Lipid rafts are specialized, liquid-ordered subdomains of the plasma membrane. Through their ability to promote specific compartmentalization of lipids and membrane proteins, lipid rafts have emerged as membrane platforms specialized in signal transduction organization. The authors studied the spatio-temporal formation of signaling clusters in immune signaling after T cell activation. JURKAT cells were nucleofected with a GPI-EYFP fusion protein (a lipid raft marker) to localize the formation of clusters containing e.g. ZAP-70, LAT, Grb2, Gads. GPI was not found associated with lipid raft formation.
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.