Dynein separately partners with NDE1 and dynactin to orchestrate T cell focused secretion

摘要

Helper and cytotoxic T cells accomplish focused secretion through the clustering of vesicles around the MTOC and translocation of the MTOC to the target contact site. Here, using Jurkat cells and OT-I T cell receptor (TcR) transgenic primary murine CTLs, we show that the dynein-binding proteins NDE1 and dynactin (as represented by p150^Glued) form mutually exclusive complexes with dynein, exhibit non-overlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-EGFP fusion) were activated by SEE-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150^Glued, which depleted the alternative dynein-dynactin complex, resulted in impaired accumulation of CTLA-4 and granzyme-B containing intracellular vesicles at the IS, while MTOC translocation was not affected. Depletion of p150^Glued in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lis1 and dynactin complexes separately mediate two key components of T cell focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.