Forced expression of Lck at the plasma membrane in MAL-deficient cells corrects TCR targeting to the IS. JTIM cells were transfected with the CD4/Lck or the LAT/Lck plasmids. After 48 h at 37°C, cells were conjugated to SEE-loaded Raji cells for 20 min and fixed. Transfected cells were detected with antibodies to mouse CD4 or to the c-Myc tag to detect expression of the CD4/Lck or LAT/Lck chimeras, respectively. (A) The distribution of TCR and MTOC was determined by immunofluorescence analysis. In the case of MTOC analysis, the contour of the cells has been drawn with a dotted line to facilitate the identification of the cells. The arrows indicate the position of the TCR or MTOC in the T cells expressing the chimera. (B) The distribution of ICAM-3, ZAP-70, and PKC-θ was determined by immunofluorescence analysis. Conjugates in A and B formed by untransfected JTIM cells serve as internal controls. Bars, 5 μm. (C) Quantitative analysis of the polarization of TCR, MTOC, ZAP-70, and PKC-θ in JTIM cells expressing the CD4/Lck or LAT Lck chimeras. Three independent experiments were performed. n > 100 T cells/experiment. The histogram shows the mean percentage ± SEM of APC conjugates with TCR, MTOC, ZAP-70, or PKC-θ polarized to the IS. (D and E) Jurkat cells and JTIM cells transfected or not with the CD4/Lck or LAT/Lck chimeras were transfected with CD4ΔCyt-GFP (D) or pEGFP-C1 (E) and treated with activating anti-CD3 antibodies or PMA as indicated. After 15 min (D) or 16 h (E) at 37°C, EGFP-expressing cells were analyzed for phosphorylated Erk (D) or CD69 (E) expression by flow cytometry. The cotransfection efficiency was >80% as determined by immunofluorescence microscopy. The histograms shows the percentage ± SEM of the mean fluorescence of phospho-Erk and CD69 obtained in each case relative to that of Jurkat cells stimulated with anti-CD3 antibodies. Three independent experiments were performed.