Figure 2.

The Immune Synapse Undergoes Membrane Specialization as It Forms

(A–C) CTLs expressing EGFP-Tubby and Lifeact-mApple upon first contact with EL4-blue target (t = 0), initial depletion, and recovery of probes with time (min:s); 100% conjugates, n = 27; 14 independent experiments.

(A) Single confocal slices from Video S2A shown as merged and separate channels.

(B) En-face views across 3 μm slice of the synapse as single and merged channels with intensity plot for PI(4,5,)P2 (Tubby).

(C) Pixel intensity (y axis) plots against distance across the synapse (x axis, μm) for boxed region shown in (A).

(D–F) CTLs expressing EGFP-tagged probes (as shown) and Lifeact-mApple upon first contact between CTL and EL4-blue target (t = 0), showing initial clustering and depletion of probes with time (min:s) taken from Videos S2B–S2F.

(D) Single confocal slices taken from Video S2B (PA, PASS; 100% conjugates n = 9; all independent experiments), Video S2C (PI(4)P, Osh2p-PHx2; 100% conjugates, n = 16; all independent experiments), Video S2D (PS, Lactdherin-C2; 61% conjugates, n = 29; 14 independent experiments), Video S2E (PI(3,4,5)P3, Grp1-PH; 100% conjugates, n = 27; 15 independent experiments), and Video S2F (PI(3,4)P2, Bam32-PH; 70% conjugates, n = 10; all independent experiments) showing probes in green and Lifeact in red; EL4-blue targets in merged channel only.

(E) Corresponding en-face views across 3 μm slice of the synapse as single and merged channels with intensity plot for the phosphoinositide probe.

(F) En-Face Lifeact and phospholipid probe with pixel intensity plots of probes for boxed region shown in (D).

See also Figures S1 and S2.