Abstract

Binding of T cells to antigen-presenting cells triggers translocation of the T cell microtubule organizing center (MTOC) and secretory vesicles to the immunological synapse, processes that are essential for T cell effector function. Data from localization and functional studies indicate that MTOC translocation and secretory vesicle movement rely on discrete pools of dynein. For example, we show that siRNA knockdown of dynactin blocks secretory vesicle accumulation but not MTOC translocation. In order to dissect how dynein is involved, we used in vivo molecular trap technology to sequester dynein intermediate and light chains (DIC and DLCs, respectively). In Jurkat cells expressing the traps, we demonstrated that the target protein co-immunoprecipitated with the trap in the presence but not absence of AP20187 (Ariad). In scoring MTOC translocation in activated Jurkat-Target pairs, addition of AP20187 reduced the values to 37% of positive controls in cells expressing the DIC trap and to 52% of control values in cells expressing the DLC LC8 trap. No effect was observed with the DLC TcTex trap, with or without the drug. Finally, we show that Lissencephaly1 protein (Lis1), a dynein-binding protein that is involved in polarized nuclear movements and spindle orientation, also appears to be important for MTOC polarization. Lis1 is recruited to the immunological synapse and co-immunoprecipitates with dynein. To test whether Lis1 is important for MTOC translocation, we prepared truncated Lis1 constructs containing GFP linked to either the N-terminal homodimerization domain or the C-terminal WD repeat domain. In activated Jurkat-Target pairs, MTOC translocation was reduced to 26% of the positive controls in Jurkat cells expressing the N-terminal domain and to 10% of the positive controls in cells expressing the WD repeat domain. Ongoing studies aim to determine the impact of Lis1 and DLCs on vesicle movements.

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