Dynamics of Subsynaptic Vesicles and Surface Microclusters at the Immunological Synapse

Vesicles dynamically deliver the T cell adaptor protein LAT to sites of T cell signalosomes. Submariner Adaptor Engagement of the T cell antigen receptor (TCR) on the surface of a T cell with peptide-loaded major histocompatibility complex on the surface of an antigen-presenting cell occurs at a specialized contact point known as the immunological synapse (IS). Stimulation of the TCR triggers the activation of the proximal kinases Lck and ZAP-70, which leads to the formation at the IS of microclusters of kinases and adaptor molecules that are required for T cell signaling. Two of these adaptor molecules are the cytosolic protein SLP-76 and the plasma membrane–associated protein LAT. SLP-76 and LAT form distinct microclusters, but how they interact to propagate T cell signals is unclear (see the Perspective by Billadeau). Purbhoo et al. used imaging techniques to show that a fraction of LAT was enriched in intracellular vesicles that resided below the IS and that these vesicles moved to the IS, where they interacted with SLP76-containing microclusters. LAT-containing vesicles were corralled by TCR–ZAP-70 microclusters in the IS and were more mobile than the SLP-76 microclusters, but they slowed when they interacted with SLP-76. Cells that contained a mutant LAT that could not interact with SLP-76 (through the associated protein GADS) contained fewer SLP-76 microclusters, and LAT-containing vesicles interacted less with them. Together, these data suggest that vesicular LAT interacts with protein microclusters at the IS and contributes to the propagation of T cell signaling. Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor–bound protein–related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell–APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76–containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.