The density of PS is substantially reduced during early stages of synapse formation. (A) T cells expressing a PS probe (Lact-C2-mRFP) and DAG probe (C1-PKC-eGFP) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Dotted line depicts cross section through cell for quantification shown in B. Images representative of n = 3 experiments. Bar, 5 µm. (B and D) Cross section of fluorescence signal intensity for cells shown in (A and C) at bead–cell contact as well as irrelevant area of PM. Red, Lact-C2; blue, HA-DR4 (SA-647); green, C1-PKC. Asterisk in B indicates Lact-C2 signal from intracellular compartment. (C and D) T cells expressing a PS probe (Lact-C2-mRFP) were stimulated with control CLIP-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Dotted line depicts cross section through cell for graph in D. Images representative of n = 3 experiments. Bar, 5 µm. (E) Population analysis (n ≥ 30 cells) of relative fluorescence of Lact-C2-mRFP at synapse for cells stimulated with HA-DR4 or control CLIP-DR4 beads. SEM is shown; *, P < 0.0001. n > 10 cells per experiment. (F) DR4-expressing Priess cells were pulsed with HA peptide (1 µM) for 1 h. T cells were added and imaged by confocal microscopy. Distribution of Lact-C2 and C1-PKC was assessed in stable cell–cell conjugates. Gray dotted line depicts cross section used to generate graph in (G); cyan-colored dotted line depicts Priess cell (APC). Images representative of n = 3 experiments. Bars, 10 µm. (G) Fluorescence signal intensity of probes at cell–cell contact and irrelevant area of cell surface (red, Lact-C2; green, C1-PKC).