title: Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.
abstract: 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.
authors.affiliation: Section of Hepatology and Gastroenterology, Department of Medicine, Medical School Building, Imperial College London, London W2 1PG, UK.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Purbhoo
authors.forename: Hebin
authors.initials: H
authors.lastname: Liu
authors.forename: Stephane
authors.initials: S
authors.lastname: Oddos
authors.forename: Dylan M
authors.initials: DM
authors.lastname: Owen
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Sophie V
authors.initials: SV
authors.lastname: Pageon
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Rudd
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Davis
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology; D006801:Humans; D054992:Immunological Synapses / Q000276:immunology*; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D022142:Secretory Vesicles / Q000276:immunology*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D017252:CD3 Complex; C409587:GRAP2 protein, human; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human