Figure 1.

cSMAC Formation and CD45 Exclusion from Microclusters

(A-E) TCRs were visualized by TIRFM of Alexa-488-H57-Fab-labeled AND T cells forming IS on glass-supported bilayers containing ICAM-1 and 10 agonist MHCp/μm². (B)-(E) represent TIRFM images of the box around the cSMAC in (A). The cSMAC region starts out as a collection of micro-clusters (B), and these microclusters fuse to form the cSMAC structure. Images in (B) and (C) were scaled relative to (D) and (E) for visualization of microclusters.

(F-I) Each image in these panels represent three sequential frames in a time course observing cSMAC formation color coded in red, green, and blue and overlayed that are 6 s apart. Structures that appear white are those that are not undergoing motion, while portions that appear colored are those that are undergoing substantial motion. 5-6 min after contact formation (F and G), there is more relative motion among clusters than at 10-12 min (H and I). Scale bar in (E) equals 2 μm. Similar data were obtained from at least four independent experiments.

(J-O) CD45 and TCR were visualized by TIRFM of Alexa-488-I3/2.3-Fab and Alexa-568-H57-Fab-labeled AND T cells forming IS on glass-supported bilayers containing ICAM-1 and 10 agonist MHCp/μm². (J-L) Representative images of cells forming TCR microclusters (K) 30 s post contact formation that exclude CD45. (M-O) Representative images of cells 30 min post synapse formation. Microclusters formed at 30 min (N) continue to exclude CD45 (M); however, 93% of the cSMACs (see insets) are now enriched in CD45 (three independent experiments, total n = 45 cSMACs measured). Scale bar in (L) equals 4 μm.