Regulation of Sustained Actin Dynamics by the TCR and Costimulation as a Mechanism of Receptor Localization

Abstract

The localization of receptors, signaling intermediates, and cytoskeletal components at the T cell/APC interface is thought to be a major determinant of efficient T cell activation. However, important questions remain open. What are the dynamics of the T cell cytoskeleton as a potential mediator of such localization? How are they regulated by the TCR and costimulatory receptors? Do they actually mediate receptor localization? In this study, we have addressed these questions. Even under limiting T cell activation conditions, actin accumulated immediately and transiently at the T cell/APC interface, the microtubule organizing center reoriented toward it. In contrast, sustained (>5 min) actin accumulation in highly dynamic patterns depended on an optimal T cell stimulus: high concentrations of the strong TCR ligand agonist peptide/MHC and engagement of the costimulatory receptors CD28 and LFA-1 were required in an overlapping, yet distinct, fashion. Intact sustained actin dynamics were required for interface accumulation of TCR/MHC in a central pattern and for efficient T cell proliferation, as established using a novel approach to selectively block only the sustained actin dynamics. These data suggest that control of specific elements of actin dynamics by TCR and costimulatory receptors is a mechanism to regulate the efficiency of T cell activation.

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…In cells treated with colchicine to disrupt microtubules, STIM1 localized to blebs that protruded from the top of the cell instead of forming a normal cap (Supplemental Figure S4, A and B). Evaluating the effects of disrupting the actin cytoskeleton was problematic because complete disassembly inhibits TCR activation, Ca 2ϩ flux and blocks spreading of T-cells (Valitutti et al, 1995;Bunnell et al, 2001;Tskvitaria-Fuller et al, 2003). We used a low dose of latrunculin to disrupt the actin network as visualized by actin-GFP (data not shown).…”

A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

“…Internal -chain accumulation was defined as a spherical area of increased fluorescence (Ն40% above cellular background) at the center of the interface that was removed at least 1 area diameter from the T cell-APC interface, which did not have any overlap with the APC and did not last longer than two frames (i.e., 40 s). Both tat SEC7 and tat SEC7mt were constructed, expressed in Escherichia coli, and purified in analogy to the previously described tat WASP VCA domain (17) using the published SEC7 inactivating mutation and domain boundaries (18). The tat dynein L chain 1 (DLC1) peptide was the same as published (19) with the exception of a disulfide bond linking the tat and the DLC1 moieties instead of a peptide bond.…”

“…For two-color three-dimensional imaging, an actin-mCherry (21) fusion protein was generated and retrovirally expressed as actin-GFP (17). During image acquisition, both red and green images were acquired at each z plane before moving to the next plane.…”

“…The tat dynein L chain 1 (DLC1) peptide was the same as published (19) with the exception of a disulfide bond linking the tat and the DLC1 moieties instead of a peptide bond. The tat SEC7mt and tat DLC1 were used at the indicated concentrations as previously described (17). Briefly, the T cells were preincubated for 30 min at 37°C with the tat fusion reagent, and the reagent was present during the T cell to APC interactions at the same concentration.…”

“…The microscopy system and image acquisition have been described in detail (17). Briefly, primary T cells and peptide incubated APCs were allowed to interact at 37°C on the microscope stage.…”