The density of PS is substantially reduced in TCR microclusters during early synapse formation. (A and C) T cells expressing Lact-C2-mRFP were imaged by TIRFM on bilayers displaying HA-DR4/ICAM-1 at 30-s intervals. Early synapse A showing pMHC microclusters and Lact-C2 labeling of PS on inner leaflet of PM. Late synapse C showing cSMAC. Higher magnifications (inset boxes) shown beneath each image. Images representative of n > 3 experiments. Bar, 5 µm. (B and D) Inset magnifications used to calculate Pearson’s correlation coefficient (r), displayed as dot plots. Line tool used to quantify signal intensity of HA-DR4 (green) and Lact-C2 (red), see dotted lines in A and C. (E) T cells expressing C1-PKC-eGFP imaged by TIRFM on bilayers displaying HA-DR4/ICAM-1 (30-s intervals). Early synapse showing pMHC microclusters and C1-PKC labeling of DAG on inner leaflet of PM (top). Higher magnification (inset boxes) shown beneath each image. Images representative of n = 3 experiments. Bar, 5 µm. (F) Pearson’s correlation coefficient (inset box; r), displayed as dot plot. Line tool used to quantify signal intensity of HA-DR4 (blue) and C1-PKC (green) for dotted line in E.