RPMI 1640 and G418 were obtained from GIBCO BRL. 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 proteinCGPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70Cmade up of clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 constantly dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results Dauricine 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. = 6). (D) Mean fluorescence recovery traces from cells plated for 20 min before photobleaching (= 6). (E) The extent of fluorescence recovery in bleached ROIs as a function of the age of the observed contacts at the time of photobleaching. In each case, half-maximal recovery is usually observed within 7.5 to 10 s. A nonphosphorylated lipid raft marker is not retained in signaling complexes Several reports have suggested that TCR ligation results in the coordinated accumulation of lipid rafts with the aggregated TCR (Janes et al., 1999). As a lipid raft component, LAT could be passively stabilized in signaling complexes through interactions with rafts associated with the activated TCR. To test this hypothesis we tracked the localization of LAT-EGFP and EYFP-GPI, a lipid raft marker (Keller et al., 2001). As shown above, LAT-EGFP transiently clusters upon T cell activation (Fig. 8 A; Video 12, available at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1). In contrast, EYFP-GPI does not cluster in this manner, suggesting that raft association is usually insufficient to direct clustering (Fig. 8 B; Video 13, available at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1). Fixed cell studies confirmed that LAT accumulates within signaling clusters identified by immunofluorescent staining for phosphotyrosine (Fig. 8 C). YFP-GPI, in contrast, does not colocalize with these signaling clusters, and appears to form aggregates alongside the phosphotyrosine-rich signaling clusters (Fig. 8 D). These data indicate that lipid raft components do not nonspecifically accumulate in signaling clusters. Furthermore, the inhibition of Src-family kinases by PP2 revealed that this clustering of LAT is usually strictly tyrosine kinase-dependent (Fig. 8, E and Dauricine F). These data indicate that the accumulation of LAT in signaling clusters is usually unlikely to be stabilized solely through interactions with lipid rafts, but does require phosphotyrosine-dependent interactions. Open in a separate window Physique 8. LAT is usually selectively retained in signaling complexes. (A and B) Jurkat cells expressing either LAT-EGFP or EYFP-GPI were plated on stimulatory coverslips and imaged using the Ultraview system. Five-image, 2.5 m deep Z stacks were collected every 20 s. The bright spot observed in the EYFP-GPICexpressing cells corresponds to a membrane fold, not a cluster. Comparable structures are also occasionally observed with Tac-EGFP, a nonraft membrane protein. (C and D) Jurkat cells expressing either LAT-EGFP or EYFP-GPI (green) were plated on stimulatory coverslips, fixed after Rabbit Polyclonal to GRP94 2 min, and stained for phosphotyrosine (red). (E and F) Jurkat cells expressing either LAT-EGFP or EYFP-GPI were treated with 10 M PP2, and then plated and imaged in C and D. Characterizing the formation and movement of SLP-76Crich structures To demonstrate that the unique movement of SLP-76 requires activation of the TCR, we plated SLP-76-YFPCexpressing cells on coverslips coated with Dauricine nonstimulatory antibodies. SLP-76 is usually recruited into moving clusters in response to TCR-ligation (see above; Fig. 9 A; Video 14, available at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1), but Dauricine not in response to either CD45 (Fig. 9 B; Video 15, available at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1) or CD43 ligation (Fig. 9 C; Video 16, available at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1). Furthermore, resting SLP-76-YFPCexpressing cells contain few or no detectable vesicle-like accumulations of SLP-76 (unpublished data). One property of the movement of SLP-76Crich structures observed in Videos 9 and 14 is usually striking: entire SLP-76Cmade up of.