ABSTRACT
Polarization of the T cell microtubule-organizing center (MTOC) to the immunological synapse between the T cell and an antigen-presenting cell (APC) maintains the specificity of T cell effector responses by enabling directional secretion toward the APC. The reorientation of the MTOC is guided by a sharp gradient of the second messenger diacylglycerol (DAG), which is centered at the immunological synapse. We used a single-cell photoactivation approach to demonstrate that diacylglycerol kinase α (DGK-α), which catalyzes the conversion of DAG to phosphatidic acid, determined T cell polarity by limiting the diffusion of DAG. DGK-α-deficient T cells exhibited enlarged accumulations of DAG at the immunological synapse, as well as impaired reorientation of the MTOC. In contrast, T cells lacking the related isoform DGK-ζ did not display polarization defects. We also found that DGK-α localized preferentially to the periphery of the immunological synapse, suggesting that it constrained the area over which DAG accumulated. Phosphoinositide 3-kinase activity was required for the peripheral localization pattern of DGK-α, which suggests a link between DAG and phosphatidylinositol signaling during T cell activation. These results reveal a previously unappreciated function of DGK-α and provide insight into the mechanisms that determine lymphocyte polarity.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , Cell Polarity/immunology , Diacylglycerol Kinase/metabolism , Diglycerides/metabolism , Immunological Synapses/metabolism , Microtubule-Organizing Center/metabolism , Animals , Diacylglycerol Kinase/genetics , Diacylglycerol Kinase/immunology , Enzyme-Linked Immunosorbent Assay , Image Processing, Computer-Assisted , Mice , Mice, Transgenic , Phosphatidylinositol 3-Kinases/metabolism , Single-Cell Analysis , Statistics, NonparametricABSTRACT
The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.
