T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.
Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D008563:Membrane Lipids / Q000502:physiology*; D021962:Membrane Microdomains; D008565:Membrane Proteins / Q000502:physiology; D018613:Microscopy, Confocal; D019308:Palmitic Acid / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; D008164:Luminescent Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D002784:Cholesterol; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D002118:Calcium