of a specialized contact site known as the immunological synapse (IS), clustering of vesicles around the microtubule organizing center (MTOC) and translocation of the MTOC up to the IS. Ultimately, this leads to focused secretion towards the cognate target. In an effort to understand how MTOC translocation takes place, we have investigated the roles of dynein, p150Glued, Lis1,NDE1 and NDEL1 using Jurkat cells as a model system. Using expressed molecular traps aimed at dynein intermediate chain (DIC) or LC8 (a dynein light chain), and Jurkat cells expressing fluorescent CTLA-4 constructs targeting secretory vesicles, we have shown that dynein is required for both vesicle clustering and MTOC translocation. When p150Glued expression was reduced, vesicles became dispersed but MTOC translocation was unaffected suggesting that dynactin was needed for vesicle movements but not MTOC translocation. MTOC translocation was potently blocked when Lis1 expression was reduced. We also find that NDE1 accumulates at the IS and pulls down with DIC and Lis1. Overexpression of GFP-NDE1 blocks MTOC polarization. The combination of localization and functional data suggests that there are two separate dynein complexes, a dynein-Lis1-NDE1/NDEL complex that mediates MTOC translocation and a dynein-dynactin complex that mediates vesicle clustering.