LAT and TCRζ vesicle fusion at the immunological synapse is required for signal amplification. (A–F) Primary CD4 T cells were transfected with a siRNA control (siCtr; A–C) or a siRNA MAL (siMAL; D–F), and allowed to form conjugates with superantigen-loaded Raji cells for 30 min. Cells were stained for Lck and pLck (A and D), Lck and pLAT (B and E), or TCRζ and pTCRζ (C and F) and analyzed by 3D confocal microscopy. (G–I) Population analysis of primary CD4 T cells transfected with siRNA control (siCtr), or siRNA MAL (siMAL) of pLck (G), pLAT (H) and pTCRζ (I) fluorescence intensity at the immunological synapse of at least 20 conjugates per group. (J–O) Primary CD4 T cells were transfected with a siRNA control (siCtr; J–L) or siRNA Syt7 (siSyt7; M–O) and allowed to form conjugates with superantigen-loaded Raji cells. Cells were stained for Lck and pLck (J and M), Lck and pLAT (K and N), or TCRζ and pTCRζ (L and O) and analyzed by 3D confocal microscopy. (P–R) Population analysis of pLck (P), pLAT (Q), and pTCRζ (R) fluorescence intensity at the synapse of at least 20 conjugates per group, processed as in J–O. Confocal images were post-treated by deconvolution. A 1-µm-thick medial stack is shown. Synaptic clustering and intracellular compartments are highlighted by arrows and arrowheads, respectively. Each dot in plots represents one conjugate. . ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.