Figure 1.
Figure 1. Overexpression of p50-dynamitin-GFP impairs MTOC translocation upon TCR stimulation. (A and B) Dynein–dynactin components are found at the contact area between T and APC. The conjugates of J77 Jurkat T cells and SEE-pulsed Raji APC (A) or CH7C17 T cells and HA-pulsed Hom2 APC (B) were fixed with paraformaldehyde. Raji (R) and Hom2 (H) APC were CMAC-labeled (blue). Dynein–dynactin components were detected with specific antibodies (Ab; α-p150, p150-glued; α-p50, p50-dynamitin; α-p74, p74 dynein intermediate chain, clone 74.1) followed by Alexa 568–labeled anti–mouse secondary antibody (red in merged images). α-tubulin was detected with FITC-labeled antibody (green). Confocal z slices and corresponding 3D reconstructions of SMAC area (right) are shown (arrowheads in the z slice mark the cell–cell junction selected for 3D projection; arrows indicate MTOC). Bars, 10 μm. (C) p50-dynamitin-GFP overexpression prevents dynein–dynactin complex formation upon TCR stimulation. p150-glued was immunoprecipitated (IP) from J77 Jurkat cells overexpressing GFP (J77-GFP) or p50-dynamitin-GFP (J77-p50-GFP), then left unstimulated or stimulated by SEE during the indicated times. Samples were processed by Western blotting (WB) using the indicated antibodies. Arrows indicate positions of endogenous (50 kD) and GFP-tagged p50 (76 kD) and IgG H (55 kD). Ab, immunoprecipitating antibody; Bd, G–agarose-coupled beads; Lys, whole lysate; Rj, Raji cells. (D) Dynein–dynactin components cluster at IS in dynamitin-disrupted cells. Conjugates of dynamitin-disrupted Jurkat T cell clones and SEE-pulsed Raji APC (CMAC-labeled, blue) were processed as in A. GFP is shown in green. Bar, 10 μm. (E) Quantification of MTOC translocation in SEE-stimulated dynamitin-disrupted clones (c1, c3, c8, and c9) and GFP control cells. A total of 600 conjugates were counted from five independent experiments. Data are means ± SD. *, P < 0.05 versus GFP (Student's t test). (F) Time-lapse confocal video microscopy sequence showing the dynamics of MTOC movement in Jurkat cells overexpressing paxillin-GFP (green, control) or p50-dynamitin-GFP (green) at the indicated times. MTOC translocation was stimulated with SEE-pulsed Raji APC loaded with CMCTR cell tracker (red/orange). Images were captured at 37°C. Arrows indicate the position of the MTOC; arrowheads indicate T cell ISs. M, movie.

Overexpression of p50-dynamitin-GFP impairs MTOC translocation upon TCR stimulation. (A and B) Dynein–dynactin components are found at the contact area between T and APC. The conjugates of J77 Jurkat T cells and SEE-pulsed Raji APC (A) or CH7C17 T cells and HA-pulsed Hom2 APC (B) were fixed with paraformaldehyde. Raji (R) and Hom2 (H) APC were CMAC-labeled (blue). Dynein–dynactin components were detected with specific antibodies (Ab; α-p150, p150-glued; α-p50, p50-dynamitin; α-p74, p74 dynein intermediate chain, clone 74.1) followed by Alexa 568–labeled anti–mouse secondary antibody (red in merged images). α-tubulin was detected with FITC-labeled antibody (green). Confocal z slices and corresponding 3D reconstructions of SMAC area (right) are shown (arrowheads in the z slice mark the cell–cell junction selected for 3D projection; arrows indicate MTOC). Bars, 10 μm. (C) p50-dynamitin-GFP overexpression prevents dynein–dynactin complex formation upon TCR stimulation. p150-glued was immunoprecipitated (IP) from J77 Jurkat cells overexpressing GFP (J77-GFP) or p50-dynamitin-GFP (J77-p50-GFP), then left unstimulated or stimulated by SEE during the indicated times. Samples were processed by Western blotting (WB) using the indicated antibodies. Arrows indicate positions of endogenous (50 kD) and GFP-tagged p50 (76 kD) and IgG H (55 kD). Ab, immunoprecipitating antibody; Bd, G–agarose-coupled beads; Lys, whole lysate; Rj, Raji cells. (D) Dynein–dynactin components cluster at IS in dynamitin-disrupted cells. Conjugates of dynamitin-disrupted Jurkat T cell clones and SEE-pulsed Raji APC (CMAC-labeled, blue) were processed as in A. GFP is shown in green. Bar, 10 μm. (E) Quantification of MTOC translocation in SEE-stimulated dynamitin-disrupted clones (c1, c3, c8, and c9) and GFP control cells. A total of 600 conjugates were counted from five independent experiments. Data are means ± SD. *, P < 0.05 versus GFP (Student's t test). (F) Time-lapse confocal video microscopy sequence showing the dynamics of MTOC movement in Jurkat cells overexpressing paxillin-GFP (green, control) or p50-dynamitin-GFP (green) at the indicated times. MTOC translocation was stimulated with SEE-pulsed Raji APC loaded with CMCTR cell tracker (red/orange). Images were captured at 37°C. Arrows indicate the position of the MTOC; arrowheads indicate T cell ISs. M, movie.

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